WO2018185618A1 - Anti-cdh6 antibody drug conjugates and anti-gitr antibody combinations and methods of treatment - Google Patents

Anti-cdh6 antibody drug conjugates and anti-gitr antibody combinations and methods of treatment Download PDF

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WO2018185618A1
WO2018185618A1 PCT/IB2018/052151 IB2018052151W WO2018185618A1 WO 2018185618 A1 WO2018185618 A1 WO 2018185618A1 IB 2018052151 W IB2018052151 W IB 2018052151W WO 2018185618 A1 WO2018185618 A1 WO 2018185618A1
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seq
chain variable
variable region
light chain
heavy chain
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PCT/IB2018/052151
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French (fr)
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Brandon Peter ANTONAKOS
Carl Uli BIALUCHA
Scott Collins
Deborah A. Knee
Fiona Alexandra SHARP
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Novartis Ag
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure is directed to the combination of antibody drug conjugates
  • ADC in combination with anti-GITR agonist antibodies and their uses for the treatment of cancer.
  • Cadherin-6 is a member of the cadherin superfamily of calcium-dependent cell-cell adhesion molecules. Aside from their role in mechanical adhesion, cadherins are involved in a diverse array of cellular processes relating to tissue morphogenesis (Haliata and Nelson Genes Dev. 2006; 20(23):3199-3214). This superfamily is classified into classical, desmosomal, and protocadherin groups with classical cadherins subdivided into Type 1 and Type II (Sotomayor et al., Trends Cell Biol. 2014; 34(9):524-536)).
  • CDH6 is a type II, classical cadherin, first described as K-cadherin, which was found to be preferentially expressed in fetal kidney and kidney carcinoma (Xiang et al., Cancer Res. 1994; 54(11):3034-3041; Paul et al., Cancer Res. 1997; 57(13):2741-2748), serous ovarian carcinoma (Kobel et al., PLoS Med. 2008 5(12):e232), as well as during normal renal development (Cho et al., Development 1998; 125(5):803-812; Mah et al., Dev. Biol. 2000; 223(1):38- 53).
  • ADCs Antibody drug conjugates
  • ADCs have been used for the local delivery of cytotoxic agents in the treatment of cancer (see e.g., Lambert, Curr. Opinion In Pharmacology 2005; 5:543-549).
  • ADCs allow targeted delivery of the drug moiety where maximum efficacy with minimal toxicity may be achieved.
  • ADCs delivering microtubule-disrupting payloads can induce a pro-inflammatory tumor
  • GITR Glucocorticoid-induced TNFR-related protein
  • GITR Necrosis Factor Superfamily (TNFRSF) constitutively expressed on regulatory T cells (Tregs) and at low levels on naive and memory T cells.
  • GITR is a bi-functional molecule capable of driving the expansion of CD8+ T effector (Teff) memory cell populations, while promoting the loss or inhibition of Tregs (Knee et al., Eur. J. Cancer 2016; 67: 1-10).
  • ADC induces GITR upregulation on Tregs
  • a synergistic combination of a CDH6 -targeting ADC and agonistic GITR antibody induces greater antitumor activity.
  • a combination comprising: a) an anti-CDH6 antibody drag conjugate of the formula:
  • Ab is an antibody or antigen binding fragment thereof that specifically binds to an epitope of human CDH6;
  • L is a linker
  • D is a drag moiety, wherein the drag moiety is N(2')-deacetyl-N2-(4- mercapto-4-methyl- 1 - oxopentyl)-maytansine (DM4) or N(2')- deacetyl-N(2')-(3-mercapto-l-oxopropyl)-maytansine (DM1) ; m is an integer from 1 to 8; and
  • n is an integer from 1 to 10; and b) an anti-GITR agonist antibody or antigen binding fragment thereof.
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:224, (b) a LCDR2 of SEQ ID NO:225, (c) a LCDR3 of SEQ ID NO:226; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 227, (e) a HCDR2 of SEQ ID NO: 228, and (f) a HCDR3 of SEQ ID NO:229;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:210, (b) a LCDR2 of SEQ ID NO:211, (c) a LCDR3 of SEQ ID NO:212; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:213, (e) a HCDR2 of SEQ ID NO: 214, and (f) a HCDR3 of SEQ ID NO:215;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:266, (b) a LCDR2 of SEQ ID NO:267, (c) a LCDR3 of SEQ ID NO:268; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 269, (e) a HCDR2 of SEQ ID NO:270, and (f) a HCDR3 of SEQ ID NO: 271;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:308, (b) a LCDR2 of SEQ ID NO:309, (c) a LCDR3 of SEQ ID NO:310; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:311, (e) a HCDR2 of SEQ ID NO:312, and (f) a HCDR3 of SEQ ID NO:313; (v) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 14, (b) a LCDR2 of SEQ ID NO: 15, (c) a LCDR3 of SEQ ID NO: 16; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 17, (e) a HCDR2 of SEQ ID NO: 18, and (f)
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:28, (b) a LCDR2 of SEQ ID NO:29, (c) a LCDR3 of SEQ ID NO:30; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:31, (e) a HCDR2 of SEQ ID NO:32, and (f) a HCDR3 of SEQ ID NO:33;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:42, (b) a LCDR2 of SEQ ID NO:43, (c) a LCDR3 of SEQ ID NO:44; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:45, (e) a HCDR2 of SEQ ID NO:46, and (f) a HCDR3 of SEQ ID NO:47;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:56, (b) a LCDR2 of SEQ ID NO:57, (c) a LCDR3 of SEQ ID NO:58; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:59, (e) a HCDR2 of SEQ ID NO:60, and (f) a HCDR3 of SEQ ID NO:61;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:70, (b) a LCDR2 of SEQ ID NO:71, (c) a LCDR3 of SEQ ID NO:72; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:73, (e) a HCDR2 of SEQ ID NO:74, and (f) a HCDR3 of SEQ ID NO:75;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:84, (b) a LCDR2 of SEQ ID NO:85, (c) a LCDR3 of SEQ ID NO:86; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:87, (e) a HCDR2 of SEQ ID NO: 88, and (f) a HCDR3 of SEQ ID NO: 89;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:98, (b) a LCDR2 of SEQ ID NO:99, (c) a LCDR3 of SEQ ID NO: 100; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 101, (e) a HCDR2 of SEQ ID NO: 102, and (f) a HCDR3 of SEQ ID NO: 103;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 112, (b) a LCDR2 of SEQ ID NO: 113, (c) a LCDR3 of SEQ ID NO: 114; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 115, (e) a HCDR2 of SEQ ID NO: 116, and (f) a HCDR3 of SEQ ID NO: 117; (xiii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 126, (b) a LCDR2 of SEQ ID NO: 127, (c) a LCDR3 of SEQ ID NO: 128; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 129, (e) a HCDRl of S
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 140, (b) a LCDR2 of SEQ ID NO: 141, (c) a LCDR3 of SEQ ID NO: 142; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 143, (e) a HCDR2 of SEQ ID NO: 144, and (f) a HCDR3 of SEQ ID NO: 145;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 154, (b) a LCDR2 of SEQ ID NO: 155, (c) a LCDR3 of SEQ ID NO: 156; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 157, (e) a HCDR2 of SEQ ID NO: 158, and (f) a HCDR3 of SEQ ID NO: 159;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 168, (b) a LCDR2 of SEQ ID NO: 169, (c) a LCDR3 of SEQ ID NO: 170; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 171, (e) a HCDR2 of SEQ ID NO: 172, and (f) a HCDR3 of SEQ ID NO: 173;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 182, (b) a LCDR2 of SEQ ID NO: 183, (c) a LCDR3 of SEQ ID NO: 184; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 185, (e) a HCDR2 of SEQ ID NO: 186, and (f) a HCDR3 of SEQ ID NO: 187;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 196, (b) a LCDR2 of SEQ ID NO: 197, (c) a LCDR3 of SEQ ID NO: 198; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 199, (e) a HCDR2 of SEQ ID NO:200, and (f) a HCDR3 of SEQ ID NO:201;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:238, (b) a LCDR2 of SEQ ID NO:239, (c) a LCDR3 of SEQ ID NO:240; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:241, (e) a HCDR2 of SEQ ID NO:242, and (f) a HCDR3 of SEQ ID NO:243;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:252, (b) a LCDR2 of SEQ ID NO:253, (c) a LCDR3 of SEQ ID NO:254; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:255, (e) a HCDR2 of SEQ ID NO:256, and (f) a HCDR3 of SEQ ID NO:257; (xxi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:280, (b) a LCDR2 of SEQ ID NO:281, (c) a LCDR3 of SEQ ID NO:282; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:283, (e) a HCDR2 of SEQ ID NO:28
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:294, (b) a LCDR2 of SEQ ID NO:295, (c) a LCDR3 of SEQ ID NO:296; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:297, (e) a HCDR2 of SEQ ID NO:298, and (f) a HCDR3 of SEQ ID NO:299; or
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:322, (b) a LCDR2 of SEQ ID NO:323, (c) a LCDR3 of SEQ ID NO:324; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:325, (e) a HCDR2 of SEQ ID NO:326, and (f) a HCDR3 of SEQ ID NO:327.
  • v a heavy chain variable region (vH) that comprises SEQ ID NO: 20, and a light chain variable region (vL) that comprises SEQ ID NO:21;
  • xv a heavy chain variable region (vH) that comprises SEQ ID NO: 160, and a light chain variable region (vL) that comprises SEQ ID NO: 161;
  • linker (L) is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker and a dicarboxylic acid based linker.
  • the linker is derived from a cross-linking reagent selected from the group consisting of: N-succinimidyl-4-(2-pyridyldithio)2- sulfo-butanoate (sulfo-SPDB), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N- succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxy
  • N-succinimidyl-4-(2-pyridyldithio)2-sulfo-butanoate (sulfo-SPDB).
  • the combination wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:352, (b) a LCDR2 of SEQ ID NO:353, (c) a LCDR3 of SEQ ID NO:354; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 355, (e) a HCDR2 of SEQ ID NO: 356, and (f) a HCDR3 of SEQ ID NO: 357;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:366, (b) a LCDR2 of SEQ ID NO:367, (c) a LCDR3 of SEQ ID NO:368; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 369, (e) a HCDR2 of SEQ ID NO:370, and (f) a HCDR3 of SEQ ID NO:371;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:380, (b) a LCDR2 of SEQ ID NO:381, (c) a LCDR3 of SEQ ID NO:382; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 383, (e) a HCDR2 of SEQ ID NO: 384, and (f) a HCDR3 of SEQ ID NO: 385;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:394, (b) a LCDR2 of SEQ ID NO:395, (c) a LCDR3 of SEQ ID NO:396; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 397, (e) a HCDR2 of SEQ ID NO: 398, and (f) a HCDR3 of SEQ ID NO: 399;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:408, (b) a LCDR2 of SEQ ID NO:409, (c) a LCDR3 of SEQ ID NO:410; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 411, (e) a HCDR2 of SEQ ID NO: 412, and (f) a HCDR3 of SEQ ID NO: 413; or
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:436, (b) a LCDR2 of SEQ ID NO:437, (c) a LCDR3 of SEQ ID NO:438; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 439, (e) a HCDR2 of SEQ ID NO: 440, and (f) a HCDR3 of SEQ ID NO: 441.
  • the combination wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
  • v a heavy chain variable region (vH) that comprises SEQ ID NO: 400, and a light chain variable region (vL) that comprises SEQ ID NO:401;
  • the combination wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
  • the anti-GITR agonist antibody or antigen binding fragment thereof consists of: (i) a light chain variable region that consists of (a) a LCDR1 (CDR- Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that consists of: (d) a HCDRI of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427.
  • a light chain variable region that consists of (a) a LCDR1 (CDR- Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that consists of: (d) a HCDRI of
  • anti-GITR agonist antibody or antigen binding fragment thereof is selected from the group consisting of: BMS-986156, INCAGN01876, AMG 228, TRX518, MEDI1873, MK-4166, MK-1248 and FPA-154.
  • cancer selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and
  • a method of decreasing Treg cells in a tumor comprising;
  • cancer selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma.
  • cancer is selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and
  • alkyl refers to a monovalent saturated hydrocarbon chain having the specified number of carbon atoms.
  • C ⁇ _g alkyl refers to an alkyl group having from 1 to 6 carbon atoms.
  • Alkyl groups may be straight or branched. Representative branched alkyl groups have one, two, or three branches.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, sec -butyl, and t-butyl), pentyl (n- pentyl, isopentyl, and neopentyl), and hexyl.
  • antibody refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner.
  • a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino- terminus to carboxy -terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the present disclosure).
  • the antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).
  • CDRs complementarity -determining domains
  • VHCDRl complementary -determining regions
  • HCDR1 complementary -determining regions
  • the CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity.
  • the positions of the CDRs and framework regions can be determined using various well known definitions in the art, e.g. , Kabat, Chothia, and AbM (see, e.g. , Johnson et al. , Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al, Nature, 342:877-883 (1989); Chothia et al , J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al , J.Mol.Biol., 273:927-748 (1997)).
  • antigen combining sites are also described in the following: Ruiz et al , Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al , J. Mol. Biol., 262:732-745 (1996); and Martin et al, Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al , Methods Enzymol., 203: 121-153 (1991); and Rees et al , In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).
  • variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody.
  • the N-terminus is a variable region and at the C- terminus is a constant region; the CH3 and CL domains actually comprise the carboxy -terminal domains of the heavy and light chain, respectively.
  • the term "antigen binding fragment”, as used herein, refers to a polypeptide including one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al. , Nature 341 :544-546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.
  • scFv single-chain Fvs
  • sdFv disulfide-linked Fvs
  • Fab fragments F(a
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv ("scFv"); see, e.g., Bird et al, Science 242:423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85:5879-5883, 1988).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment.”
  • antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis- scFv (see, e.g. , Hollinger and Hudson, Nature Biotechnology 23 : 1126-1136, 2005).
  • Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703, 199, which describes fibronectin polypeptide monobodies).
  • Fn3 fibronectin type III
  • Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al, Protein Eng. 8: 1057- 1062, 1995; and U.S. Pat. No. 5,641,870).
  • the term "monoclonal antibody” or “monoclonal antibody composition” as used herein refers to polypeptides, including antibodies and antigen binding fragments that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • the term "human antibody”, as used herein, includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g.
  • human germline sequences or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al , J. Mol. Biol. 296:57-86, 2000.
  • the antibodies of the present disclosure can include amino acid residues not encoded by human sequences (e.g. , mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).
  • the term “recognize” as used herein refers to an antibody or antigen binding fragment thereof that finds and interacts (e.g. , binds) with its epitope, whether that epitope is linear or conformational.
  • epitope refers to a site on an antigen to which an antibody or antigen binding fragment of the disclosure specifically binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include techniques in the art, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
  • a "paratope” is the part of the antibody which recognizes the epitope of the antigen.
  • a biological sample e.g. , a blood, serum, plasma or tissue sample.
  • the antibody or binding agent with a particular binding specificity binds to a particular antigen at least ten (10) times the background and does not substantially bind in a significant amount to other antigens present in the sample.
  • Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein. As desired or appropriate, this selection may be achieved by subtracting out antibodies that cross-react with molecules from other species (e.g. , mouse or rat) or other subtypes. Alternatively, in some aspects, antibodies or antibody fragments are selected that cross-react with certain desired molecules.
  • affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities.
  • An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • corresponding human germline sequence refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences.
  • the corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences.
  • the corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art.
  • the corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least 10 to 100 times over the background.
  • Equilibrium dissociation constant refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M-l). Equilibrium dissociation constants can be measured using any known method in the art.
  • the antibodies of the present disclosure generally will have an equilibrium dissociation constant of less than about 10 "7 or 10 "8 M, for example, less than about 10 "9 M or 10 "10 M, in some aspects, less than about 10 "11 M, 10 "12 M or 10 "13 M.
  • bioavailability refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.
  • the phrase “consisting essentially of” refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any excipients inactive for the intended purpose of the methods or compositions. In some aspects, the phrase “consisting essentially of expressly excludes the inclusion of one or more additional active agents other than an antibody drug conjugate of the present disclosure. In some aspects, the phrase “consisting essentially of expressly excludes the inclusion of one or more additional active agents other than an antibody drug conjugate of the present disclosure and a second co-administered agent.
  • amino acid refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • silent variations are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule.
  • each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • conservatively modified variants include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles.
  • the following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the term "conservative sequence modifications” are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
  • the term "optimized” as used herein refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell.
  • the optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence.
  • percent identical in the context of two or more nucleic acids or polypeptide sequences, refers to the extent to which two or more sequences or subsequences that are the same. Two sequences are “identical” if they have the same sequence of amino acids or nucleotides over the region being compared.
  • Two sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g. , by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al , J. Mol. Biol. 215:403-410, 1990, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive -valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. , supra).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al , (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al, (1994) Mol. Cell. Probes 8:91-98).
  • operably linked in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g. , DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • immunoconjugate or "antibody drug conjugate” or “ADC” as used herein refers to the linkage of an antibody or an antigen binding fragment thereof with another agent, such as a pay load, drug moiety, chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, and the like.
  • the linkage can be covalent bonds, or non-covalent interactions such as through electrostatic forces.
  • linkers known in the art, can be employed in order to form the immunoconjugate.
  • the immunoconjugate can be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate.
  • fusion protein refers to proteins created through the joining of two or more genes or gene fragments which originally coded for separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.
  • the term "subject” includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g. , mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
  • toxin refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.
  • anti-cancer agent refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.
  • drug moiety or “payload” as used herein refers to a chemical moiety that is conjugated to an antibody or antigen binding fragment, and can include any therapeutic or diagnostic agent, for example, an anti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic, anti-viral), or an anesthetic agent.
  • an anti-cancer for example, an anti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic, anti-viral), or an anesthetic agent.
  • a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HD AC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
  • a payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharide or a polysaccharide.
  • the term "maytansinoid drug moiety” means the substructure of an antibody -drug conjugate that has the structure of a maytansinoid compound.
  • Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4, 151,042). Synthetic maytansinol and maytansinol analogues have been reported. See U.S. Pat. Nos.
  • Tumor refers to neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • anti-tumor activity means a reduction in the rate of tumor cell proliferation, viability, or metastatic activity.
  • a possible way of showing anti-tumor activity is to show a decline in growth rate of tumor cells, tumor size stasis or tumor size reduction.
  • Such activity can be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to investigate anti-tumor activity.
  • malignancy refers to a non-benign tumor or a cancer.
  • cancer includes a malignancy characterized by deregulated or uncontrolled cell growth.
  • Exemplary cancers include: carcinomas, sarcomas, leukemias and lymphomas.
  • cancer includes primary malignant tumors (e.g. , those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).
  • primary malignant tumors e.g. , those whose cells have not migrated to sites in the subject's body other than the site of the original tumor
  • secondary malignant tumors e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.
  • ICD Immunogenic cell death
  • CDH6 refers to a cell adhesion molecule that is a member of the cadherin family of cell-cell adhesion molecules.
  • the nucleic acid and amino acid sequences of CDH6 are known, and have been published in GenBank Accession Nos. AK291290 (protein accession number BAF83979.1) See also SEQ ID NO: 1 for the human CDH6 cDNA sequence and SEQ ID NO:2 for the human CDH6 protein sequence.
  • CDH6 receptor is a type II cadherin with five extracellular cadherin repeats and has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:2.
  • a CDH6 nucleic acid sequence has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO 1.
  • GITR glucocorticoid-induced tumor necrosis factor receptor family -related protein
  • TNFRSF18 TNFRSF18
  • the nucleic acid and amino acid sequences of GITR are known, and have been published in GenBank Accession Nos. BC152381 (protein accession number AAI52382) See also SEQ ID NO: 336 for the human GITR protein sequence.
  • GITR has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 336.
  • P-cadherin also known as Pcad, PCad, or CDH3 refers to the nucleic acid and amino acid sequence of P-cadherin, which have been published in GenBank Accession Nos. NP_ 001784, NP 001784.2 (amino acid sequence), and NM OO 1793.4, GenBank Accession Nos. AA14462, NG 009096, and NG_009096.1 (nucleotide sequences). Sequence information for human P-cadherin domains 1-5 are extracellular and are published in GenBank Acession Nos. NM 001793.4 and NP OO 1784.
  • CDH6 expressing cancer or “CDH6 positive cancer” refers to a cancer that expresses CDH6 and/or a mutant form of CDH6 on the surface of cancer cells.
  • the terms “treat,” “treating,” or “treatment” of any disease or disorder refer in one aspect, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g. , stabilization of a physical parameter), or both.
  • “treat,” “treating,” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.
  • therapeutically acceptable amount or “therapeutically effective dose” interchangeably refers to an amount sufficient to effect the desired result (i.e., a reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infection). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects.
  • a therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.
  • a “prophylactically effective dosage,” and a “therapeutically effective dosage,” of the molecules of the present disclosure can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with cancer.
  • co-administer refers to the simultaneous presence of two active agents in the blood of an individual. Active agents that are co-administered can be concurrently or sequentially delivered.
  • combination or “pharmaceutical combination” is defined as the combined administration of an antibody drug conjugate and an anti-GITR antibody, or a binding fragment of each respective antibody thereof to a patient in need.
  • the combination may be administered independently, at the same time or separately, or sequentially within time intervals that allow the combination partners show a cooperative, e.g., additive or synergistic, effect.
  • the combination can be a fixed or non-fixed combination.
  • fixed combination means that the active ingredients or therapeutic agents, are administered to a patient simultaneously in the form of a single entity or dosage form.
  • non-fixed combination means that the active ingredients or therapeutic agents, are both administered to a patient as separate entities or dosage forms either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the three compounds in the body of the subject, e.g., a mammal or human, in need thereof.
  • combination therapy refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients.
  • such administration encompasses co-administration in multiple, or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to a desired dose prior to administration.
  • such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
  • the combination therapy can provide "synergy” and prove “synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g. , by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
  • Figure 1 shows in vivo efficacy of anti-CDH6 antibodies as sulfo-SPDB-DM4 conjugates as a single agent in a xenograft mouse model of ovarian cancer.
  • Figure 2 shows in vivo efficacy of anti-GITR antibodies as a single agent in a mouse model of colon cancer.
  • Figure 3 depicts the generation of the RENCA murine CDH6 mouse model.
  • Figure 4 demonstrates the increase in Tregs following treatment with CDH6 ADC in the RENCA mouse model.
  • Figure 5 shows a reduction in tumor volume of RENCAm CDH6 tumors when treated with the CDH6 ADC/GITR combination.
  • Figure 6 demonstrates that the CDH6 ADC/GITR combination increases survival in a
  • Figure 7 is a graph of a tumor re-challenge in the RENCAmCDH6 mouse model.
  • Figure 8 is a graph of individual tumor volume from control mice during the tumor re- challenge.
  • Figure 9 is a graph of TEE survival data in the RENCAm CDH6 mouse model.
  • Figure 10 shows the increase of IFN-gamma signal in the presence of splenocytes from re-challenged mice incubated with target cells compared to control splenocytes ( pO.0001, 2 way ANOVA).
  • Figure 11 depicts a colon tumor mouse xenograft model treated with CDH6 ADC as a single agent.
  • Figure 12 shows the increase in expression in GITR on T reg cells following
  • CDH6ADC treatment in the colon tumor mouse model was assessed for CDH6ADC treatment in the colon tumor mouse model.
  • the present disclosure provides for CDH6 antibody drug conjugates in combination with anti-GITR antibodies.
  • the present disclosure is directed to the combination of CDH6 antibody drug conjugates (ADC), and agonistic anti-GITR antibodies.
  • ADC CDH6 antibody drug conjugates
  • the combination of antibodies and antibody fragments (e.g. , antigen binding fragments) of the present disclosure can be used in the treatment of cancer.
  • cancer for example, in the treatment of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma.
  • the present disclosure further provides pharmaceutical compositions comprising the combination and methods dosing of the combination for the treatment of cancer.
  • the present disclosure provides antibody drug conjugates, where an antibody, antigen binding fragment or its functional equivalent that specifically binds to CDH6 is linked to a drug moiety.
  • the antibodies, antigen binding fragments or their functional equivalents are linked, via covalent attachment by a linker, to a drug moiety that is an anti-cancer agent.
  • the antibody drug conjugates can selectively deliver an effective dose of an anti-cancer agent (e.g., a cytotoxic agent) to tumor tissues expressing CDH6, whereby greater selectivity (and lower efficacious dose) may be achieved.
  • an anti-cancer agent e.g., a cytotoxic agent
  • the disclosure provides for an immunoconjugate of Formula (I):
  • Ab represents a CDH6 binding antibody or antibody fragment (e.g., antigen binding fragment) described herein;
  • L is a linker
  • D is a drug moiety
  • n is an integer from 1-8;
  • n is an integer from 1-20. In one aspect, n is an integer from 1 to 10, 2 to 8, or 2 to 5. In a specific aspect, n is 3 to 4. In some aspects, m is 1. In some aspects, m is 2, 3 or 4.
  • the drug moiety to antibody ratio has an exact integer value for a specific conjugate molecule (e.g., n multiplied by m in Formula (I)), it is understood that the value will often be an average value when used to describe a sample containing many molecules, due to some degree of inhomogeneity, typically associated with the conjugation step.
  • the average loading for a sample of an immunoconjugate is referred to herein as the drug moiety to antibody ratio, or "DAR.”
  • DAR drug moiety to antibody ratio
  • MAR maytansinoid to antibody ratio
  • the DAR is between about 1 and about 5, and typically is about 3, 3.5, 4, 4.5, or 5.
  • At least 50% of a sample by weight is compound having the average DAR plus or minus 2, and preferably at least 50% of the sample is a conjugate that contains the average DAR plus or minus 1.
  • Other aspects include immunoconjugates wherein the DAR is about 3.5.
  • a DAR of 'about n' means the measured value for DAR is within 20% of n.
  • the present disclosure provides immunoconjugates comprising the antibodies, antibody fragments (e.g. , antigen binding fragments) and their functional equivalents as disclosed herein, linked or conjugated to a drug moiety.
  • the drug moiety D is a maytansinoid drug moiety, including those having the structure:
  • the drug moiety D can be linked to the antibody Ab through a linker L.
  • L is any chemical moiety that is capable of linking the antibody Ab to the drug moiety D.
  • the linker, L attaches the antibody Ab to the drug moiety D through covalent bond(s).
  • the linker reagent is a bifunctional or multifunctional moiety which can be used to link a drug moiety D and an antibody Ab to form antibody drug conjugates.
  • Antibody drug conjugates can be prepared using a linker having a reactive functionality for binding to the drug moiety D and to the antibody Ab.
  • a cysteine, thiol or an amine, e.g. N-terminus or amino acid side chain such as lysine of the antibody can form a bond with a functional group of a linker reagent.
  • L is a cleavable linker. In another aspect, L is a non-cleavable linker.
  • L is an acid-labile linker, photo-labile linker, peptidase cleavable linker, esterase cleavable linker, a disulfide bond reducible linker, a hydrophilic linker, a procharged linker, or a dicarboxylic acid based linker.
  • Suitable cross-linking reagents that form a non-cleavable linker between the drug moiety D, for example maytansinoid, and the antibody Ab are well known in the art, and can form non-cleavable linkers that comprise a sulfur atom (such as SMCC) or those that are without a sulfur atom.
  • Preferred cross-linking reagents that form non-cleavable linkers between the drug moiety D, for example maytansinoid, and the antibody Ab comprise a maleimido- or haloacetyl-based moiety. According to the present disclosure, such non-cleavable linkers are said to be derived from maleimido- or haloacetyl-based moieties.
  • Cross-linking reagents comprising a maleimido-based moiety include but not limited to, A i -succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane-l-carboxylate (sulfo-SMCC), jV-succinimidyl-4- (maleimidomethyl)cyclohexane-l-carboxy-(6-amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC), K-maleimidoundeconoic acid jV-succinimidyl ester (KMUA), ⁇ -maleimidobutyric acid jV-succinimidyl ester (GMBS), ⁇ -maleimidocaproic acid -succinimidyl ester (EMCS), m
  • linker L is derived from iV-succinimidyl-4-
  • SMCC maleimidomethylcyclohexanecarboxylate
  • sulfo-SMCC sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate
  • MAL-PEG-NHS MAL-PEG-NHS
  • Cross-linking reagents comprising a haloacetyle-based moiety include N-succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP). These cross-linking reagents form a non-cleavable linker derived from haloacetyl-based moieties. Representative structures of haloacetyl-based cross-linking reagents are shown below.
  • the linker L is derived from N-succinimidyl iodoacetate (SIA) or N- succinimidyl(4-iodoacetyl)aminobenzoate (SIAB).
  • Disulfide containing linkers are linkers cleavable through disulfide exchange, which can occur under physiological conditions. According to the present disclosure, such cleavable linkers are said to be derived from disulfide-based moieties.
  • Suitable disulfide cross-linking reagents include N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N- succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and iV-succinimidyl-4-(2-pyridyldithio)2-sulfo- butanoate (sulfo-SPDB), the structures of which are shown below.
  • These disulfide cross-linking reagents form a cleavable linker derived from disulfide-based moieties.
  • SPDP N-succinimidyl-3 -(2-py ridyldithio)propionate
  • SPDB N-succimmidyl-4-(2-pyridyldithio)butanoate
  • the linker L is derived from N-succinimidyl-4-(2-pyridyldithio)2-sulfo- butanoate (sulfo-SPDB).
  • the conjugate is represented by any one of the following structural formulae:
  • Ab is an antibody or antigen binding fragment thereof that specifically binds to human CDH6; n, which indicates the number of D-L groups attached the Ab through the formation of an amide bond with a primary amine of the Ab, is an integer from 1 to 20. In one aspect, n is an integer from 1 to 10, 2 to 8 or 2 to 5. In a specific aspect, n is 3 or 4.
  • the average molar ratio of drug moiety (e.g., DM1 or DM4) to the antibody in the conjugate is about 1 to about 10, about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,
  • drug moiety e.g., DM1 or DM4
  • average w value also known as Maytansinoid Antibody Ratio (MAR)
  • MAR Maytansinoid Antibody Ratio
  • the conjugate has substantially high purity and has one or more of the following features: (a) greater than about 90% (e.g. , greater than or equal to about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), preferably greater than about 95%, of conjugate species are monomelic, (b) unconjugated linker level in the conjugate preparation is less than about 10% (e.g., less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%) (relative to total linker), (c) less than 10% of conjugate species are crosslinked (e.g.
  • free drug moiety (e.g., DM1 or DM4) level in the conjugate preparation is less than about 2% (e.g., less than or equal to about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) (mol/mol relative to total cytotoxic agent).
  • the term "unconjugated linker” refers to the antibody that is covalently linked with a linker derived from a cross-linking reagent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1), wherein the antibody is not covalently coupled to the drug moiety (e.g., DM1 or DM4) through a linker (i.e., the "unconjugated linker" can be represented by Ab-SMCC, Ab-SPDB, Ab-sulfo-SPDB, or Ab-CXl-1).
  • the present disclosure provides immunoconjugates that specifically bind to CDH6.
  • the immunoconjugates of the present disclosure comprise anti-CDH6 antibodies, antibody fragments (e.g., antigen binding fragments) or functional equivalents that are conjugated to a drug moiety, e.g., an anti-cancer agent, anti-hematological disorder agent, an autoimmune treatment agent, an antiinflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent.
  • a drug moiety e.g., an anti-cancer agent, anti-hematological disorder agent, an autoimmune treatment agent, an antiinflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent.
  • the antibodies, antibody fragments (e.g., antigen binding fragments) or functional equivalents can be conjugated to several identical or different drug moieties using any methods known in the art.
  • the drug moiety of the immunoconjugates of the present disclosure is selected from a group consisting of: a maytansinoid, a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor
  • the drug moiety of the immunoconjugates of the present disclosure is a maytansinoid drug moiety, such as but not limited to, DM1, DM3, or DM4.
  • the antibodies, antibody fragments (e.g. , antigen binding fragments) or functional equivalents of the present disclosure may be conjugated to a drug moiety that modifies a given biological response.
  • Drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein such as tumor necrosis factor, a- interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response modifier such as, for example, a lymphokine.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, a- interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response
  • the antibodies, antibody fragments (e.g., antigen binding fragments) or functional equivalents of the present disclosure are conjugated to a drug moiety, such as a cytotoxin, a drug (e.g. , an immunosuppressant) or a radiotoxin.
  • a drug moiety such as a cytotoxin, a drug (e.g. , an immunosuppressant) or a radiotoxin.
  • cytotoxin include but are not limited to, taxanes (see, e.g., International (PCT) Patent Application Nos. WO 01/38318 and
  • DNA-alkylating agents e.g., CC-1065 analogs
  • anthracychnes e.g., tubuly sin analogs
  • duocarmycin analogs e.g., auristatin E
  • auristatin F e.g., maytansinoids
  • cytotoxic agents comprising a reactive polyethylene glycol moiety (see, e.g. , Sasse et al , J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et al , Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et al , J. Antibiot.
  • WO 01/49698 taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents also include, for example, anti-metabolites (e.g. , methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thiotepa chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • the antibodies, antibody fragments (e.g. , antigen binding fragments) or functional equivalents of the present disclosure can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, referred to as radioimmunoconjugates.
  • radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177.
  • Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including ZevalinTM (IDEC Pharmaceuticals) and BexxarTM (Corixa
  • the macrocyclic chelator is 1,4,7, 10- tetraazacyclododecane- ⁇ , ⁇ ' , ⁇ ' ' ,N" ' -tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • linker molecules are commonly known in the art and described in Denardo et al , (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al, (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al , (1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.
  • the antibodies, antibody fragments (e.g. , antigen binding fragments) or functional equivalents of the present disclosure can also conjugated to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • a heterologous protein or polypeptide or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids
  • the present disclosure provides fusion proteins comprising an antibody fragment (e.g., antigen binding fragment) described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide.
  • an antibody fragment e.g., antigen binding fragment
  • a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR
  • a heterologous protein e.g., polypeptide, or peptide.
  • DNA shuffling may be employed to alter the activities of antibodies of the present disclosure or fragments thereof (e.g. , antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol.
  • Antibodies or fragments thereof, or the encoded antibodies or fragments thereof may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • a polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • a "linker” is any chemical moiety that is capable of linking an antibody, antibody fragment (e.g. , antigen binding fragments) or functional equivalent to another moiety, such as a drug moiety.
  • Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active.
  • linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker).
  • the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid based linker.
  • the linker used is derived from a crosslinking reagent such as N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), V-succinimidyl-4-(2-pyridyldithio)-2- sulfo-butanoate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl(4- iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-sulfo
  • SPDP N- suc
  • the linker used is derived from a cross-linking agent such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N- succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-sulfosuccinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (sulfo-SMCC), -succinimidyl-4-(2-pyridyldithio)-2- sulfo-butanoate (sulfo-SPDB) or 2,5-dioxopyrrolidin-l-yl 17-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)- 5,8, 11, 14-tetraoxo-4,7, 10,13-tetraazaheptadecan-l-oate (CXl-1).
  • SPDP
  • Non-cleavable linkers are any chemical moiety capable of linking a drug moiety, such as a maytansinoid, to an antibody in a stable, covalent manner and does not fall off under the categories listed above for cleavable linkers.
  • non-cleavable linkers are substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage.
  • non-cleavable refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, photolabile- cleaving agent, a peptidase, an esterase, or a chemical or physiological compound that cleaves a disulfide bond, at conditions under which the drug moiety, such as maytansinoid or the antibody does not lose its activity.
  • Acid-labile linkers are linkers cleavable at acidic pH.
  • certain intracellular compartments such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid-labile linkers.
  • Photo-labile linkers are linkers that are useful at the body surface and in many body cavities that are accessible to light. Furthermore, infrared light can penetrate tissue. [00139] Some linkers can be cleaved by peptidases, i.e. peptidase cleavable linkers. Only certain peptides are readily cleaved inside or outside cells, see e.g. Trout et al., 79 Proc. Natl.
  • peptides are composed of a-amino acids and peptidic bonds, which chemically are amide bonds between the carboxylate of one amino acid and the amino group of a second amino acid.
  • Other amide bonds such as the bond between a carboxylate and the ⁇ -amino group of lysine, are understood not to be peptidic bonds and are considered non-cleavable.
  • linkers can be cleaved by esterases, i.e. esterase cleavable linkers. Again, only certain esters can be cleaved by esterases present inside or outside of cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters produced with simple alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols.
  • Procharged linkers are derived from charged cross-linking reagents that retain their charge after incorporation into an antibody drug conjugate. Examples of procharged linkers can be found in US 2009/0274713.
  • the conjugates of the present disclosure can be prepared by any methods known in the art, such as those described in US Patent Nos. 7,811,572, 6,411, 163, 7,368,565, and 8, 163,888, and US application publications 2011/0003969, 2011/0166319, 2012/0253021 and 2012/0259100. The entire teachings of these patents and patent application publications are herein incorporated by reference.
  • the conjugates of the present disclosure can be prepared by a one-step process.
  • the process comprises combining the antibody, drug and cross-linking agent in a substantially aqueous medium, optionally containing one or more co-solvents, at a suitable pH.
  • the process comprises the step of contacting the antibody of the present disclosure with a drug (e.g., DM1 or DM4) to form a first mixture comprising the antibody and the drug, and then contacting the first mixture comprising the antibody and the drug with a cross-linking agent (e.g.
  • SMCC Sulfo- SMCC
  • SPDB Sulfo-SPDB or CXl-1
  • a solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the conjugate (e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Sulfo-SPDB-DM4, or Ab-CXl-l-DMl), (ii) free drug (e.g. , DM1 or DM4), and (iii) reaction by-products.
  • the conjugate e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Sulfo-SPDB-DM4, or Ab-CXl-l-DMl
  • free drug e.g. , DM1 or DM4
  • the one-step process comprises contacting the antibody with the drug
  • the process comprises contacting a cell-binding agent with the drug (DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) in a solution having a pH of about 6 or greater (e.g. , about 6 to about 9, about 6 to about 7, about 7 to about 9, about 7 to about 8.5, about 7.5 to about 8.5, about 7.5 to about 8.0, about 8.0 to about 9.0, or about 8.5 to about 9.0).
  • the process comprises contacting a cell-binding agent with the drug (DM1 or DM4) and then the cross-linking agent (e.g.
  • the process comprises contacting a cell-binding agent with the drug (e.g.
  • DM1 or DM4 the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) in a solution having a pH of about 7.8 (e.g. , a pH of 7.6 to 8.0 or a pH of 7.7 to 7.9).
  • the cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the one-step process i.e., contacting the antibody with the drug (e.g. , DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) can be carried out at any suitable temperature known in the art.
  • the drug e.g. , DM1 or DM4
  • the cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the one-step process can occur at about 20°C or less (e.g., about -10°C (provided that the solution is prevented from freezing, e.g., by the presence of organic solvent used to dissolve the cytotoxic agent and the bifunctional crosslinking reagent) to about 20°C, about 0°C to about 18°C, about 4°C to about 16°C), at room temperature (e.g., about 20°C to about 30°C or about 20°C to about 25°C), or at an elevated temperature (e.g., about 30°C to about 37°C).
  • room temperature e.g., about 20°C to about 30°C or about 20°C to about 25°C
  • an elevated temperature e.g., about 30°C to about 37°C.
  • the one-step process occurs at a temperature of about 16°C to about 24°C (e.g., about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, or about 25°C).
  • the one-step process is carried out at a temperature of about 15°C or less (e.g., about -10°C to about 15°C, or about 0°C to about 15°C).
  • the process comprises contacting the antibody with the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) at a temperature of about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, about 2°C, about 1°C, about 0°C, about -1°C, about -2°C, about -3°C, about -4°C, about -5°C, about -6°C, about -7°C, about -8°C, about -9°C, or about -10°C, provided that the solution is prevented from freezing, e.g., by the presence of organic solvent(s) used to dissolve the cross-linking agent (e.g.,
  • the process comprises contacting the antibody with the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) at a temperature of about -10°C to about 15°C, about 0°C to about 15°C, about 0°C to about 10°C, about 0°C to about 5°C, about 5°C to about 15°C, about 10°C to about 15°C, or about 5°C to about 10°C.
  • the drug e.g., DM1 or DM4
  • the cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the process comprises contacting the antibody with the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) at a temperature of about 10°C (e.g., a temperature of 8°C to 12°C or a temperature of 9°C to 11°C).
  • the contacting described above is effected by providing the antibody, then contacting the antibody with the drug (e.g. , DM1 or DM4) to form a first mixture comprising the antibody and the drug (e.g.
  • the antibody is provided in a reaction vessel, the drug (e.g.
  • the antibody is provided in a reaction vessel, and the drug (e.g. , DM1 or DM4) is added to the reaction vessel immediately following providing the antibody to the vessel.
  • the cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the drug e.g., DM1 or DM4
  • the antibody is provided in a reaction vessel, and the drug (e.g. , DM1 or DM4) is added to the reaction vessel immediately following providing the antibody to the vessel.
  • the antibody is provided in a reaction vessel, and the drug (e.g., DM1 or DM4) is added to the reaction vessel after a time interval following providing the antibody to the vessel (e.g., about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1 day or longer after providing the cell-binding agent to the space).
  • the drug e.g. , DM1 or DM4
  • the mixture comprising the antibody and the drug (e.g., DM1 or DM4) can then be contacted with the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) either immediately after contacting the antibody with the drug (e.g. , DM1 or DM4) or at some later point (e.g., about 5 minutes to about 8 hours or longer) after contacting the antibody with the drug (e.g., DM1 or DM4).
  • the cross-linking agent e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the drug e.g., DM1 or DM4 immediately after the addition of the drug (e.g. , DM1 or DM4) to the reaction vessel comprising the antibody.
  • the mixture comprising the antibody and the drug can be contacted with the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo- SPDB or CXl-1) at about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, or longer after contacting the antibody with the drug (e.g. , DM1 or DM4).
  • the cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo- SPDB or CXl-1
  • the reaction is allowed to proceed for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or longer (e.g., about 30 hours, about 35 hours, about 40 hours, about 45 hours, or about 48 hrs).
  • the cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the one-step process further comprises a quenching step to quench any unreacted drug (e.g. , DM 1 or DM4) and/or unreacted cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1).
  • the quenching step is typically performed prior to purification of the conjugate.
  • the mixture is quenched by contacting the mixture with a quenching reagent.
  • the "quenching reagent” refers to a reagent that reacts with the free drug (e.g., DM1 or DM4) and/or cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1).
  • the free drug e.g., DM1 or DM4
  • cross-linking agent e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • maleimide or haloacetamide quenching reagents such as 4-maleimidobutyric acid, 3- maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, or iodoacetamidopropionic acid, can be used to ensure that any unreacted group (such as thiol) in the drug (e.g.
  • DM1 or DM4 is quenched.
  • the quenching step can help prevent the dimerization of the drug (e.g., DM1).
  • the dimerized DM1 can be difficult to remove.
  • polar, charged thiol-quenching reagents such as 4- maleimidobutyric acid or 3-maleimidopropionic acid
  • the excess, unreacted DM1 is converted into a polar, charged, water-soluble adduct that can be easily separated from the covalently- linked conjugate during the purification step. Quenching with non-polar and neutral thiol-quenching reagents can also be used.
  • the mixture is quenched by contacting the mixture with a quenching reagent that reacts with the unreacted cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1).
  • a quenching reagent that reacts with the unreacted cross-linking agent
  • nucleophiles can be added to the mixture in order to quench any unreacted SMCC.
  • the nucleophile preferably is an amino group containing nucleophile, such as lysine, taurine and hydroxylamine.
  • the reaction i.e., contacting the antibody with the drug (e.g. , DM1 or DM4) and then cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1)) is allowed to proceed to completion prior to contacting the mixture with a quenching reagent.
  • the quenching reagent is added to the mixture about 1 hour to about 48 hours (e.g.
  • the mixture is quenched by lowering the pH of the mixture to about 5.0
  • the mixture is quenched by lowering the pH to less than 6.0, less than 5.5, less than 5.0, less than 4.8, less than 4.6, less than 4.4, less than 4.2, less than 4.0.
  • the pH is lowered to about 4.0 (e.g., 3.8, 3.9, 4.0, 4.1 or 4.2) to about 6.0 (e.g., 5.8, 5.9, 6.0, 6.1 or 6.2), about 4.0 to about 5.0, about 4.5 (e.g., 4.3, 4.4, 4.5, 4.6 or 4.7) to about 5.0.
  • the mixture is quenched by lowering the pH of the mixture to 4.8. In another aspect, the mixture is quenched by lowering the pH of the mixture to 5.5.
  • the one-step process further comprises a holding step to release the unstably bound linkers from the antibody.
  • the holding step comprises holding the mixture prior to purification of the conjugate (e.g. , after the reaction step, between the reaction step and the quenching step, or after the quenching step).
  • the process comprises (a) contacting the antibody with the drug (e.g.
  • DM1 or DM4 to form a mixture comprising the antibody and the drug (e.g., DM1 or DM4); and then contacting the mixture comprising the antibody and drug (e.g. , DM1 or DM4) with the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1), in a solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the conjugate (e.g., Ab-MCC- DM1, Ab-SPDB-DM4, Sulfo-SPDB-DM4 or Ab-CXl-l-DMl), (ii) free drug (e.g.
  • the conjugate e.g., Ab-MCC- DM1, Ab-SPDB-DM4, Sulfo-SPDB-DM4 or Ab-CXl-l-DMl
  • free drug e.g.
  • step (b) holding the mixture prepared in step (a) to release the unstably bound linkers from the cell-binding agent, and (c) purifying the mixture to provide a purified conjugate.
  • the process comprises (a) contacting the antibody with the drug
  • DM1 or DM4 a mixture comprising the antibody and the drug (e.g., DM1 or DM4); and then contacting the mixture comprising the antibody and the drug (e.g. , DM1 or DM4) with the cross- linking agent (e.g.
  • SMCC conjugate, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • a solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the conjugate, (ii) free drug (e.g., DM1 or DM4), and (iii) reaction by-products, (b) quenching the mixture prepared in step (a) to quench any unreacted drug (e.g. , DM 1 or DM4) and/or unreacted cross-linking agent (e.g.
  • SMCC Sulfo-SMCC
  • SPDB Sulfo-SPDB or CXl-1
  • a purified conjugate e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl.
  • the holding step can be performed after purification of the conjugate, followed by an additional purification step.
  • the reaction is allowed to proceed to completion prior to the holding step.
  • the holding step can be performed about 1 hour to about 48 hours (e.g. , about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or about 24 hours to about 48 hours) after the mixture comprising the antibody and the drug (e.g. , DM1 or DM4) is contacted with the cross- linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1).
  • the cross- linking agent e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1
  • the holding step comprises maintaining the solution at a suitable temperature (e.g. , about 0°C to about 37°C) for a suitable period of time (e.g. , about 1 hour to about 1 week, about 1 hour to about 24 hours, about 1 hour to about 8 hours, or about 1 hour to about 4 hours) to release the unstably bound linkers from the antibody while not substantially releasing the stably bound linkers from the antibody.
  • a suitable temperature e.g. , about 0°C to about 37°C
  • a suitable period of time e.g. , about 1 hour to about 1 week, about 1 hour to about 24 hours, about 1 hour to about 8 hours, or about 1 hour to about 4 hours
  • the holding step comprises maintaining the solution at about 20 °C or less (e.g., about 0°C to about 18°C, about 4°C to about 16°C), at room temperature (e.g., about 20°C to about 30°C or about 20°C to about 25°C), or at an elevated temperature (e.g., about 30°C to about 37°C).
  • the holding step comprises maintaining the solution at a temperature of about 16°C to about 24°C (e.g., about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, or about 25°C).
  • the holding step comprises maintaining the solution at a temperature of about 2°C to about 8°C (e.g., about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C).
  • the holding step comprises maintaining the solution at a temperature of about 37°C (e.g., about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C).
  • the duration of the holding step depends on the temperature and the pH at which the holding step is performed.
  • the duration of the holding step can be substantially reduced by performing the holding step at elevated temperature, with the maximum temperature limited by the stability of the cell-binding agent-cytotoxic agent conjugate.
  • the holding step can comprise maintaining the solution for about 1 hour to about 1 day (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours), about 10 hours to about 24 hours, about 12 hours to about 24 hours, about 14 hours to about 24 hours, about 16 hours to about 24 hours, about 18 hours to about 24 hours, about 20 hours to about 24 hours, about 5 hours to about 1 week, about 20 hours to about 1 week, about 12 hours to about 1 week (e.g., about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days), or about 1 day to about 1 week.
  • about 1 hour to about 1 day e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 days, about
  • the holding step comprises maintaining the solution at a temperature of about 2 °C to about 8 °C for a period of at least about 12 hours for up to a week. In another aspect, the holding step comprises maintaining the solution at a temperature of about 2 °C to about 8 °C overnight (e.g., about 12 to about 24 hours, preferably about 20 hours).
  • the pH value for the holding step preferably is about 4 to about 10.
  • the pH value for the holding step is about 4 or more, but less than about 6 (e.g., 4 to 5.9) or about 5 or more, but less than about 6 (e.g. , 5 to 5.9).
  • the pH values for the holding step range from about 6 to about 10 (e.g., about 6.5 to about 9, about 6 to about 8).
  • pH values for the holding step can be about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.
  • the holding step can comprise incubating the mixture at 25°C at a pH of about 6-7.5 for about 12 hours to about 1 week, incubating the mixture at 4°C at a pH of about 4.5- 5.9 for about 5 hours to about 5 days, or incubating the mixture at 25°C at a pH of about 4.5-5.9 for about 5 hours to about 1 day.
  • the one-step process can optionally include the addition of sucrose to the reaction step to increase solubility and recovery of the conjugates.
  • sucrose is added at a concentration of about 0.1% (w/v) to about 20% (w/v) (e.g. , about 0.1% (w/v), 1% (w/v), 5% (w/v), 10% (w/v), 15% (w/v), or 20% (w/v)).
  • sucrose is added at a concentration of about 1% (w/v) to about 10% (w/v) (e.g., about 0.5% (w/v), about 1% (w/v), about 1.5% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), or about 11% (w/v)).
  • the reaction step also can comprise the addition of a buffering agent. Any suitable buffering agent known in the art can be used.
  • Suitable buffering agents include, for example, a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.
  • the buffering agent is selected from the group consisting of HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), POPSO (piperazine- l,4-bis-(2-hydroxy-propane-sulfonic acid) dehydrate), HEPES (4-(2-hydroxyethyl)piperazine-l- ethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazine-l-propanesulfonic acid), TES (N- [tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and a combination thereof.
  • the one-step process can further comprise the step of purifying the mixture to provide purified conjugate (e.g., Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl).
  • purified conjugate e.g., Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl.
  • Any purification methods known in the art can be used to purify the conjugates of the present disclosure.
  • the conjugates of the present disclosure use tangential flow filtration (TFF), non-adsorptive chromatography, adsorptive chromatography, adsorptive filtration, selective precipitation, or any other suitable purification process, as well as combinations thereof.
  • the conjugates prior to subjecting the conjugates to purification process described above, are first filtered through one or more PVDF membranes.
  • the conjugates are filtered through one or more PVDF membranes after subjecting the conjugates to the purification process described above.
  • the conjugates are filtered through one or more PVDF membranes and then purified using tangential flow filtration.
  • the conjugates are purified using tangential flow filtration and then filtered through one or more PVDF membranes.
  • TFF systems Any suitable TFF systems may be utilized for purification, including a Pellicon® type system (MiUipore, Billerica, MA), a Sartocon® Cassette system (Sartorius AG, Edgewood, NY), and a Centrasette® type system (Pall Corp., East Hills, NY).
  • Pellicon® type system MiUipore, Billerica, MA
  • Sartocon® Cassette system Sartorius AG, Edgewood, NY
  • Centrasette® type system Pall Corp., East Hills, NY.
  • Any suitable adsorptive chromatography resin may be utilized for purification.
  • Preferred adsorptive chromatography resins include hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (DVIAC), dye ligand chromatography, affinity chromatography, reversed phase chromatography, and combinations thereof.
  • suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT Type I and Type II, Bio-Rad Laboratories, Hercules, CA), HA
  • HCIC resin Ultrogel® hydroxyapatite (Pall Corp., East Hills, NY), and ceramic fluoroapatite (CFT Type I and Type II, Bio-Rad Laboratories, Hercules, CA).
  • An example of a suitable HCIC resin is MEP
  • Hypercel® resin (Pall Corp., East Hills, NY).
  • suitable HIC resins include Butyl- Sepharose, Hexyl-Sepaharose, Phenyl-Sepharose, and Octyl Sepharose resins (all from GE Healthcare, Piscataway, NJ), as well as Macro-prep® Methyl and Macro-Prep® t-Butyl resins (Biorad
  • Suitable ion exchange resins include SP-Sepharose®, CM- Sepharose®, and Q-Sepharose® resins (all from GE Healthcare, Piscataway, NJ), and Unosphere® S resin (Bio-Rad Laboratories, Hercules, CA).
  • suitable mixed mode ion exchangers include Bakerbond® ABx resin (JT Baker, Phillipsburg NJ).
  • suitable IMAC resins include Chelating Sepharose ® resin (GE Healthcare, Piscataway, NJ) and Profinity® IMAC resin (Bio-Rad Laboratories, Hercules, CA).
  • Suitable dye ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, NJ) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, CA).
  • suitable affinity resins include Protein A Sepharose resin (e.g. , MabSelect, GE
  • lectin affinity resins e.g. Lentil Lectin Sepharose® resin (GE Healthcare, Piscataway, NJ), where the antibody bears appropriate lectin binding sites.
  • suitable reversed phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, CA).
  • Any suitable non-adsorptive chromatography resin may be utilized for purification.
  • non-adsorptive chromatography resins examples include, but are not limited to,
  • SEPHADEXTM G-25, G-50, G-100, SEPHACRYLTM resins e.g. , S-200 and S-300
  • SUPERDEXTM resins e.g., SUPERDEXTM 75 and SUPERDEXTM 200
  • BIO-GEL® resins e.g., P-6, P-10, P-30, P- 60, and P-100
  • the conjugates of the present disclosure can be prepared as described in the U.S. Patent 7,811,572 and U.S. Patent Application Publication No. 2006/0182750.
  • the process comprises the steps of (a) contacting the antibody of the present disclosure with the cross-linking agent (e.g.
  • SMCC SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) to covalently attach the linker (i.e., Ab-SMCC, Ab-SPDB or Ab-CXl-1) to the antibody and thereby prepare a first mixture comprising the antibody having the linker bound thereto; (b) optionally subjecting the first mixture to a purification process to prepare a purified first mixture of the antibody having the linker bound thereto; (c) conjugating the drug (e.g. , DM1 or DM4) to the antibody having the linker bound thereto in the first mixture by reacting the antibody having the linker bound thereto with the drug (e.g.
  • the drug e.g. DM1 or DM4
  • a second mixture comprising (i) conjugate (e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl), (ii) free drug (e.g. , DM1 or DM4); and (iii) reaction by-products; and (d) subjecting the second mixture to a purification process to purify the conjugate from the other components of the second mixture.
  • conjugate e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl
  • free drug e.g. , DM1 or DM4
  • reaction by-products e.g. DM1 or DM4
  • the purification step (b) can be omitted. Any purification methods described herein can be used for steps (b) and (d). In one embodiment, TFF is used for both steps (b) and (d). In another embodiment, TFF is used for step (b) and absorptive chromatography (e.g. , CHT) is used for step (d).
  • absorptive chromatography e.g. , CHT
  • the conjugates of the present disclosure can be prepared by conjugating pre-formed drug-linker compound (e.g. , SMCC-DM1, Sulfo-SMCC-DMl, SPDB-DM4, Sulfo-SPDB- DM4 or CXl-l-DMl) to the antibody of the present disclosure, as described in U.S. Patent 6,441, 163 and U.S. Patent Application Publication Nos. 2011/0003969 and 2008/0145374, followed by a purification step. Any purification methods described herein can be used.
  • pre-formed drug-linker compound e.g. , SMCC-DM1, Sulfo-SMCC-DMl, SPDB-DM4, Sulfo-SPDB- DM4 or CXl-l-DMl
  • the drug-linker compound is prepared by reacting the drug (e.g., DM1 or DM4) with the cross-linking agent (e.g., SMCC, Sulfo- SMCC, SPDB, Sulfo-SPDB or CXl-1).
  • the drug-linker compound e.g. , SMCC-DM1, Sulfo-SMCC- DMl, SPDB-DM4, Sulfo-SPDB-DM4 or CXl-l-DMl
  • the antibodies, antibody fragments (e.g. , antigen binding fragments) or antibody drug conjugates of the present disclosure can be characterized and selected for their physical/chemical properties and/or biological activities by various assays known in the art.
  • an antibody of the present disclosure can be tested for its antigen binding activity by known methods such as ELISA, FACS, Biacore or Western blot.
  • Transgenic animals and cell lines are particularly useful in screening antibody drug conjugates (ADCs) that have potential as prophylactic or therapeutic treatments of cancer overexpression of tumor-associated antigens and cell surface receptors.
  • Screening for a useful ADC may involve administering a candidate ADC over a range of doses to the transgenic animal, and assaying at various time points for the effect(s) of the ADC on the disease or disorder being evaluated.
  • the drug can be administered prior to or simultaneously with exposure to an inducer of the disease, if applicable.
  • the candidate ADC may be screened serially and individually, or in parallel under medium or high-throughput screening format.
  • One aspect is a screening method comprising (a) transplanting cells from a stable cancer cell line or human patient tumor expressing CDH6 (e.g., an ovarian cell line or tumor fragment, a renal cell line or tumor fragment, a hepatic cell line or tumor fragment, a thyroid cell line or tumor fragment, a CNS cancer cell line or tumor fragment, a cholangiocarcinoma cancer cell line or tumor fragment, ovarian, renal, hepatic, soft tissue, CNS, thyroid, or cholangiocarcinoma primary cells) into a non-human animal, (b) administering an ADC drag candidate to the non-human animal and (c) determining the ability of the candidate to inhibit the growth of tumors from the transplanted cell line.
  • CDH6 e.g., an ovarian cell line or tumor fragment, a renal cell line or tumor fragment, a hepatic cell line or tumor fragment, a thyroid cell line or tumor fragment, a CNS cancer cell line or tumor fragment, a cholangio
  • the present disclosure also encompasses a method of screening ADC candidates for the treatment of a disease or disorder characterized by the overexpression of CDH6 comprising (a) contacting cells from a stable cancer cell line expressing CDH6 with a drag candidate, and (b) evaluating the ability of the ADC candidate to inhibit the growth of the stable cell line.
  • a further aspect is a screening method comprising (a) contacting cells from a stable cancer cell line expressing CDH6 with an ADC drag candidate and (b) evaluating the ability of the ADC candidate to induce immunogenic cell death. In one aspect the ability of the ADC candidate to induce apoptosis is evaluated.
  • Candidate ADC can be screened by being administered to the transgenic animal over a range of doses, and evaluating the animal's physiological response to the compounds over time. In some cases, it can be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound. If cell lines derived from the subject transgenic animals are used to screen for ADCs useful in treating various disorders associated with overexpression of CDH6, the test ADCs are added to the cell culture medium at an appropriate time, and the cellular response to the ADCs is evaluated over time using the appropriate biochemical and/or histological assays.
  • the present disclosure provides for antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to human CDH6.
  • Antibodies or antibody fragments (e.g., antigen binding fragments) of the present disclosure include, but are not limited to, the human monoclonal antibodies or fragments thereof, isolated as described, in the Examples below.
  • the present disclosure in certain aspects provides antibodies or antibody fragments
  • said antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind CDH6, said antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VH domain having an amino acid sequence of SEQ ID NO: 20, 34, 48, 62, 76, 90, 104, 118, 132, 146, 160, 174, 188, 202, 216, 230, 244, 258, 272, 286, 300, 314 or 328 (Table 5).
  • the present disclosure also provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 5.
  • the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to CDH6, said antibodies comprising (or alternatively, consist of) one, two, three, four, five or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 5.
  • the present disclosure provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VL domain having an amino acid sequence of SEQ ID NO: 21, 35, 49, 63, 77, 91, 105, 119, 133, 147, 161, 175, 189, 203, 217, 231, 245, 259, 273, 287, 301, 315 or 329 (Table 5).
  • the present disclosure also provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g.
  • antigen binding fragments comprise a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 5, infra.
  • the disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise (or alternatively, consist of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 5.
  • antibodies or antibody fragments include amino acids that have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity in the CDR regions with the CDR regions depicted in the sequences described in Table 5. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared with the CDR regions depicted in the sequence described in Table 5.
  • the present disclosure also provides nucleic acid sequences that encode VH, VL, the full length heavy chain, and the full length light chain of the antibodies that specifically bind to CDH6. Such nucleic acid sequences can be optimized for expression in mammalian cells.
  • antibodies of the present disclosure include those where the amino acids or nucleic acids encoding the amino acids have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity to the sequences described in Table 5. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared with the variable regions depicted in the sequence described in Table 5, while retaining substantially the same therapeutic activity.
  • each of these antibodies can bind to CDH6, the VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other CDH6-binding antibodies.
  • Such "mixed and matched" CDH6 -binding antibodies can be tested using the binding assays known in the art (e.g. , ELISAs, and other assays described in the Example section). When these chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence.
  • a full length heavy chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence.
  • a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence.
  • a full length light chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length light chain sequence.
  • the disclosure provides for an isolated monoclonal antibody or antigen binding region thereof having: a heavy chain variable region (vH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 34, 48, 62, 76, 90, 104, 1 18, 132, 146, 160, 174, 188, 202, 216, 230, 244, 258, 272, 286, 300, 314 or 328 (Table 5); and a light chain variable region (vL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 35, 49, 63, 77, 91, 105, 119, 133, 147, 161, 175, 189, 203, 217, 231, 245, 259, 273, 287, 301, 315 or 329 (Table 5); wherein the antibody specifically binds to CDH6.
  • vH heavy chain variable region
  • VL light chain variable region
  • the disclosure provides (i) an isolated monoclonal antibody having: a full length heavy chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 24, 38, 52, 66, 80, 94, 108, 122, 136, 150, 164, 178, 192, 206, 220, 234, 248, 262, 276, 290, 304, 318 or 332; and a full length light chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 25, 39, 53, 67, 81, 95, 109, 123, 137, 151, 165, 179, 193, 207, 221, 235, 249, 263, 277, 291, 305, 319 or 333 ; or (ii) a functional protein comprising an antigen binding portion thereof.
  • the present disclosure provides CDH6 -binding antibodies that comprise the heavy chain and light chain CDRls, CDR2s and CDR3s as described in Table 5, or combinations thereof.
  • the amino acid sequences of the VH CDRls of the antibodies are shown in SEQ ID NOs: 17, 31, 45, 59, 73, 87, 101, 115, 129, 143, 157, 171, 185, 199, 213, 227, 241, 255, 269,
  • amino acid sequences of the VH CDR3s of the antibodies are shown in SEQ ID NOs: 19, 33, 47, 61, 75, 89, 103, 117, 131, 145, 159, 173, 187, 201, 215, 229, 243, 257, 271,
  • VL CDRls The amino acid sequences of the VL CDRls of the antibodies are shown in SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182, 196, 210, 224, 238, 252, 266,
  • VL CDR2s of the antibodies are shown in SEQ ID NOs: 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267,
  • amino acid sequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268,
  • VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and match, although each antibody must contain a VH CDR1, 2 and 3 and a VL CDR1, 2 and 3 to create other CDH6-binding binding molecules.
  • Such "mixed and matched" CDH6-binding antibodies can be tested using the binding assays known in the art and those described in the Examples (e.g., ELISAs).
  • VH CDR sequences When VH CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular VH sequence should be replaced with a structurally similar CDR sequence(s).
  • VL CDR sequences when VL CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular VL sequence should be replaced with a structurally similar CDR sequence(s). It will be readily apparent to the ordinarily skilled artisan that novel VH and VL sequences can be created by substituting one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences shown herein for monoclonal antibodies of the present disclosure.
  • the present disclosure provides an isolated monoclonal antibody or antigen binding region thereof comprising a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 31, 45, 59, 73, 87, 101, 115, 129, 143, 157, 171, 185, 199, 213, 227, 241, 255, 269, 283, 297, 311 and 325; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 32, 46, 60, 74, 88, 102,116, 130, 144, 158, 172, 186, 200, 214, 228, 242, 256, 270, 284, 298, 312 and 326; a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 33, 47, 61, 75, 89, 103, 117, 131, 145, 159, 173, 187, 201
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:224, (b) a LCDR2 of SEQ ID NO:225, (c) a LCDR3 of SEQ ID NO:226; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 227, (e) a HCDR2 of SEQ ID NO: 228, and (f) a HCDR3 of SEQ ID NO:229;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:210, (b) a LCDR2 of SEQ ID NO:211, (c) a LCDR3 of SEQ ID NO:212; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:213, (e) a HCDR2 of SEQ ID NO: 214, and (f) a HCDR3 of SEQ ID NO:215;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:266, (b) a LCDR2 of SEQ ID NO:267, (c) a LCDR3 of SEQ ID NO:268; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 269, (e) a HCDR2 of SEQ ID NO:270, and (f) a HCDR3 of SEQ ID NO: 271;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:308, (b) a LCDR2 of SEQ ID NO:309, (c) a LCDR3 of SEQ ID NO:310; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:311, (e) a HCDR2 of SEQ ID NO:312, and (f) a HCDR3 of SEQ ID NO:313;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 14, (b) a LCDR2 of SEQ ID NO: 15, (c) a LCDR3 of SEQ ID NO: 16; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 17, (e) a HCDR2 of SEQ ID NO: 18, and (f) a HCDR3 of SEQ ID NO: 19;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:28, (b) a LCDR2 of SEQ ID NO:29, (c) a LCDR3 of SEQ ID NO:30; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:31, (e) a HCDR2 of SEQ ID NO:32, and (f) a HCDR3 of SEQ ID NO:33;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:42, (b) a LCDR2 of SEQ ID NO:43, (c) a LCDR3 of SEQ ID NO:44; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:45, (e) a HCDR2 of SEQ ID NO:46, and (f) a HCDR3 of SEQ ID NO:47;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:56, (b) a LCDR2 of SEQ ID NO:57, (c) a LCDR3 of SEQ ID NO:58; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:59, (e) a HCDR2 of SEQ ID NO:60, and (f) a HCDR3 of SEQ ID NO:61;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:70, (b) a LCDR2 of SEQ ID NO:71, (c) a LCDR3 of SEQ ID NO:72; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:73, (e) a HCDR2 of SEQ ID NO:74, and (f) a HCDR3 of SEQ ID NO:75;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:84, (b) a LCDR2 of SEQ ID NO:85, (c) a LCDR3 of SEQ ID NO:86; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:87, (e) a HCDR2 of SEQ ID NO: 88, and (f) a HCDR3 of SEQ ID NO: 89;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:98, (b) a LCDR2 of SEQ ID NO:99, (c) a LCDR3 of SEQ ID NO: 100; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 101, (e) a HCDR2 of SEQ ID NO: 102, and (f) a HCDR3 of SEQ ID NO: 103;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 112, (b) a LCDR2 of SEQ ID NO: 113, (c) a LCDR3 of SEQ ID NO: 114; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 115, (e) a HCDR2 of SEQ ID NO: 116, and (f) a HCDR3 of SEQ ID NO: 117;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 126, (b) a LCDR2 of SEQ ID NO: 127, (c) a LCDR3 of SEQ ID NO: 128; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 129, (e) a HCDR2 of SEQ ID NO: 130, and (f) a HCDR3 of SEQ ID NO: 131;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 140, (b) a LCDR2 of SEQ ID NO: 141, (c) a LCDR3 of SEQ ID NO: 142; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 143, (e) a HCDR2 of SEQ ID NO: 144, and (f) a HCDR3 of SEQ ID NO: 145;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 154, (b) a LCDR2 of SEQ ID NO: 155, (c) a LCDR3 of SEQ ID NO: 156; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 157, (e) a HCDR2 of SEQ ID NO: 158, and (f) a HCDR3 of SEQ ID NO: 159;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 168, (b) a LCDR2 of SEQ ID NO: 169, (c) a LCDR3 of SEQ ID NO: 170; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 171, (e) a HCDR2 of SEQ ID NO: 172, and (f) a HCDR3 of SEQ ID NO: 173;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 182, (b) a LCDR2 of SEQ ID NO: 183, (c) a LCDR3 of SEQ ID NO: 184; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 185, (e) a HCDR2 of SEQ ID NO: 186, and (f) a HCDR3 of SEQ ID NO: 187;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 196, (b) a LCDR2 of SEQ ID NO: 197, (c) a LCDR3 of SEQ ID NO: 198; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 199, (e) a HCDR2 of SEQ ID NO:200, and (f) a HCDR3 of SEQ ID NO:201;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:238, (b) a LCDR2 of SEQ ID NO:239, (c) a LCDR3 of SEQ ID NO:240; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:241, (e) a HCDR2 of SEQ ID NO:242, and (f) a HCDR3 of SEQ ID NO:243;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:252, (b) a LCDR2 of SEQ ID NO:253, (c) a LCDR3 of SEQ ID NO:254; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:255, (e) a HCDR2 of SEQ ID NO:256, and (f) a HCDR3 of SEQ ID NO:257;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:280, (b) a LCDR2 of SEQ ID NO:281, (c) a LCDR3 of SEQ ID NO:282; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:283, (e) a HCDR2 of SEQ ID NO:284, and (f) a HCDR3 of SEQ ID NO:285;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:294, (b) a LCDR2 of SEQ ID NO:295, (c) a LCDR3 of SEQ ID NO:296; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:297, (e) a HCDR2 of SEQ ID NO:298, and (f) a HCDR3 of SEQ ID NO:299; or
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:322, (b) a LCDR2 of SEQ ID NO:323, (c) a LCDR3 of SEQ ID NO:324; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:325, (e) a HCDR2 of SEQ ID NO:326, and (f) a HCDR3 of SEQ ID NO:327.
  • the present disclosure provides for antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to human GITR.
  • Antibodies or antibody fragments (e.g. , antigen binding fragments) of the present disclosure include, but are not limited to, the human monoclonal antibodies or fragments thereof, isolated as described in the Examples below.
  • the present disclosure in certain aspects provides antibodies or antibody fragments
  • antibodies or antibody fragments that specifically bind GITR
  • said antibodies or antibody fragments comprise a VH domain having an amino acid sequence of SEQ ID NO: 348, 358, 372, 386, 400, 414, 428 or 442 (Table 6).
  • the present disclosure also provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 6.
  • the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to GITR, said antibodies comprising (or alternatively, consist of) one, two, three, four, five or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 6.
  • the present disclosure provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise a VL domain having an amino acid sequence of SEQ ID NO: 349, 359, 373, 387, 401, 415, 429 or 443 (Table 6).
  • the present disclosure also provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 6, infra.
  • the disclosure provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise (or alternatively, consist of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 6.
  • antibodies or antibody fragments include amino acids that have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity in the CDR regions with the CDR regions depicted in the sequences described in Table 6. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared with the CDR regions depicted in the sequence described in Table 6.
  • the present disclosure also provides nucleic acid sequences that encode VH, VL, the full length heavy chain, and the full length light chain of the antibodies that specifically bind to GITR. Such nucleic acid sequences can be optimized for expression in mammalian cells.
  • antibodies of the present disclosure include those where the amino acids or nucleic acids encoding the amino acids have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity to the sequences described in Table 6. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared with the variable regions depicted in the sequence described in Table 6, while retaining substantially the same therapeutic activity.
  • each of these antibodies can bind to GITR, the VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other GITR binding antibodies.
  • Such "mixed and matched" GITR binding antibodies can be tested using the binding assays known in the art (e.g., ELISAs, and other assays described in the Example section). When these chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence.
  • a full length heavy chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence.
  • a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence.
  • a full length light chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length light chain sequence.
  • the disclosure provides for an isolated monoclonal antibody or antigen binding region thereof having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 348, 358, 372, 386, 400, 414, 428 or 442 (Table 6); and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 349, 359, 373, 387, 401, 415, 429 or 443 (Table 6); wherein the antibody specifically binds to GITR.
  • a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 348, 358, 372, 386, 400, 414, 428 or 442
  • a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 349, 359, 373, 387, 401, 415, 429 or 443
  • the disclosure provides (i) an isolated monoclonal antibody having: a full length heavy chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 362, 376, 390, 404, 418, 432 or 446; and a full length light chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 363, 377, 391, 405, 419, 433 or 447; or (ii) a functional protein comprising an antigen binding portion thereof .
  • the present disclosure provides anti-GITR antibodies that comprise the heavy chain and light chains.
  • the present disclosure provides anti-GITR antibodies that comprise the heavy chain and light chain CDRls, CDR2s and CDR3s as described in Table 6, or combinations thereof.
  • the combination comprises GITR.MAB7.
  • an antibody or antibody fragment that specifically binds to GITR comprising:
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:352, (b) a LCDR2 of SEQ ID NO:353, (c) a LCDR3 of SEQ ID NO:354; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 355, (e) a HCDR2 of SEQ ID NO: 356, and (f) a HCDR3 of SEQ ID NO: 357;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:366, (b) a LCDR2 of SEQ ID NO:367, (c) a LCDR3 of SEQ ID NO:368; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 369, (e) a HCDR2 of SEQ ID NO:370, and (f) a HCDR3 of SEQ ID NO:371;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:380, (b) a LCDR2 of SEQ ID NO:381, (c) a LCDR3 of SEQ ID NO:382; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 383, (e) a HCDR2 of SEQ ID NO: 384, and (f) a HCDR3 of SEQ ID NO: 385;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:394, (b) a LCDR2 of SEQ ID NO:395, (c) a LCDR3 of SEQ ID NO:396; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 397, (e) a HCDR2 of SEQ ID NO: 398, and (f) a HCDR3 of SEQ ID NO: 399;
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:408, (b) a LCDR2 of SEQ ID NO:409, (c) a LCDR3 of SEQ ID NO:410; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 411, (e) a HCDR2 of SEQ ID NO: 412, and (f) a HCDR3 of SEQ ID NO: 413; and
  • a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:436, (b) a LCDR2 of SEQ ID NO:437, (c) a LCDR3 of SEQ ID NO:438; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 439, (e) a HCDR2 of SEQ ID NO: 440, and (f) a HCDR3 of SEQ ID NO: 441.
  • the present disclosure provides antibodies and antibody fragments (e.g., antigen binding fragments) that bind to an epitope of the extracellular domain of CDH6 or the extracellular domain of GITR.
  • antibody fragments e.g., antigen binding fragments
  • the present disclosure also provides antibodies and antibody fragments (e.g., antigen binding fragments) that bind to the same epitope as do the CDH6 antibodies described in Table 5 and the GITR antibodies described in Table 6. Additional antibodies and antibody fragments (e.g., antigen binding fragments) can therefore be identified based on their ability to cross-compete (e.g. , to competitively inhibit the binding of, in a statistically significant manner) with other antibodies in antibody binding assays.
  • the ability of a test antibody to inhibit the binding of antibodies and antibody fragments (e.g., antigen binding fragments) of the present disclosure to a protein (e.g., human CDH6 or human GITR) demonstrates that the test antibody can compete with that antibody or antibody fragment (e.g.
  • antigen binding fragments for binding to CDH6 or GITR; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the respective extracellular domain as the antibody or antibody fragment (e.g., antigen binding fragments) with which it competes.
  • the antibody that binds to the same epitope on CDH6 or GITR as the antibodies or antibody fragments (e.g. , antigen binding fragments) of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.
  • the present disclosure provides site-specific labeled immunoconjugates.
  • These immunoconjugates can comprise modified antibodies or antigen binding fragments thereof that further comprise modifications to framework residues within VH and/or VL, e.g. to improve the properties of the antibody.
  • framework modifications are made to decrease the immunogenicity of the antibody.
  • one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence.
  • an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived.
  • Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • the somatic mutations can be "back-mutated" to the germline sequence by, for example, site-directed mutagenesis.
  • Such "back-mutated” antibodies are also intended to be encompassed.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 2003/0153043 by Carr et al.
  • antibodies can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody can be chemically modified (e.g. , one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g. , increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc -hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in, e.g. , U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g. , the PCT Publication WO 94/29351 by Bodmer et al.
  • one or more amino acids of an antibody or antigen binding fragment thereof of the present disclosure are replaced by one or more allotypic amino acid residues, for the IgGl subclass and the kappa isotype.
  • Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgGl, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al. , MAbs. 1 :332-338 (2009).
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta.
  • the binding sites on human IgGl for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).
  • the glycosylation of an antibody is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen.”
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • Such an approach is described in, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. For example, EP 1, 176,195 by Hang et al.
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al. , (2002) J. Biol. Chem. 277:26733-26740).
  • PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g.
  • beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17: 176-180, 1999).
  • the antibody is modified to increase its biological half-life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6, 121,022 by Presta et al.
  • the "IgG Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions.
  • the human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl- terminus of the IgG antibody.
  • the numbering of residues in the Fc region is that of the EU index of Kabat.
  • the C- terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody.
  • Silenced effector functions can be obtained by mutation in the Fc region of the antibodies and have been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181 : 6664-69) see also Heusser et al., WO2012065950.
  • Examples of silent Fc lgGl antibodies are the LALA mutant comprising L234A and L235A mutation in the lgGl Fc amino acid sequence.
  • Another example of a silent lgGl antibody is the DAPA (D265A, P329A) mutation (US 6,737,056).
  • Another silent lgGl antibody comprises the N297A mutation, which results in aglycosylated/non-glycosylated antibodies.
  • Fc silent antibodies result in no or low ADCC activity, meaning that an Fc silent antibody exhibits an ADCC activity that is below 50% specific cell lysis, No ADCC activity means that the Fc silent antibody exhibits an ADCC activity (specific cell lysis) that is below 1 %.
  • the CDH6 or GITR antibodies and antibody fragments (e.g., antigen binding fragments) thereof can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g. , hybridoma or recombinant production.
  • Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.
  • the disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions as described herein.
  • the polynucleotide encoding the CDH6 antibody heavy chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162,
  • the polynucleotide encoding the CDH6 antibody light chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 23, 37, 51, 65, 79, 93, 107, 121, 135, 149, 163,
  • the polynucleotide encoding the CDH6 antibody heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 26, 40, 54, 68, 82, 96, 110, 124, 138, 152, 166, 180, 194, 208, 222, 236, 250, 264, 278, 292, 306, 320, and 334.
  • the polynucleotide encoding the CDH6 antibody light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 27, 41, 55, 69, 83, 97, 111, 125, 139, 153, 167, 181, 195, 209, 223, 237, 251, 265, 279, 293, 307, 321, and 335.
  • the polynucleotide encoding the GITR antibody heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:364, 378, 392, 406, 420, 434 or 448.
  • the polynucleotide encoding the GITR antibody light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:365, 379, 393, 407, 421, 435 or 449.
  • the polynucleotide encoding the GITR antibody heavy chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 350, 360, 374, 388, 402, 416, 430 and 444.
  • the polynucleotide encoding the GITR antibody light chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 351, 361, 375, 389, 403, 417, 431 and 445.
  • the polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in the Examples below) encoding an anti-CDH6 antibody or its binding fragment.
  • Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al. , Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown e/ al, Meth. Enzymol. 68: 109, 1979; the diethylphosphoramidite method of Beaucage et al , Tetra.
  • Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al, Nat Genet 15:345, 1997).
  • nonviral vectors useful for expression of the antibody polynucleotides and polypeptides in mammalian (e.g. , human) cells include pThioHis A, B & C, pcDNA3.1 His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPS V vectors, and numerous other vectors known in the art for expressing other proteins.
  • Useful viral vectors include vectors based on retroviruses, adenoviruses, adenoassociated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al, supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.
  • the choice of expression vector depends on the intended host cells in which the vector is to be expressed.
  • the expression vectors contain a promoter and other regulatory sequences (e.g. , enhancers) that are operably linked to the polynucleotides encoding an antibody chain or fragment.
  • an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions.
  • Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells.
  • promoters In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an antibody chain or fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al, Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al , Meth. Enzymol., 153 :516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.
  • the expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted anti-CDH6 or anti-GITR antibody sequences. More often, the inserted anti-CDH6 or anti-GITR antibody sequences are linked to a signal sequences before inclusion in the vector.
  • Vectors to be used to receive sequences encoding antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.
  • the host cells for harboring and expressing the anti-CDH6 or anti-GITR antibody chains can be either prokaryotic or eukaryotic.
  • E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • expression vectors which typically contain expression control sequences compatible with the host cell (e.g. , an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Other microbes, such as yeast can also be employed to express anti-CDH6 or anti-GITR polypeptides. Insect cells in combination with baculovirus vectors can also be used.
  • mammalian host cells are used to express and produce the anti-CDH6 or anti-GITR polypeptides of the present disclosure.
  • they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., a myeloma hybridoma clone) or a mammalian cell line harboring an exogenous expression vector (e.g. , SP2/0 myeloma cells). These include any normal mortal or normal or abnormal immortal animal or human cell.
  • a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B- cells and hybridomas.
  • the use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987.
  • Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g. , Queen et al, Immunol. Rev.
  • expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable.
  • Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.
  • Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al, supra). Other methods include, e.g.
  • cell lines which stably express anti-CDH6 or anti-GITR antibody chains or binding fragments can be prepared using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media.
  • Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • the antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates of the present disclosure are useful in a variety of applications including, but not limited to, treatment of cancer, such as solid cancers.
  • the antibodies, antibody fragments (e.g., antigen binding fragments), and antibody drug conjugates are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and/or reducing the tumorigenicity of a tumor.
  • the methods of use can be in vitro, ex vivo, or in vivo methods.
  • the disclosure provides for a method of treating, preventing or ameliorating a disease comprising administering the antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates to a patient, thereby treating the disease.
  • the disease treated with the antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates is a cancer.
  • diseases which can be treated and/or prevented include, but are not limited to, ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma.
  • the cancer is characterized by CDH6 expressing cells to which the antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates can specifically bind.
  • the present disclosure provides for methods of treating cancer comprising administering a therapeutically effective amount of the CDH6 ADC and GITR antibody in combination.
  • the cancer is a solid cancer.
  • the subject is a human.
  • the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of the CDH6 ADC and GITR antibody.
  • the subject is a human.
  • the subject has a tumor or has had a tumor removed.
  • the subject has a reoccurrence of the tumor.
  • the tumor expresses the CDH6 to which the anti-CDH6 ADC binds.
  • the tumor overexpresses the human CDH6.
  • the administration of the anti-CDH6 ADC results in an increase of GITR expression on Treg cells.
  • the appropriate dosage of the CDH6 ADC and GITR antibody combination depends on various factors, such as the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, previous therapy, patient's clinical history, and so on.
  • the CDH6 ADC and GITR antibody combination can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size).
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody, antibody fragment (e.g., antigen binding fragment), or antibody drug conjugates.
  • dosage is from 0.0 lmg to 10 mg (e.g., 0.01 mg, 0.05mg, O.lmg, 0.5mg, lmg, 2mg, 3mg, 4mg, 5mg, 7mg, 8mg, 9mg, or lOmg) per kg of body weight, and can be given once or more daily, weekly, monthly or yearly.
  • the CDH6 ADC and GITR antibody combination of the present disclosure is given once every two weeks or once every three weeks.
  • the CDH6 ADC is given as a single agent, and the anti-GTIR antibody is administered a certain time afterward. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • the CDH6 ADC/anti-GITR combination of the present disclosure is combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, antinausea agents (or anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.
  • other therapeutic agents such as other anti-cancer agents, anti-allergic agents, antinausea agents (or anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.
  • General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy- 5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC- Dome®), d
  • an antibody, antibody fragment (e.g. , antigen binding fragment), or antibody drug conjugate of the present disclosure is combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties.
  • the second compound of the pharmaceutical combination formulation or dosing regimen can have complementary activities to the antibody or immunoconjugate of the combination such that they do not adversely affect each other.
  • an antibody, antibody fragment (e.g., antigen binding fragment), or antibody drug conjugate of the present disclosure can be administered in combination with, but not limited to, a chemotherapeutic agent, a tyrosine kinase inhibitor, for example, Imatinib.
  • the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody drug conjugate in combination with one or more tyrosine kinase inhibitors, including but not limited to, EGFR inhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors.
  • one or more tyrosine kinase inhibitors including but not limited to, EGFR inhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors.
  • tyrosine kinase inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-lH-indazol-4-yl)phenyl]-N'-(2-fluoro-5- methylphenyl)urea, also known as ABT 869, available from Genentech); Sunitinib malate (Sutent®); Bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-l- yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606, and described in US Patent No.
  • Epidermal growth factor receptor (EGFR) inhibitors include but are not limited to,
  • Erlotinib hydrochloride (Tarceva®), Gefitnib (Iressa®); N-[4-[(3-Chloro-4-fluorophenyl)amino]-7- [[(3"S'') etrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®); (3R,4R)-4-Amino-l-((4-((3- methoxyphenyl)amino)pyrrolo[2, 1-f] [l,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514);
  • Canertinib dihydrochloride (CI-1033); 6-[4-[(4-Ethyl-l-piperazinyl)methyl]phenyl]-N-[(lR)-l- phenylethyl]- 7H-Pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569); Afatinib (BIBW2992); Neratinib (HKI-272); N-[4-[[l-[(3- Fluoropheny l)methyl] - lH-indazol-5-y ljamino] -5-methy lpyrrolo [2, 1 -f] [ 1 ,2,4]triazin-6-y 1] -carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626); N-(3,4-Dichloro-2-
  • EGFR antibodies include but are not limited to, Cetuximab (Erbitux®); Panitumumab
  • HER2 receptor Human Epidermal Growth Factor Receptor 2 (HER2 receptor) (also known as Neu,
  • ErbB-2, CD340, or pl85 inhibitors include but are not limited to, Trastuzumab (Herceptin®);
  • Pertuzumab (Omnitarg®); Neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridin-2- yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide, and described PCT Publication No.
  • WO 05/028443 Lapatinib or Lapatinib ditosylate (Tykerb®); (3R,4R)-4-amino- 1 -((4-((3 -methoxypheny l)amino)pyrrolo [2, 1-f] [ 1 ,2,4]triazin-5-yl)methyl)piperidin- 3-ol (BMS690514); (2E)-N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-
  • HER3 inhibitors include but are not limited to, LJM716, MM-121, AMG-888,
  • RG7116 REGN-1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.
  • MET inhibitors include but are not limited to, Cabozantinib (XL184, CAS 849217-68-
  • Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); Tivantinib (ARQ197, CAS 1000873-98-2); l-(2 -Hydroxy -2 -methylpropyl)- ⁇ -(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5- methyl-3-oxo-2-phenyl-2,3-dihydro-l f-pyrazole-4-carboxamide (AMG 458); Cryzotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-Dihydro-lH-indol-l-ylsulfonyl)-3-( ⁇ 3,5-dimethyl-4-[(4-methylpiperazin- l-yl)carbonyl]-lH-pyrrol-2-yl ⁇ methylene)-l,3-dihydro-2H
  • IGF1R inhibitors include but are not limited to, BMS-754807, XL-228, OSI-906,
  • GSK0904529A A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. See e.g., Yee, JNCI, 104; 975 (2012) for review.
  • the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody drug conjugate in combination with one or more FGF downstream signaling pathway inhibitors, including but not limited to, MEK inhibitors, Braf inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTor inhibitors.
  • FGF downstream signaling pathway inhibitors including but not limited to, MEK inhibitors, Braf inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTor inhibitors.
  • mitogen-activated protein kinase (MEK) inhibitors include but are not limited to, XL-518 (also known as GDC-0973, Cas No. 1029872-29-4, available from ACC Corp.); 2- [(2-Chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as CI- 1040 or PD184352 and described in PCT Publication No.
  • N-[3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-l-[(2R)-2,3- dihydroxypropyl]- cyclopropanesulfonamide also known as RDEAl 19 or BAY869766 and described in PCT Publication No.
  • Pimasertib AS-703026, CAS 1204531-26-9
  • Trametinib dimethyl sulfoxide GSK-1120212, CAS 1204531-25-80.
  • Phosphoinositide 3 -kinase (PI3K) inhibitors include but are not limited to, 4-[2-(lH-
  • mTor inhibitors include but are not limited to, Temsirolimus (Torisel®);
  • Ridaforolimus (formally known as deferolimus, (lR,2R,4S)-4-[(2R)-2
  • the present disclosure provides a method of treating cancer by administering to a subject in need thereof a CDH6 ADC and anti-GITR combination with one or more pro-apoptotics, including but not limited to, IAP inhibitors, Bcl2 inhibitors, MCll inhibitors, Trail agents, Chk inhibitors.
  • a CDH6 ADC and anti-GITR combination with one or more pro-apoptotics, including but not limited to, IAP inhibitors, Bcl2 inhibitors, MCll inhibitors, Trail agents, Chk inhibitors.
  • IAP inhibitors include but are not limited to, NVP-LCL161, GDC-
  • IAP inhibitors include but are not limited to those disclosed in WO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118, WO 06/017295, and WO08/134679, all of which are inco ⁇ orated herein by reference.
  • BCL-2 inhibitors include but are not limited to, 4-[4-[[2-(4-Chlorophenyl)-5,5- dimethyl-l-cyclohexen-l-yl]methyl]-l-piperazinyl]-N-[[4-[[(lR)-3-(4-mo ⁇ holinyl)-l- [(phenyltWo)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide (also known as ABT-263 and described in PCT Publication No.
  • PARAs Proapoptotic receptor agonists
  • DR4 TRAILR1
  • DR5 Proapoptotic receptor agonists
  • TRAILR2 including but are not limited to, Dulanermin (AMG-951, RhApo2L/TRAIL);
  • Mapatumumab (HRS-ETR1, CAS 658052-09-6); Lexatumumab (HGS-ETR2, CAS 845816-02-6); Apomab (Apomab®); Conatumumab (AMG655, CAS 896731-82-1); and Tigatuzumab (CS1008, CAS 946415-34-5, available from Daiichi Sankyo).
  • Checkpoint Kinase (CHK) inhibitors include but are not limited to, 7-
  • the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody drug conjugate in combination with one or more
  • FGFR inhibitors include but are not limited to, Brivanib alaninate
  • the present disclosure provides a method of treating cancer by administering to a subject in need thereof a CDH6
  • ADC and anti-GITR antibody combination with an FGFR2 inhibitor such as 3-(2,6-dichloro-3,5- dimethoxyphenyl)- 1 -(6((4-(4-ethy lpiperazin- 1 -yl)pheny l)amino)pyrimidin-4-yl)- 1-methy lurea (also known as BGJ-398); or 4-amino-5-fluoro-3-(5-(4-methylpiperazinl-yl)-l /-benzo[if
  • an FGFR2 inhibitor such as 3-(2,6-dichloro-3,5- dimethoxyphenyl)- 1 -(6((4-(4-ethy lpiperazin- 1 -yl)pheny l)amino)pyrimidin-4-yl)- 1-meth
  • AZD4547 (Gavine et al, 2012, Cancer Research 72, 2045-56, N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3R,5S)- diemthylpiperazin-l-yl)benzamide), Ponatinib (AP24534; Gozgit e/ a/.
  • a CDH6 ADC and anti-GITR antibody combination is used with a PD-1 inhibitor, e.g., as described in WO2015/026684 or WO2016/057846.
  • WO2016/057846 in particular discloses that treatment with an anti-GITR antagonist antibody causes an increase in PD-1 expression on CD8+ T cells in a mouse colon tumor model.
  • the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab.
  • the anti-PD-1 antibody is Nivolumab. Alternative names for
  • Nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558.
  • the anti-PD- 1 antibody is Nivolumab (CAS Registry Number: 946414-94-4).
  • Nivolumab is a fully human IgG4 monoclonal antibody which specifically blocks PD 1.
  • Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US 8,008,449 and WO2006/121168.
  • the inhibitor of PD-1 is Nivolumab, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).
  • the anti-PD-1 antibody is Pembrolizumab. Pembrolizumab
  • the inhibitor of PD-1 is Pembrolizumab disclosed in, e.g., US
  • the anti-PD-1 antibody is Pidilizumab. Pidilizumab (CT-011;
  • Cure Tech is a humanized IgGlk monoclonal antibody that binds to PD1.
  • Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009/101611.
  • Other anti-PDl antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD l antibodies disclosed in US 8,609,089, US 2010028330, and/or US 20120114649.
  • the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-1 inhibitor is AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 and
  • WO2011/066342 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and
  • the CDH6 ADC is in combination with an anti-GITR antibody, wherein the anti-GITR antibody is BMS-986156, INCAGN01876, AMG 228, TRX518, MEDI1873, MK-4166, MK-1248 or FPA-154.
  • the combination comprises an anti-GITR antibody or agonist described in any of the following publications: WO2015187835, WO2015031667, WO2015026684, WO2015184099, WO2016196792, WO2016126781,
  • WO2006105021 WO2005007190, US20070098719, US20050014224, or US20140072566.
  • the anti-GITR antibody is in combination with an ADC.
  • the ADC NOV169N31Q-MCC-DM1 is a P-cadherin antibody conjugated to DM1, and is disclosed in US 2016/0137730.
  • DM1 as well as DM4 can induce immunogenic cell death, and the combination of NOV169N31Q-MCC-DM1 and anti-GITR antibody can be used for the treatment of P-cadherin positive cancers.
  • compositions prepared pharmaceutical or sterile compositions the CDH6 ADC and GITR antibody of the present disclosure are mixed with a pharmaceutically acceptable carrier or excipient.
  • the CDH6 ADC and GITR antibody can be prepared separately or as an admixture.
  • the compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing cancer, for example, ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma.
  • Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g. , lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g. , Hardman et al. , Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al.
  • an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix.
  • an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects.
  • the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub.
  • CDH6 ADC and GITR antibody in pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.
  • compositions comprising the CDH6 ADC and GITR antibody combination can be provided by continuous infusion, or by doses at intervals of, e.g. , one day, one week, or 1-7 times per week.
  • Doses can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation.
  • a specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.
  • the dosage of each respective CDH6 ADC and GITR antibody administered to a patient can be 0.0001 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage can be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight.
  • the dosage of each CDH6 ADC and GITR antibody of the combination can thereof be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
  • Doses of the CDH6 ADC and GITR antibody combination can be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.
  • doses of the CDH6 ADC and GITR antibody combination of the present disclosure are repeated every 3 weeks.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route and dose of administration and the severity of side effects (see, e.g., Maynard et al, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).
  • the route of administration may be by, e.g. , topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g. , Sidman et al, Biopolymers 22:547-556, 1983; Langer e/ a/., J. Biomed. Mater. Res. 15: 167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al. , Proc. Natl. Acad. Sci.
  • composition may also include a solubilizing agent or a local anesthetic such as lidocaine to ease pain at the site of the injection, or both.
  • pulmonary administration can also be employed, e.g. , by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g. , U.S. Pat. Nos.
  • a composition of the present disclosure can also be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes and/or mode of administration will vary depending upon the desired results.
  • Selected routes of administration for the immunoconjugates include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • a composition of the present disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the CDH6 ADC and GITR antibody combination of the present disclosure is administered by infusion.
  • the CDH6 ADC and GITR antibody combination is administered subcutaneously.
  • a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald e/ a/., Surgery 88:507, 1980; Saudek e/ a/., N. Engl. J. Med. 321 :574, 1989).
  • Polymeric materials can be used to achieve controlled or sustained release of the therapies of the
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly (methacry lie acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).
  • CDH6 ADC and anti-GITR combination of the disclosure can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995).
  • viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed.
  • Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts
  • Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle.
  • a pressurized volatile e.g., a gaseous propellant, such as freon
  • humectants can also be added to
  • compositions comprising the immunoconjugates are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops.
  • prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the CDH6 ADC and GITR antibody combination as disclosed herein can be administered together or separately.
  • the CDH6 ADC and GITR antibody can be administered separately less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart.
  • the CDH6 ADC and GITR antibody combination can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the disclosure cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g. , U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685).
  • Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannosides (Umezawa e/ a/., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (Bloeman e/ al, (1995) FEBS Lett. 357: 140; Owais et al , (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al, (1995) Am. J. Physiol. 1233 : 134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
  • the present disclosure provides protocols for the administration of a CDH6 ADC and
  • the CDH6 ADC and GITR antibody combination can be administered concomitantly or sequentially to a subject.
  • the CDH6 ADC and GITR antibody combination can also be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g. , CDH6 ADC) for a period of time, followed by the
  • a second therapy e.g. , anti-GITR antibody
  • administration of a second therapy e.g. , anti-GITR antibody
  • this sequential administration i.e., the cycle, in order to reduce the development of resistance to one of the therapies to avoid or reduce the side effects of one of the therapies, and/or to improve the efficacy of the therapies.
  • the CDH6 ADC and GITR antibody combination of the disclosure can be administered to a subject concurrently.
  • the term "concurrently” is not limited to the administration of the combination at exactly the same time, but rather it is meant that a pharmaceutical composition comprising the combination is administered to a subject in a sequence and within a time interval such that the combination can act together to provide an increased benefit than if they were administered otherwise.
  • each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.
  • Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route.
  • the CDH6 ADC and GITR antibody combination can be administered to a subject in the same pharmaceutical composition.
  • the CDH6 ADC and GITR antibody combination can be administered concurrently to a subject in separate pharmaceutical compositions.
  • HuCAL PLATINUM ® Pannings Antibodies were identified by the selection of clones that bound to CDH6-ECD. As a source of antibody variants a commercially available phage display library, the Morphosys HuCAL PLATINUM® library, was used. The phagemid library is based on the HuCAL® concept (Knappik et al., . J Mol Biol. 2000: 296(l):57-86; Prassler et al., J Mol Biol. 2011 : 413(l):261-78 2011) and employs the CysDisplayTM technology for displaying the Fab on the phage surface (Rothe et al., J Mol Biol. 2008: 376(4): 1182-200). For isolation of anti-CDH6 antibodies, standard panning strategies were performed using solid phase and solution panning approaches.
  • CDH6 proteins and CDH6-expressing cell lines used as antigens for pannings and subsequent screening and characterization is given in Table 1.
  • TREX expression system (Invitrogen, Carlsbad, CA) was used according to the manufacturer's instructions. Briefly, CHO-TREx cells (Invitrogen, R718-07, Carlsbad, CA) were grown in DMEM media (Invitrogen 11995-085) withl0% FBS (Invitrogen, 10082-147, Carlsbad, CA), and 10 ⁇ g/ml Blasticidin (Invitrogen Al 1139-02, Carlsbad, CA). Transfection was performed in 6 well plates when cell reach 90% confluence.
  • the transfected cells were split from 6 well plate to T175 flask next day and grew in selection DMEM media with 10% FBS, 10 ⁇ g/ml Blasticidin, 800-1000 ⁇ g/ml Geneticin (Invitrogen 10131-027).
  • CDH6 expression was induced with final 1 ⁇ g/ml Tetracycline in selection media for 20-24 hours.
  • Positive cells were labeled with final 5 ⁇ g/ml anti-CDH6 primary monoclonal antibody (MAB2715 from R&D Systems, Minneapolis, MN), and then labeled with PE conjugated anti-mouse secondary antibody (cat #12- 4010-87, eBioscience, San Diego, CA) and sorted by FACS.
  • Stable CHO cell lines featuring exogenous expression of CDH6 from mouse, rat and cynomolgus origin were generated by transfection of CHO-K1 cells (Invitrogen, Carlsbad, CA) with the respective cDNAs cloned into a mammalian expression vector (pcDNA6.1, Invitrogen, Carlsbad, CA). Transfection was performed in 6 well plates when cell reach 90% confluence.
  • the transfected cells were split from 6 well plate to T175 flask next day and grew in selection DMEM media with 10% FBS, 10 ⁇ g/ml Blasticidin, 800-1000 ⁇ g/ml Geneticin (Invitrogen 10131-027 Carlsbad, CA).
  • CDH6 expression was induced with final 1 ⁇ g/ml Tetracycline in selection media for 20-24 hours. Positive cells were labeled with final 5 ⁇ g/ml anti- CDH6 primary monoclonal antibody (MAB2715 from R&D Systems, Minneapolis, MN), and then labeled with PE conjugated anti-mouse secondary antibody (cat #12-4010-87, eBioscience, San Diego, CA) and sorted by FACS.
  • An alternative commercially available anti-CDH6 antibody is the 2B6 antibody (#GWB-E8FDF3 - Genway, San Diego, CA). This antibody was also used to confirm CDH6 expression in cells.
  • IQATKRLDREEKPVYILRAQAINRRTGRP APP-Avi SEQ ID NO:3
  • Protein was expressed in Rosetta® 2 De3 pLysS cells (Millipore, Billerica, MA) in pET vector using 2xYT + 50 ⁇ g/ml Kanamycin and 30 ⁇ g/ml chloramphenicol. Cells were induced with 0.1 mM IPTG at 30°C. The pellet resuspended in 400 ml 20mM Tris-Cl, pH 8.0, 150 mM NaCl, 3mM CaC12, 20 mM Imidazole, + PI tabs (1 per 50 ml) + lmg/ml lysozyme added fresh, homogenized to break up clumps and passed through microfluidizer 3 times.
  • the soluble fraction of supernatant was used for purification over a 2 ml Ni-NTA (Qiagen superflow resin, Qiagen, Venlo, Netherlands).
  • the lysate was passed over the column at 2.0 ml/min, washed with 10 CV (column volume) 20mM Tris-Cl, pH 8.0, 150 mM NaCl, 3mM CaC12, 20 mM Imidazole and protein was eluted with 4 x 3.0 ml elution fractions using 20mM Tris-Cl, pH 8.0, 500 mM NaCl, 3mM CaC12, 250 mM Imidazole.
  • hCDH6aa54-615 APPavi, hCDH6aa54-615 Fc, cynoCDH6 FL, moCDH6 FL, ratCDH6 FL
  • the protein was expressed in HEK293 (ATCC CRL-1573) derived cell lines previously adapted to suspension culture and grown in a Novartis proprietary serum-free medium. Small scale expression verification was undertaken in transient 6-well-plate transfection assays on the basis of lipofection. Large-scale protein production via transient transfection and was performed at the 10-20 L scale in the WaveTM bioreactor system (GE Healthcare, Pittsburgh, PA). DNA Polyethylenimine (Polysciences, Warrington, PA) was used as a plasmid carrier at a ratio of 1 :3 (w:w). The cell culture supernatants were harvested 7-10 days post transfection and concentrated by cross- flow filtration and diafiltration prior to purification.
  • Non- Avi-tagged proteins were dialyzed using Slide-A-Lyzer Mini Dialysis units,
  • Nanodrop® device (Thermo Scientific, Rockford, IL). Samples were stored at 4°C until usage.
  • the Fab fragment displaying phages are incubated with antigen that has been bound to a surface support by direct immobilization.
  • a coating check ELISA was performed to determine the optimal antigen coating concentration. For this purpose a 2 fold dilution series of hCDH6aa54-615 APPavi (see Table 1) covering 24 to 0.19 ⁇ g/mL was generated. The individual dilutions were used to coat wells on a 96-well MaxisorpTM plate (#442404 Nunc, Rochester, NY) via direct immobilization. After coating the plate was washed thrice with 300 ⁇ , PBS and subsequently blocked with lx blocking buffer (2.5% milk, 2.5% BSA, 0.05% Tween 20) for 2h at room temperature.
  • lx blocking buffer (2.5% milk, 2.5% BSA, 0.05% Tween 20
  • an anti-APP antibody (generated in-house) was added to 1 ⁇ , 0.18 ⁇ or 0.04 ⁇ . Secondary detection was done using AP -labeled anti-mouse IgG-F(ab)2 specific antibody in a 1 :5000 dilution Ref. 115-056-006 Jackson-Immunoresearch, West Grove, PA). The antigen concentration at which signal saturation was observed was chosen as coating concentrations for SP pannings.
  • the mix of E. coli TG1F+ and phage eluate was incubated for 45 min in a water bath at 37°C for phage infection.
  • the bacterial pellets were re- suspended in 2xYT medium, plated on LB agar plates supplemented with 34 ⁇ g/mL chloramphenicol and incubated overnight at 37°C.
  • Colonies were scraped off the plates and were used for polyclonal amplification of enriched clones and phage production. With purified phage the next panning round was started. The second round of pannings was performed according to the protocol of the first round except for a more stringent washing condition. A third panning round was omitted due to the low second round output titers of -104 cfu/mL.
  • Table 1 was coated on a 96-well MaxisorpTM plate (Nunc) via direct immobilization as described for pancodes 1038.1-8.
  • a 96-well MaxisorpTM plate (Nunc) via direct immobilization as described for pancodes 1038.1-8.
  • the antigen coated and a third panning round the pannings were done as described for pancodes 1038.1-8.
  • the Fab fragment displaying phage and biotinylated antigens are incubated in solution which is expected to increase accessibility of the antigen by the phage.
  • Antigen biotinylation was done as described above.
  • Pancodes 1038.17-22 [00300] Each phage library subpool was blocked with an equal volume of 2x Chemiblocker for 2h at room temperature on a turning wheel. To avoid selection of antibodies against the APPavi and biotin tag unrelated FLT3-APPAvi and biotinylated anti-Cyclosporin msIgG was added to 7.5 and 18 ⁇ g/mL in the blocking step, respectively. For removal of phage particles binding to Streptavidin- beads, pre-adsorption of blocked phage particles was performed using blocked Streptavidin beads (# 112.06D Invitrogen, Carlsbad, CA).
  • pancodes 1038.1-8 were done as described for pancodes 1038.1-8 but using biotinylated hCDH6aa54-615-Fc (see Table 1, SEQ ID NO:6) as antigen.
  • the Fab encoding inserts of the selected HuCAL® PLATINUM phage particles were subcloned from the pMORPH®30 display vector into the pMORPH® 11 FH expression vector.
  • Glycerol stocks containing E. coli that were infected with the final panning round output phages were used to inoculate cultures for pMORPH®30 DNA purification using the Nucleobond® Xtra Midi Plus kit according to the manufacturers manual (#740.412.50 Machery Nagel, Bethlehem, PA).
  • each pMORPH®30 DNA output pool was triple digested via EcoRI/Xbal/Bmtl (all restriction enzymes were purchased from New England Biolabs, Ipswich, MA).
  • EcoRI/Xbal Fab encoding 1485 bp fragment was gel purified using the Wizard SV Gel/PCR clean-up kit according to the manufacturers manual (A9282, Promega, Madison, WI) and ligated into the EcoRI/Xbal cut pMORPH® 11 FH vector backbone.
  • RapCLONE® is a two-step cloning method for the batch conversion of a large amount of Fab expression vectors into IgG expression vectors.
  • a eukaryotic expression cassette was introduced into the pMORPH® 11 expression vectors via BsiWI/Mfel (for pools) or Hpal Mfel (for ⁇ pools) digestion and subsequent ligation.
  • BsiWI/Mfel for pools
  • Hpal Mfel for ⁇ pools
  • pMORPH®4_IgGlf acceptor vector for expression in mammalian cells.
  • Electroporation-competent E. coli TG1F- aliquots of 50 were thawed on ice, mixed with 50 pg/ ⁇ pMORPH® 11 FH plasmid DNA and transferred into pre-cooled electroporation cuvettes.
  • the cells were electroporated (Bio-Rad Gene Pulser; settings: 1.75 kV, 200 ⁇ , and 25 ⁇ (Bio-Rad, Hercules, CA), transferred into 950 ⁇ pre-warmed SOB medium and incubated for 1 h at 37 °C shaking at 220 rpm.
  • An appropriate volume of the transformation samples were plated onto LB agar plates supplemented with 34 ⁇ g/mL Chloramphenicol and incubated overnight at 37°C to obtain single colonies.
  • Chloramphenicol resistant single clones were picked into the wells of a sterile 384- well microtiter plate (Nunc) pre-filled with 60 ⁇ 2xYT medium supplemented with 34 ⁇ g/ml of Chloramphenicol and 1 % glucose and grown overnight at 37°C. Next morning, 20 ⁇ sterile 2xYT media containing 60 % glycerol and 1 % glucose were added into each well of the master plates. Plates were sealed with aluminum foil and stored at -8 °C.
  • the plate was centrifuged for 10 min at 1200g and the supernatant discarded.
  • the bacterial pellets were frozen overnight at -20°C to facilitate the lysis step.
  • the thawed pellets were re-suspended in 200 ⁇ . of BEL lysis buffer (see above) and incubated for 1 h at 22°C and 250 rpm.
  • Resulting Fab lysates were centrifuged to remove cellular debris for 10 min at 1200 g.
  • Fab containing supernatants were used for screening purposes.
  • E. coli TG1F- transformants containing pMORPH®l 1 Fab FH DNA were singled out on 2xYT supplemented with 34 ⁇ g/mL Chloramphenicol and 1% glucose. Individual clones were picked and transferred into 3 mL 2xYT seed cultures supplemented with 34 ⁇ g/mL Chloramphenicol and 1% glucose and incubated at 37°C, 220 rpm for 4h.
  • the seed culture was used to inoculate 50 mL main cultures with 2xYT supplemented with 34 ⁇ g/mL Chloramphenicol and 1% glucose and incubated in 250 mL shake flasks at 30°C until the OD600 reached a value of 0.6.
  • Fab expression was induced by addition of IPTG to a final concentration of 0.75 mM and cultures were further incubated overnight at 25°C and 220 rpm. The next day cells were harvested and the cell pellets frozen overnight at -20°C.
  • His6-tagged Fab fragments were isolated via immobilized metal ion affinity chromatography (Ni-NTA Superflow® beads, #30430 Qiagen, Venlo, Netherlands) and eluted using imidazole. Buffer exchange to lx PBS was performed using PD-10 columns (# 17-0851-01 GE Healthcare, Pittsburgh, PA). Samples were sterile-filtered and the protein concentrations were determined by UV- spectrophotometry. The purity of the samples was analyzed in denaturing, reducing 15% SDS-PAGE. The identity of the samples was confirmed by MS.
  • Eukaryotic HEK293cl8 cells (ATCC CRL-10852) were used in a 96-well expression system for the generation of conditioned cell culture supematants containing full-length IgG for the subsequent use in specificity and/or functional assays.
  • Eukaryotic HEK293 cl8 cells were re- suspended in transfection medium (D-MEM supplemented with 2% L-glutamine (#25030-024 Gibco, Grand Island, NY) 10 % FCS (#3302 PAN Biotech, Aidenbach, Germany) and 1 %
  • Opti-MEM® I medium (#31985-047 Invitrogen, Carlsbad, CA). The master mix was incubated for 15 min at room temperature. On a new plate 20 ⁇ . of Opti- MEM® I medium was added to wells containing 300 ng DNA and gently mixed.
  • the DNA was combined with the pre-incubated Lipofectamine® 2000, mixed gently and incubated for 20 min at room temperature. ⁇ of the pre-incubated Lipofectamine® 2000 DNA complexes were then transferred to each well of the plates with the cells and gently mixed. Plates were incubated for 40 h at 37°C and 6 % C02 for transient expression.
  • the culture supematants were transferred to 96-well V-bottom plates and cleared by centrifugation. The resulting IgG-containing supematants were tested by an anti-Fd capture ELISA for assessment of IgG protein concentration in reference to a known standard and stored at -80°C for later use.
  • AttoPhos fluorescence substrate (#1484281 Roche, Nutley, NJ) was added according to the manufacturer's instructions and the fluorescence emission at 535 nm was recorded with excitation at 430 nm with an ELISA reader.
  • CAP-T® cells (CEVEC Pharmaceuticals, Cologne, Germany) were transiently transfected with pMORPH®4 IgG expression plasmid in FreeStyle293® expression medium (#12338 Invitrogen, Carlsbad, CA) using 40 kDa linear PEI ((PEI Max (#24765-2 Polysciences Warrington, PA)) as gene delivery vehicle.
  • transfection cells One day prior to the transfection cells were diluted to -0.8E+06 cells/ml to ensure exponential growth. At the day of transfection cells were diluted in 9 mL pre-warmed Freestyle® (Invitrogen, Carlsbad, CA) medium to ⁇ 0.5xl0 7 cells/ml and transferred into a 125ml shake flask. 30 ⁇ g DNA was diluted in 500 ⁇ OptiMEM. 1200 ⁇ g PEI Max (#24765-2 Polysciences Warrington, PA) was diluted in 8.80 mL OptiMEM medium. The DNA solution was added drop wise to the cells and gently mixed. After that, 500 ⁇ . PEI Max solution was added to the cells and gently mixed.
  • IgG F(ab')2 (#109-055-097 Jackson Immunoresearch, West Grove, PA) was added in a 1/2500 dilution. After lh at room temperature plates were washed 5x with TBST and AttoPhos substrate was added according to the manufacturer's specifications. Plates were read in an ELISA reader 5 minutes after adding the substrate.
  • Fab fragments binding to cell surface expressed CDH6 antigen were identified from the panning output.
  • lxlO 5 cells/well were transferred into U bottom 96 well plates and mixed with 40 ⁇ /well of the Fab-containing bacterial lysates. Plates were incubated shaking at 4°C for 1 h. After the incubation, 100 ⁇ /well ice-cold FACS buffer (3% FCS, 0.02% NaN 3 , 2 mM EDTA in PBS) was added, cells were spun down at 4°C for 5 min at 250 g and washed twice with 180 ⁇ /well ice-cold FACS buffer. After each washing step, cells were centrifuged and carefully re-suspended.
  • cells were re-suspended in 50 ⁇ , of 1/200 diluted secondary detection antibody (PE-conjugated goat anti-human IgG (#109-116-088, Jackson Immuno research, West Grove, PA). After lh incubation at 4°C cells were again washed twice in 180 ⁇ ice-cold FACS buffer. Finally, cell pellets were re-suspended in 120 ⁇ /well FACS buffer with 0.4 % paraformaldehyde and analyzed in a FACSCalibur® (BD Biosciences, San Jose, CA) equipped with an HTS plate reader.
  • FACSCalibur® BD Biosciences, San Jose, CA
  • VL and VH primers M13rev (5' CAGGAAACAGCTATGAC 3' (SEQ ID NO: 10) and HuCAL VH for ((5' GATAAGCATGCGTAGGAGAAA 3 (SEQ ID NO: 11)) were used, respectively.
  • CTCTAGCGCCACCATGAAACA 3 ' (SEQ ID NO: 13) were used, respectively.
  • OVCAR3 cells (ATCC HTB-161) were grown in T150 tissue culture flasks to approximately 90% confluency. Media was discarded and cells flushed with 10 mL PBS. Cells were detached by incubation with 4 mL cell dissociation buffer for 5 min at 37°C. Detached cells were re- suspended thoroughly in growth medium and transferred to a 50 mL tube. After counting the cells on a Vi-Cell® analyzer (Beckman Coulter, Brea, CA) the suspension was adjusted to 10 5 cells/mL in growth medium. 100 ⁇ ⁇ of the cell suspension was seeded per well of a 96-well microtiter plate with transparent bottom. Plates were incubated for 24 h at 37°C in 5% C02 to allow the cells to adhere and to expand.
  • IgG containing cell culture supernatants were generated as described and diluted in
  • 100 ⁇ of each sample dilution was dispensed per well of the cell containing microtiter plate and incubated for 2h at 37°C to permit IgG internalization.
  • cells were fixed by adding 100 ⁇ lx CellFix® reagent (#340181 BD Biosciences, San Jose, CA) per well. After 10 minutes plates were washed twice with PBS and cells then permeabilized by adding 100 ⁇ 0.1% Triton X-100 per well.
  • a surrogate ADC assay was performed mixing anti-human Fab-DMl reagent (AffiniPure® Fab Fragment Goat Anti-Human IgG H+L conjugated with SMCC-DM1) with purified IgGs at a fixed 1:2 ratio. Cytotoxic potential was tested on the cancer cell line OVCAR3 (ovarian serous carcinoma, cultured in McCoys + 20% FCS) as these cells show high expression of CDH6.
  • OVCAR3 ovarian serous carcinoma, cultured in McCoys + 20% FCS
  • a 10-point, 1 :3 serial dilution was prepared in a 384-well deep-well plate (Brandtech Scientific Inc #701355, Essex, CT ) and 25 ⁇ 1 were transferred per assay plate (triplicates) to yield a highest starting concentration of FAB-DMl/human IgG of 66nM and 33nM, respectively.
  • Cellular activity of the primary antibody /Fab- DM1 complexes was determined using CellTiter-Glo® reagent (#G7571 Promega, Madison, WI) according to the manufacturer's instructions. Viability was normalized to the cells only control.
  • the HuCAL® PLATINUM phagemid library was used to select specific Fab fragments against human CDH6-ECD antigens. Recombinant human APPAvi and Fc fusion as well as truncated domain APPAvi fusion proteins were used for the pannings. HuCAL® PLATINUM antibody -phage particles were subjected in different subpool combinations to a total of 8 different panning strategies resulting in 28 panning output pools. In summary, six out of eight strategies have been productive and resulted in 771 ELISA positive screening hits.
  • the Fab antibody clones were converted into the IgG format, yielding 47 unique IgG clones of which 44 were successfully expressed at screening scale.
  • the purified, unique IgGs were subjected to a series of characterization assays including human/cy no/rat/mouse cross-reactivity by FACS, affinity ranking by Octet, cellular internalization assays as well as surrogate ADC assays using an anti-human IgG Fab-SMCC-DMl secondary reagent. Based on these assays, human IgGs were selected for scaled up production, subsequent direct conjugation to ADC linker/pay loads and testing as ADCs in in vitro and in vivo experiments.

Abstract

The present invention relates to anti-CDH6 ADCs in combination with an anti-GITR antibody, and their uses for the treatment of cancer.

Description

ANTI-CDH6 ANTIBODY DRUG CONJUGATES AND ANTI-GITR ANTIBODY COMBINATIONS AND METHODS OF TREATMENT
FIELD
[0001] The present disclosure is directed to the combination of antibody drug conjugates
(ADC) in combination with anti-GITR agonist antibodies and their uses for the treatment of cancer.
BACKGROUND
[0002] Cadherin-6 (CDH6) is a member of the cadherin superfamily of calcium-dependent cell-cell adhesion molecules. Aside from their role in mechanical adhesion, cadherins are involved in a diverse array of cellular processes relating to tissue morphogenesis (Halbleib and Nelson Genes Dev. 2006; 20(23):3199-3214). This superfamily is classified into classical, desmosomal, and protocadherin groups with classical cadherins subdivided into Type 1 and Type II (Sotomayor et al., Trends Cell Biol. 2014; 34(9):524-536)). CDH6 is a type II, classical cadherin, first described as K-cadherin, which was found to be preferentially expressed in fetal kidney and kidney carcinoma (Xiang et al., Cancer Res. 1994; 54(11):3034-3041; Paul et al., Cancer Res. 1997; 57(13):2741-2748), serous ovarian carcinoma (Kobel et al., PLoS Med. 2008 5(12):e232), as well as during normal renal development (Cho et al., Development 1998; 125(5):803-812; Mah et al., Dev. Biol. 2000; 223(1):38- 53).
[0003] Antibody drug conjugates ("ADCs") have been used for the local delivery of cytotoxic agents in the treatment of cancer (see e.g., Lambert, Curr. Opinion In Pharmacology 2005; 5:543-549). ADCs allow targeted delivery of the drug moiety where maximum efficacy with minimal toxicity may be achieved. In addition to inducing tumor cell death, it has been demonstrated that ADCs delivering microtubule-disrupting payloads can induce a pro-inflammatory tumor
microenvironment and promote anti-tumor immunity (Gerber et al., Biochem.Pharma. 2016; 102:(1- 6).
[0004] Glucocorticoid-induced TNFR-related protein ("GITR") is a member of the Tumor
Necrosis Factor Superfamily (TNFRSF) constitutively expressed on regulatory T cells (Tregs) and at low levels on naive and memory T cells. GITR is a bi-functional molecule capable of driving the expansion of CD8+ T effector (Teff) memory cell populations, while promoting the loss or inhibition of Tregs (Knee et al., Eur. J. Cancer 2016; 67: 1-10). Herein, we disclose the discovery of a unique mechanism of action, wherein administration of an ADC induces GITR upregulation on Tregs, and a synergistic combination of a CDH6 -targeting ADC and agonistic GITR antibody induces greater antitumor activity. SUMMARY
[0005] A combination comprising: a) an anti-CDH6 antibody drag conjugate of the formula:
Ab-(L-(D)m) ':n
or a pharmaceutically acceptable salt thereof; wherein Ab is an antibody or antigen binding fragment thereof that specifically binds to an epitope of human CDH6;
L is a linker;
D is a drag moiety, wherein the drag moiety is N(2')-deacetyl-N2-(4- mercapto-4-methyl- 1 - oxopentyl)-maytansine (DM4) or N(2')- deacetyl-N(2')-(3-mercapto-l-oxopropyl)-maytansine (DM1) ; m is an integer from 1 to 8; and
n is an integer from 1 to 10; and b) an anti-GITR agonist antibody or antigen binding fragment thereof.
[0006] The combination, wherein in the antibody drag conjugate said n is 3 or 4.
[0007] The combination, wherein in said antibody drag conjugate said antibody or antigen binding fragment thereof comprises:
(i) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:224, (b) a LCDR2 of SEQ ID NO:225, (c) a LCDR3 of SEQ ID NO:226; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 227, (e) a HCDR2 of SEQ ID NO: 228, and (f) a HCDR3 of SEQ ID NO:229;
(ii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:210, (b) a LCDR2 of SEQ ID NO:211, (c) a LCDR3 of SEQ ID NO:212; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:213, (e) a HCDR2 of SEQ ID NO: 214, and (f) a HCDR3 of SEQ ID NO:215;
(iii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:266, (b) a LCDR2 of SEQ ID NO:267, (c) a LCDR3 of SEQ ID NO:268; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 269, (e) a HCDR2 of SEQ ID NO:270, and (f) a HCDR3 of SEQ ID NO: 271;
(iv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:308, (b) a LCDR2 of SEQ ID NO:309, (c) a LCDR3 of SEQ ID NO:310; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:311, (e) a HCDR2 of SEQ ID NO:312, and (f) a HCDR3 of SEQ ID NO:313; (v) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 14, (b) a LCDR2 of SEQ ID NO: 15, (c) a LCDR3 of SEQ ID NO: 16; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 17, (e) a HCDR2 of SEQ ID NO: 18, and (f) a HCDR3 of SEQ ID NO: 19;
(vi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:28, (b) a LCDR2 of SEQ ID NO:29, (c) a LCDR3 of SEQ ID NO:30; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:31, (e) a HCDR2 of SEQ ID NO:32, and (f) a HCDR3 of SEQ ID NO:33;
(vii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:42, (b) a LCDR2 of SEQ ID NO:43, (c) a LCDR3 of SEQ ID NO:44; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:45, (e) a HCDR2 of SEQ ID NO:46, and (f) a HCDR3 of SEQ ID NO:47;
(viii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:56, (b) a LCDR2 of SEQ ID NO:57, (c) a LCDR3 of SEQ ID NO:58; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:59, (e) a HCDR2 of SEQ ID NO:60, and (f) a HCDR3 of SEQ ID NO:61;
(ix) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:70, (b) a LCDR2 of SEQ ID NO:71, (c) a LCDR3 of SEQ ID NO:72; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:73, (e) a HCDR2 of SEQ ID NO:74, and (f) a HCDR3 of SEQ ID NO:75;
(x) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:84, (b) a LCDR2 of SEQ ID NO:85, (c) a LCDR3 of SEQ ID NO:86; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:87, (e) a HCDR2 of SEQ ID NO: 88, and (f) a HCDR3 of SEQ ID NO: 89;
(xi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:98, (b) a LCDR2 of SEQ ID NO:99, (c) a LCDR3 of SEQ ID NO: 100; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 101, (e) a HCDR2 of SEQ ID NO: 102, and (f) a HCDR3 of SEQ ID NO: 103;
(xii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 112, (b) a LCDR2 of SEQ ID NO: 113, (c) a LCDR3 of SEQ ID NO: 114; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 115, (e) a HCDR2 of SEQ ID NO: 116, and (f) a HCDR3 of SEQ ID NO: 117; (xiii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 126, (b) a LCDR2 of SEQ ID NO: 127, (c) a LCDR3 of SEQ ID NO: 128; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 129, (e) a HCDR2 of SEQ ID NO: 130, and (f) a HCDR3 of SEQ ID NO: 131;
(xiv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 140, (b) a LCDR2 of SEQ ID NO: 141, (c) a LCDR3 of SEQ ID NO: 142; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 143, (e) a HCDR2 of SEQ ID NO: 144, and (f) a HCDR3 of SEQ ID NO: 145;
(xv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 154, (b) a LCDR2 of SEQ ID NO: 155, (c) a LCDR3 of SEQ ID NO: 156; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 157, (e) a HCDR2 of SEQ ID NO: 158, and (f) a HCDR3 of SEQ ID NO: 159;
(xvi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 168, (b) a LCDR2 of SEQ ID NO: 169, (c) a LCDR3 of SEQ ID NO: 170; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 171, (e) a HCDR2 of SEQ ID NO: 172, and (f) a HCDR3 of SEQ ID NO: 173;
(xvii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 182, (b) a LCDR2 of SEQ ID NO: 183, (c) a LCDR3 of SEQ ID NO: 184; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 185, (e) a HCDR2 of SEQ ID NO: 186, and (f) a HCDR3 of SEQ ID NO: 187;
(xviii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 196, (b) a LCDR2 of SEQ ID NO: 197, (c) a LCDR3 of SEQ ID NO: 198; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 199, (e) a HCDR2 of SEQ ID NO:200, and (f) a HCDR3 of SEQ ID NO:201;
(xix) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:238, (b) a LCDR2 of SEQ ID NO:239, (c) a LCDR3 of SEQ ID NO:240; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:241, (e) a HCDR2 of SEQ ID NO:242, and (f) a HCDR3 of SEQ ID NO:243;
(xx) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:252, (b) a LCDR2 of SEQ ID NO:253, (c) a LCDR3 of SEQ ID NO:254; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:255, (e) a HCDR2 of SEQ ID NO:256, and (f) a HCDR3 of SEQ ID NO:257; (xxi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:280, (b) a LCDR2 of SEQ ID NO:281, (c) a LCDR3 of SEQ ID NO:282; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:283, (e) a HCDR2 of SEQ ID NO:284, and (f) a HCDR3 of SEQ ID NO:285;
(xxii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:294, (b) a LCDR2 of SEQ ID NO:295, (c) a LCDR3 of SEQ ID NO:296; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:297, (e) a HCDR2 of SEQ ID NO:298, and (f) a HCDR3 of SEQ ID NO:299; or
(xxiii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:322, (b) a LCDR2 of SEQ ID NO:323, (c) a LCDR3 of SEQ ID NO:324; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:325, (e) a HCDR2 of SEQ ID NO:326, and (f) a HCDR3 of SEQ ID NO:327.
[0008] The combination, wherein in said antibody drug conjugate said antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region (vH) that comprises SEQ ID NO: 230, and a light chain variable region (vL) that comprises SEQ ID NO:231;
(ii) a heavy chain variable region (vH) that comprises SEQ ID NO: 216, and a light chain variable region (vL) that comprises SEQ ID NO:217;
(iii) a heavy chain variable region (vH) that comprises SEQ ID NO: 272, and a light chain variable region (vL) that comprises SEQ ID NO:273;
(iv) a heavy chain variable region (vH) that comprises SEQ ID NO:314, and a light chain variable region (vL) that comprises SEQ ID NO:315;
(v) a heavy chain variable region (vH) that comprises SEQ ID NO: 20, and a light chain variable region (vL) that comprises SEQ ID NO:21;
(vi) a heavy chain variable region (vH) that comprises SEQ ID NO: 34, and a light chain variable region (vL) that comprises SEQ ID NO:35;
(vii) a heavy chain variable region (vH) that comprises SEQ ID NO:48, and a light chain variable region (vL) that comprises SEQ ID NO:49;
(viii) a heavy chain variable region (vH) that comprises SEQ ID NO:62, and a light chain variable region (vL) that comprises SEQ ID NO:63; (ix) a heavy chain variable region (vH) that comprises SEQ ID NO:76, and a light chain variable region (vL) that comprises SEQ ID NO:77;
(x) a sasy chain variable region (vH) that comprises SEQ ID NO:90, and a light chain variable region (vL) that comprises SEQ ID NO:91;
(xi) a heavy chain variable region (vH) that comprises SEQ ID NO: 104. and a light chain variable region (vL) that comprises SEQ ID NO: 105;
(xii) a easy chain variable region (vH) that comprises SEQ ID NO: 118, and a light chain variable region (vL) that comprises SEQ ID NO: 119;
(xiii) a heavy chain variable region (vH) that comprises SEQ ID NO: 132, and a light chain variable region (vL) that comprises SEQ ID NO: 133;
(xiv) a 1ιε3λ chain variable region (vH) that comprises SEQ ID NO: 146, and a light chain variable region (vL) that comprises SEQ ID NO: 147;
(xv) a heavy chain variable region (vH) that comprises SEQ ID NO: 160, and a light chain variable region (vL) that comprises SEQ ID NO: 161;
(xvi) a 1ιε3λ chain variable region (vH) that comprises SEQ ID NO: 174, and a light chain variable region (vL) that comprises SEQ ID NO: 175;
(xvii) a heavy chain variable region (vH) that comprises SEQ ID NO: 188, and a light chain variable region (vL) that comprises SEQ ID NO: 189;
(xviii) a essy chain variable region (vH) that comprises SEQ ID NO: 202, and a light chain variable region (vL) that comprises SEQ ID NO:203;
(xix) a heavy chain variable region (vH) that comprises SEQ ID NO: 244, and a light chain variable region (vL) that comprises SEQ ID NO:245;
(xx) a 1ιε3λ chain variable region (vH) that comprises SEQ ID NO:258, and a light chain variable region (vL) that comprises SEQ ID NO:259;
(xxi) a heavy chain variable region (vH) that comprises SEQ ID NO:286, and a light chain variable region (vL) that comprises SEQ ID NO:287;
(xxii) a heavy chain variable region (vH) that comprises SEQ ID NO.300, and a light chain variable region (vL) that comprises SEQ ID NO:301; or (xxiii) a heavy chain variable region (vH) that comprises SEQ ID NO:328, and a light chain variable region (vL) that comprises SEQ ID NO:329.
[0009] The combination, wherein in said antibody drug conjugate the linker (L) is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker and a dicarboxylic acid based linker.
[0010] The combination, wherein in said antibody drug conjugate the linker is derived from a cross-linking reagent selected from the group consisting of: N-succinimidyl-4-(2-pyridyldithio)2- sulfo-butanoate (sulfo-SPDB), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N- succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N- sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfo-SMCC) or 2,5- dioxopyrrolidin-l-yl 17-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-5,8,l l, 14-tetraoxo-4,7,10, 13- tetraazaheptadecan-l-oate (CXl-1).
[0011] The combination, wherein in said antibody drug conjugate the linker is derived from
N-succinimidyl-4-(2-pyridyldithio)2-sulfo-butanoate (sulfo-SPDB).
[0012] The combination, wherein in said antibody drug conjugate D and L consist of:
Figure imgf000009_0001
or a pharmaceutically acceptable salt thereof. [0013] The combination, wherein the anti-GITR agonist antibody or antigen binding fragment thereof, specifically binds to SEQ ID NO:336.
[0014] The combination, wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
(i) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427;
(ii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:352, (b) a LCDR2 of SEQ ID NO:353, (c) a LCDR3 of SEQ ID NO:354; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 355, (e) a HCDR2 of SEQ ID NO: 356, and (f) a HCDR3 of SEQ ID NO: 357;
(iii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:366, (b) a LCDR2 of SEQ ID NO:367, (c) a LCDR3 of SEQ ID NO:368; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 369, (e) a HCDR2 of SEQ ID NO:370, and (f) a HCDR3 of SEQ ID NO:371;
(iv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:380, (b) a LCDR2 of SEQ ID NO:381, (c) a LCDR3 of SEQ ID NO:382; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 383, (e) a HCDR2 of SEQ ID NO: 384, and (f) a HCDR3 of SEQ ID NO: 385;
(vi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:394, (b) a LCDR2 of SEQ ID NO:395, (c) a LCDR3 of SEQ ID NO:396; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 397, (e) a HCDR2 of SEQ ID NO: 398, and (f) a HCDR3 of SEQ ID NO: 399;
(vii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:408, (b) a LCDR2 of SEQ ID NO:409, (c) a LCDR3 of SEQ ID NO:410; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 411, (e) a HCDR2 of SEQ ID NO: 412, and (f) a HCDR3 of SEQ ID NO: 413; or
(viii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:436, (b) a LCDR2 of SEQ ID NO:437, (c) a LCDR3 of SEQ ID NO:438; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 439, (e) a HCDR2 of SEQ ID NO: 440, and (f) a HCDR3 of SEQ ID NO: 441.
[0015] The combination, wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region (vH) that comprises SEQ ID NO: 428, and a light chain variable region (vL) that comprises SEQ ID NO:429;
(ii) a heavy chain variable region (vH) that comprises SEQ ID NO: 358, and a light chain variable region (vL) that comprises SEQ ID NO:359;
(iii) a heavy chain variable region (vH) that comprises SEQ ID NO: 372, and a light chain variable region (vL) that comprises SEQ ID NO:373;
(iv) a heavy chain variable region (vH) that comprises SEQ ID NO: 386, and a light chain variable region (vL) that comprises SEQ ID NO:387;
(v) a heavy chain variable region (vH) that comprises SEQ ID NO: 400, and a light chain variable region (vL) that comprises SEQ ID NO:401;
(vi) a heavy chain variable region (vH) that comprises SEQ ID NO: 414, and a light chain variable region (vL) that comprises SEQ ID NO:415; or
(vii) a heavy chain variable region (vH) that comprises SEQ ID NO: 442, and a light chain variable region (vL) that comprises SEQ ID NO:443.
[0016] The combination, wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
(i) a heavy chain that comprises SEQ ID NO: 432, and a light chain that comprises SEQ ID NO:433;
(ii) a heavy chain that comprises SEQ ID NO: 362, and a light chain that comprises SEQ ID NO:363;
(iii) a heavy chain that comprises SEQ ID NO: 376, and a light chain that comprises SEQ ID NO:377;
(iv) a heavy chain that comprises SEQ ID NO: 390, and a light chain that comprises SEQ ID NO:391;
(v) a heavy chain that comprises SEQ ID NO: 404, and a light chain that comprises SEQ ID NO:405;
(vi) a heavy chain that comprises SEQ ID NO: 418, and a light chain that comprises SEQ ID NO:419; or (vii) a heavy chain that comprises SEQ ID NO: 446, and a light chain that comprises SEQ ID NO:447.
[0017] The combination, wherein the anti-GITR agonist antibody or antigen binding fragment thereof consists of: (i) a light chain variable region that consists of (a) a LCDR1 (CDR- Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that consists of: (d) a HCDRI of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427.
[0018] The combination, wherein the anti-GITR agonist antibody or antigen binding fragment thereof is selected from the group consisting of: BMS-986156, INCAGN01876, AMG 228, TRX518, MEDI1873, MK-4166, MK-1248 and FPA-154.
[0019] The combination, wherein the antibody drug conjugate and anti-GITR antibody or antigen binding fragment thereof are in separate forms.
[0020] The combination, wherein the antibody drug conjugate and the anti-GITR antibody or antigen binding fragment thereof are in a fixed combination.
[0021] A method of treating a CDH6 positive cancer in a patient in need thereof, comprising administering to said patient the combination of any one of the above.
[0022] The method, wherein said cancer is selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and
cholangiocarcinoma.
[0023] The method, wherein the antibody drug conjugate and the anti-GITR antibody or antigen binding fragment thereof are administered simultaneously or separately.
[0024] The method, wherein the antibody drug conjugate is administered prior to the administration of the anti-GITR antibody or antigen binding fragment thereof.
[0025] A method of decreasing Treg cells in a tumor, the method comprising;
a) administration of an antibody drug conjugate, wherein the administration of the antibody drug conjugate induces GITR expression on Treg cells;
b) administration of an anti-GITR agonist antibody or antigen binding fragment thereof, which decreases the number of Treg cells associated with the tumor.
[0026] The method, which decreases reoccurrence of the tumor in a patient.
[0027] The method, which increases progression-free survival in a patient.
[0028] Combination for use in treating cancer, wherein the combination is as in any one of the above.
[0029] Combination for use in treating cancer according to the above, wherein said cancer is selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma.
[0030] Use of the combination according to any one of the above in the manufacture of a medicament for the treatment of cancer. [0031] Use according, wherein said cancer is selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and
cholangiocarcinoma.
Definitions
[0032] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
[0033] The term "alkyl" refers to a monovalent saturated hydrocarbon chain having the specified number of carbon atoms. For example, C\ _g alkyl refers to an alkyl group having from 1 to 6 carbon atoms. Alkyl groups may be straight or branched. Representative branched alkyl groups have one, two, or three branches. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, sec -butyl, and t-butyl), pentyl (n- pentyl, isopentyl, and neopentyl), and hexyl.
[0034] The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino- terminus to carboxy -terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
[0035] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the present disclosure). The antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).
[0036] "Complementarity -determining domains" or "complementary -determining regions" ("CDRs") interchangeably refer to the hypervariable regions of VL and VH. The CDRs are the target protein-binding site of the antibody chains that harbors specificity for such target protein. There are three CDRs (CDRl-3, numbered sequentially from the N-terminus) in each human VL or VH, constituting about 15-20% of the variable domains. CDRs can be referred to by their region and order. For example, "VHCDRl" or "HCDR1" both refer to the first CDR of the heavy chain variable region. The CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity. The remaining stretches of the VL or VH, the so- called framework regions, exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).
[0037] The positions of the CDRs and framework regions can be determined using various well known definitions in the art, e.g. , Kabat, Chothia, and AbM (see, e.g. , Johnson et al. , Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al, Nature, 342:877-883 (1989); Chothia et al , J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al , J.Mol.Biol., 273:927-748 (1997)). Definitions of antigen combining sites are also described in the following: Ruiz et al , Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al , J. Mol. Biol., 262:732-745 (1996); and Martin et al, Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al , Methods Enzymol., 203: 121-153 (1991); and Rees et al , In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).
[0038] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminus is a variable region and at the C- terminus is a constant region; the CH3 and CL domains actually comprise the carboxy -terminal domains of the heavy and light chain, respectively. [0039] The term "antigen binding fragment", as used herein, refers to a polypeptide including one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al. , Nature 341 :544-546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.
[0040] Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv ("scFv"); see, e.g., Bird et al, Science 242:423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment." These antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
[0041] Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis- scFv (see, e.g. , Hollinger and Hudson, Nature Biotechnology 23 : 1126-1136, 2005). Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703, 199, which describes fibronectin polypeptide monobodies).
[0042] Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al, Protein Eng. 8: 1057- 1062, 1995; and U.S. Pat. No. 5,641,870).
[0043] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides, including antibodies and antigen binding fragments that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. [0044] The term "human antibody", as used herein, includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g. , human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al , J. Mol. Biol. 296:57-86, 2000.
[0045] The antibodies of the present disclosure can include amino acid residues not encoded by human sequences (e.g. , mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).
[0046] The term "recognize" as used herein refers to an antibody or antigen binding fragment thereof that finds and interacts (e.g. , binds) with its epitope, whether that epitope is linear or conformational. The term "epitope" refers to a site on an antigen to which an antibody or antigen binding fragment of the disclosure specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include techniques in the art, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). A "paratope" is the part of the antibody which recognizes the epitope of the antigen.
[0047] The phrase "specifically binds" or "selectively binds," when used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody -derived binding agent, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies, e.g., in a biological sample, e.g. , a blood, serum, plasma or tissue sample. Thus, under certain designated immunoassay conditions, the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample. In one aspect, under designated immunoassay conditions, the antibody or binding agent with a particular binding specificity binds to a particular antigen at least ten (10) times the background and does not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein. As desired or appropriate, this selection may be achieved by subtracting out antibodies that cross-react with molecules from other species (e.g. , mouse or rat) or other subtypes. Alternatively, in some aspects, antibodies or antibody fragments are selected that cross-react with certain desired molecules.
[0048] The term "affinity" as used herein refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody "arm" interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
[0049] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
[0050] The term "corresponding human germline sequence" refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. The corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence.
[0051] A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least 10 to 100 times over the background.
[0052] The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M-l). Equilibrium dissociation constants can be measured using any known method in the art. The antibodies of the present disclosure generally will have an equilibrium dissociation constant of less than about 10"7 or 10"8 M, for example, less than about 10"9 M or 10"10 M, in some aspects, less than about 10"11 M, 10"12 M or 10"13 M.
[0053] The term "bioavailability" refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.
[0054] As used herein, the phrase "consisting essentially of refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any excipients inactive for the intended purpose of the methods or compositions. In some aspects, the phrase "consisting essentially of expressly excludes the inclusion of one or more additional active agents other than an antibody drug conjugate of the present disclosure. In some aspects, the phrase "consisting essentially of expressly excludes the inclusion of one or more additional active agents other than an antibody drug conjugate of the present disclosure and a second co-administered agent.
[0055] The term "amino acid" refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0056] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule.
Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
[0057] For polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some aspects, the term "conservative sequence modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
[0058] The term "optimized" as used herein refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence.
[0059] The terms "percent identical" or "percent identity," in the context of two or more nucleic acids or polypeptide sequences, refers to the extent to which two or more sequences or subsequences that are the same. Two sequences are "identical" if they have the same sequence of amino acids or nucleotides over the region being compared. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
Optionally, the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
[0060] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0061] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g. , by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g. , Brent et al. , Current Protocols in Molecular Biology, 2003).
[0062] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al , J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive -valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0063] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
[0064] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0065] Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
[0066] The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0067] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, as detailed below, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al , (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al, (1994) Mol. Cell. Probes 8:91-98).
[0068] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g. , DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
[0069] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
[0070] The term "immunoconjugate" or "antibody drug conjugate" or "ADC" as used herein refers to the linkage of an antibody or an antigen binding fragment thereof with another agent, such as a pay load, drug moiety, chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, and the like. The linkage can be covalent bonds, or non-covalent interactions such as through electrostatic forces. Various linkers, known in the art, can be employed in order to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to proteins created through the joining of two or more genes or gene fragments which originally coded for separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.
[0071] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g. , mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms "patient" or "subject" are used herein interchangeably. [0072] The term "toxin," "cytotoxin" or "cytotoxic agent" as used herein, refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.
[0073] The term "anti-cancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.
[0074] The term "drug moiety" or "payload" as used herein refers to a chemical moiety that is conjugated to an antibody or antigen binding fragment, and can include any therapeutic or diagnostic agent, for example, an anti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic, anti-viral), or an anesthetic agent. In certain aspects, a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HD AC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. Methods for attaching each of these to a linker compatible with the antibodies and method of the present disclosure are known in the art. See, e.g., Singh et al., (2009) Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457. In addition, a payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharide or a polysaccharide.
[0075] The term "maytansinoid drug moiety" means the substructure of an antibody -drug conjugate that has the structure of a maytansinoid compound. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4, 151,042). Synthetic maytansinol and maytansinol analogues have been reported. See U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, and Kawai et al (1984) Chem. Pharm. Bull. 3441- 3451, each of which are expressly incorporated by reference. Specific examples of maytansinoids useful for conjugation include DM1, DM3 and DM4.
[0076] "Tumor" refers to neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
[0077] The term "anti-tumor activity" means a reduction in the rate of tumor cell proliferation, viability, or metastatic activity. A possible way of showing anti-tumor activity is to show a decline in growth rate of tumor cells, tumor size stasis or tumor size reduction. Such activity can be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to investigate anti-tumor activity.
[0078] The term "malignancy" refers to a non-benign tumor or a cancer. As used herein, the term "cancer" includes a malignancy characterized by deregulated or uncontrolled cell growth.
Exemplary cancers include: carcinomas, sarcomas, leukemias and lymphomas.
[0079] The term "cancer" includes primary malignant tumors (e.g. , those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).
[0080] "Immunogenic cell death" or "ICD" refers a process by which certain cytotoxic drugs induce apoptosis in such a way that stimulates immune cell recruitment to tumors (see Gerber et al., supra).
[0081] The term "Cadherin 6" or "CDH6" refers to a cell adhesion molecule that is a member of the cadherin family of cell-cell adhesion molecules. The nucleic acid and amino acid sequences of CDH6 are known, and have been published in GenBank Accession Nos. AK291290 (protein accession number BAF83979.1) See also SEQ ID NO: 1 for the human CDH6 cDNA sequence and SEQ ID NO:2 for the human CDH6 protein sequence. Structurally, CDH6 receptor is a type II cadherin with five extracellular cadherin repeats and has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:2. Structurally, a CDH6 nucleic acid sequence has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO 1.
[0082] The term "glucocorticoid-induced tumor necrosis factor receptor family -related protein" or "GITR" (alternatively TNFRSF18) refers to a molecule that is a member of the TNF-R superfamily. The nucleic acid and amino acid sequences of GITR are known, and have been published in GenBank Accession Nos. BC152381 (protein accession number AAI52382) See also SEQ ID NO: 336 for the human GITR protein sequence. GITR has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 336.
[0083] The term "P-cadherin" (also known as Pcad, PCad, or CDH3) refers to the nucleic acid and amino acid sequence of P-cadherin, which have been published in GenBank Accession Nos. NP_ 001784, NP 001784.2 (amino acid sequence), and NM OO 1793.4, GenBank Accession Nos. AA14462, NG 009096, and NG_009096.1 (nucleotide sequences). Sequence information for human P-cadherin domains 1-5 are extracellular and are published in GenBank Acession Nos. NM 001793.4 and NP OO 1784.
[0084] The terms "CDH6 expressing cancer" or "CDH6 positive cancer" refers to a cancer that expresses CDH6 and/or a mutant form of CDH6 on the surface of cancer cells.
[0085] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer in one aspect, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another aspect, "treat," "treating," or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g. , stabilization of a physical parameter), or both. In yet another aspect, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder.
[0086] The term "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refers to an amount sufficient to effect the desired result (i.e., a reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infection). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A "prophylactically effective dosage," and a "therapeutically effective dosage," of the molecules of the present disclosure can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with cancer.
[0087] The term "co-administer" refers to the simultaneous presence of two active agents in the blood of an individual. Active agents that are co-administered can be concurrently or sequentially delivered.
[0088] The term "combination" or "pharmaceutical combination" is defined as the combined administration of an antibody drug conjugate and an anti-GITR antibody, or a binding fragment of each respective antibody thereof to a patient in need. The combination may be administered independently, at the same time or separately, or sequentially within time intervals that allow the combination partners show a cooperative, e.g., additive or synergistic, effect. The combination can be a fixed or non-fixed combination.
[0089] The term "fixed combination" means that the active ingredients or therapeutic agents, are administered to a patient simultaneously in the form of a single entity or dosage form.
[0090] The term "non-fixed combination" means that the active ingredients or therapeutic agents, are both administered to a patient as separate entities or dosage forms either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the three compounds in the body of the subject, e.g., a mammal or human, in need thereof.
[0091] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients.
Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to a desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
[0092] The combination therapy can provide "synergy" and prove "synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g. , by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. BRIEF DESCRIPTION OF THE DRAWINGS
[0093] Figure 1 shows in vivo efficacy of anti-CDH6 antibodies as sulfo-SPDB-DM4 conjugates as a single agent in a xenograft mouse model of ovarian cancer.
[0094] Figure 2 shows in vivo efficacy of anti-GITR antibodies as a single agent in a mouse model of colon cancer.
[0095] Figure 3 depicts the generation of the RENCA murine CDH6 mouse model.
[0096] Figure 4 demonstrates the increase in Tregs following treatment with CDH6 ADC in the RENCA mouse model.
[0097] Figure 5 shows a reduction in tumor volume of RENCAm CDH6 tumors when treated with the CDH6 ADC/GITR combination.
[0098] Figure 6 demonstrates that the CDH6 ADC/GITR combination increases survival in a
RENCAmCDH6 mouse model.
[0099] Figure 7 is a graph of a tumor re-challenge in the RENCAmCDH6 mouse model.
[00100] Figure 8 is a graph of individual tumor volume from control mice during the tumor re- challenge.
[00101] Figure 9 is a graph of TEE survival data in the RENCAm CDH6 mouse model.
[00102] Figure 10 shows the increase of IFN-gamma signal in the presence of splenocytes from re-challenged mice incubated with target cells compared to control splenocytes ( pO.0001, 2 way ANOVA).
[00103] Figure 11 depicts a colon tumor mouse xenograft model treated with CDH6 ADC as a single agent.
[00104] Figure 12 shows the increase in expression in GITR on Treg cells following
CDH6ADC treatment in the colon tumor mouse model.
DETAILED DESCRIPTION
[00105] The present disclosure provides for CDH6 antibody drug conjugates in combination with anti-GITR antibodies. In particular, the present disclosure is directed to the combination of CDH6 antibody drug conjugates (ADC), and agonistic anti-GITR antibodies. The combination of antibodies and antibody fragments (e.g. , antigen binding fragments) of the present disclosure can be used in the treatment of cancer. For example, in the treatment of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma. The present disclosure further provides pharmaceutical compositions comprising the combination and methods dosing of the combination for the treatment of cancer.
Antibody Drug Conjugates
[00106] The present disclosure provides antibody drug conjugates, where an antibody, antigen binding fragment or its functional equivalent that specifically binds to CDH6 is linked to a drug moiety. In one aspect, the antibodies, antigen binding fragments or their functional equivalents are linked, via covalent attachment by a linker, to a drug moiety that is an anti-cancer agent. The antibody drug conjugates can selectively deliver an effective dose of an anti-cancer agent (e.g., a cytotoxic agent) to tumor tissues expressing CDH6, whereby greater selectivity (and lower efficacious dose) may be achieved.
[00107] In one aspect, the disclosure provides for an immunoconjugate of Formula (I):
Ab— (L— (D)m)n
Wherein Ab represents a CDH6 binding antibody or antibody fragment (e.g., antigen binding fragment) described herein;
L is a linker;
D is a drug moiety;
m is an integer from 1-8; and
n is an integer from 1-20. In one aspect, n is an integer from 1 to 10, 2 to 8, or 2 to 5. In a specific aspect, n is 3 to 4. In some aspects, m is 1. In some aspects, m is 2, 3 or 4.
[00108] While the drug moiety to antibody ratio has an exact integer value for a specific conjugate molecule (e.g., n multiplied by m in Formula (I)), it is understood that the value will often be an average value when used to describe a sample containing many molecules, due to some degree of inhomogeneity, typically associated with the conjugation step. The average loading for a sample of an immunoconjugate is referred to herein as the drug moiety to antibody ratio, or "DAR." In the aspect of maytansinoids, this can be referred to as maytansinoid to antibody ratio or "MAR." In some aspects, the DAR is between about 1 and about 5, and typically is about 3, 3.5, 4, 4.5, or 5. In some aspects, at least 50% of a sample by weight is compound having the average DAR plus or minus 2, and preferably at least 50% of the sample is a conjugate that contains the average DAR plus or minus 1. Other aspects include immunoconjugates wherein the DAR is about 3.5. In some aspects, a DAR of 'about n' means the measured value for DAR is within 20% of n.
[00109] The present disclosure provides immunoconjugates comprising the antibodies, antibody fragments (e.g. , antigen binding fragments) and their functional equivalents as disclosed herein, linked or conjugated to a drug moiety. In one aspect, the drug moiety D is a maytansinoid drug moiety, including those having the structure:
Figure imgf000030_0001
DM4
[00110] The drug moiety D can be linked to the antibody Ab through a linker L. L is any chemical moiety that is capable of linking the antibody Ab to the drug moiety D. The linker, L attaches the antibody Ab to the drug moiety D through covalent bond(s). The linker reagent is a bifunctional or multifunctional moiety which can be used to link a drug moiety D and an antibody Ab to form antibody drug conjugates. Antibody drug conjugates can be prepared using a linker having a reactive functionality for binding to the drug moiety D and to the antibody Ab. A cysteine, thiol or an amine, e.g. N-terminus or amino acid side chain such as lysine of the antibody can form a bond with a functional group of a linker reagent.
[00111] In one aspect, L is a cleavable linker. In another aspect, L is a non-cleavable linker.
In some aspects, L is an acid-labile linker, photo-labile linker, peptidase cleavable linker, esterase cleavable linker, a disulfide bond reducible linker, a hydrophilic linker, a procharged linker, or a dicarboxylic acid based linker.
[00112] Suitable cross-linking reagents that form a non-cleavable linker between the drug moiety D, for example maytansinoid, and the antibody Ab are well known in the art, and can form non-cleavable linkers that comprise a sulfur atom (such as SMCC) or those that are without a sulfur atom. Preferred cross-linking reagents that form non-cleavable linkers between the drug moiety D, for example maytansinoid, and the antibody Ab comprise a maleimido- or haloacetyl-based moiety. According to the present disclosure, such non-cleavable linkers are said to be derived from maleimido- or haloacetyl-based moieties.
[00113] Cross-linking reagents comprising a maleimido-based moiety include but not limited to, Ai-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane-l-carboxylate (sulfo-SMCC), jV-succinimidyl-4- (maleimidomethyl)cyclohexane-l-carboxy-(6-amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC), K-maleimidoundeconoic acid jV-succinimidyl ester (KMUA), γ-maleimidobutyric acid jV-succinimidyl ester (GMBS), ε-maleimidocaproic acid -succinimidyl ester (EMCS), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMSA), succinimidyl-6-( -maleimidopropionamido)hexanoate (SMPH), jV-succinimidyl-4-(p- maleimidophenyl)-butyrate (SMPB), N-(-p-maleomidophenyl)isocyanate (PMIP) and maleimido- based cross-linking reagents containing a polyethythene glycol spacer, such as MAL-PEG-NHS. These cross-linking reagents form non-cleavable linkers derived from maleimido-based moieties. Representative structures of maleimido-based cross-linking reagents are shown below.
Figure imgf000031_0001
MAL-PEG-NHS [00114] In another aspect, the linker L is derived from iV-succinimidyl-4-
(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (sulfo-SMCC) or MAL-PEG-NHS.
[00115] Cross-linking reagents comprising a haloacetyle-based moiety include N-succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP). These cross-linking reagents form a non-cleavable linker derived from haloacetyl-based moieties. Representative structures of haloacetyl-based cross-linking reagents are shown below.
Figure imgf000032_0001
or
[00116] In one aspect, the linker L is derived from N-succinimidyl iodoacetate (SIA) or N- succinimidyl(4-iodoacetyl)aminobenzoate (SIAB).
[00117] Suitable cross-linking reagents that form a cleavable linker between the drug moiety
D, for example maytansinoid, and the antibody Ab are well known in the art. Disulfide containing linkers are linkers cleavable through disulfide exchange, which can occur under physiological conditions. According to the present disclosure, such cleavable linkers are said to be derived from disulfide-based moieties. Suitable disulfide cross-linking reagents include N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N- succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and iV-succinimidyl-4-(2-pyridyldithio)2-sulfo- butanoate (sulfo-SPDB), the structures of which are shown below. These disulfide cross-linking reagents form a cleavable linker derived from disulfide-based moieties.
Figure imgf000032_0002
N-succinimidyl-3 -(2-py ridyldithio)propionate (SPDP),
Figure imgf000033_0001
Figure imgf000033_0002
N-succimmidyl-4-(2-pyridyldithio)butanoate (SPDB) and
Figure imgf000033_0003
N-succinimidyl-4-(2-pyridyldithio)2-sulfo-butanoate (sulfo-SPDB)
[00118] In one aspect, the linker L is derived from N-succinimidyl-4-(2-pyridyldithio)2-sulfo- butanoate (sulfo-SPDB).
[00119] In one aspect provided by the disclosure, the conjugate is represented by any one of the following structural formulae:
Figure imgf000033_0004
Figure imgf000034_0001
Figure imgf000034_0002
Ab-MCC-DMI
[00120] wherein: Ab is an antibody or antigen binding fragment thereof that specifically binds to human CDH6; n, which indicates the number of D-L groups attached the Ab through the formation of an amide bond with a primary amine of the Ab, is an integer from 1 to 20. In one aspect, n is an integer from 1 to 10, 2 to 8 or 2 to 5. In a specific aspect, n is 3 or 4.
[00121] In one aspect, the average molar ratio of drug moiety (e.g., DM1 or DM4) to the antibody in the conjugate (i.e., average w value, also known as Maytansinoid Antibody Ratio (MAR)) is about 1 to about 10, about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,
5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or 8.1), about 2.5 to about 7, about 3 to about 5, about 2.5 to about 4.5 (e.g., about
2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5), about 3.0 to about 4.0, about 3.2 to about 4.2, or about 4.5 to 5.5 (e.g., about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5).
[00122] In one aspect provided by the disclosure, the conjugate has substantially high purity and has one or more of the following features: (a) greater than about 90% (e.g. , greater than or equal to about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), preferably greater than about 95%, of conjugate species are monomelic, (b) unconjugated linker level in the conjugate preparation is less than about 10% (e.g., less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%) (relative to total linker), (c) less than 10% of conjugate species are crosslinked (e.g. , less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%), (d) free drug moiety (e.g., DM1 or DM4) level in the conjugate preparation is less than about 2% (e.g., less than or equal to about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) (mol/mol relative to total cytotoxic agent).
[00123] As used herein, the term "unconjugated linker" refers to the antibody that is covalently linked with a linker derived from a cross-linking reagent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1), wherein the antibody is not covalently coupled to the drug moiety (e.g., DM1 or DM4) through a linker (i.e., the "unconjugated linker" can be represented by Ab-SMCC, Ab-SPDB, Ab-sulfo-SPDB, or Ab-CXl-1).
1. Drug Moiety
[00124] The present disclosure provides immunoconjugates that specifically bind to CDH6.
The immunoconjugates of the present disclosure comprise anti-CDH6 antibodies, antibody fragments (e.g., antigen binding fragments) or functional equivalents that are conjugated to a drug moiety, e.g., an anti-cancer agent, anti-hematological disorder agent, an autoimmune treatment agent, an antiinflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent. The antibodies, antibody fragments (e.g., antigen binding fragments) or functional equivalents can be conjugated to several identical or different drug moieties using any methods known in the art.
[00125] In certain aspects, the drug moiety of the immunoconjugates of the present disclosure is selected from a group consisting of: a maytansinoid, a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
[00126] In one aspect, the drug moiety of the immunoconjugates of the present disclosure is a maytansinoid drug moiety, such as but not limited to, DM1, DM3, or DM4.
[00127] Further, the antibodies, antibody fragments (e.g. , antigen binding fragments) or functional equivalents of the present disclosure may be conjugated to a drug moiety that modifies a given biological response. Drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein such as tumor necrosis factor, a- interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response modifier such as, for example, a lymphokine.
[00128] In one aspect, the antibodies, antibody fragments (e.g., antigen binding fragments) or functional equivalents of the present disclosure are conjugated to a drug moiety, such as a cytotoxin, a drug (e.g. , an immunosuppressant) or a radiotoxin. Examples of cytotoxin include but are not limited to, taxanes (see, e.g., International (PCT) Patent Application Nos. WO 01/38318 and
PCT/US03/02675), DNA-alkylating agents (e.g., CC-1065 analogs), anthracychnes, tubuly sin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, and cytotoxic agents comprising a reactive polyethylene glycol moiety (see, e.g. , Sasse et al , J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et al , Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et al , J. Antibiot. (Tokyo), 44, 1045- 53 (1991), Francisco et al, Blood 2003 15; 102(4): 1458-65), U.S. Pat. Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application Publication No. 2001/0036923 Al, Pending U.S. patent application Ser. Nos. 10/024,290 and 10/116,053, and International (PCT) Patent Application No. WO 01/49698), taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
Therapeutic agents also include, for example, anti-metabolites (e.g. , methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thiotepa chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). (See e.g., Seattle Genetics US20090304721).
[00129] Other examples of cytotoxins that can be conjugated to the antibodies, antibody fragments (antigen binding fragments) or functional equivalents of the present disclosure include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof.
[00130] Various types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies are known in the art, see, e.g. , Saito et al, (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail et al , (2003) Cancer Immunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell 3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3: 1089- 1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53 :247-264.
[00131] The antibodies, antibody fragments (e.g. , antigen binding fragments) or functional equivalents of the present disclosure can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa
Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies disclosed herein. In certain aspects, the macrocyclic chelator is 1,4,7, 10- tetraazacyclododecane-Ν,Ν' ,Ν' ' ,N" ' -tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al , (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al, (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al , (1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.
[00132] The antibodies, antibody fragments (e.g. , antigen binding fragments) or functional equivalents of the present disclosure can also conjugated to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. In particular, the present disclosure provides fusion proteins comprising an antibody fragment (e.g., antigen binding fragment) described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide.
[00133] Additional fusion proteins may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the activities of antibodies of the present disclosure or fragments thereof (e.g. , antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol. 16(2):76-82; Hansson et al, (1999) J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308- 313 (each of these patents and publications are hereby incorporated by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
2. Linker
[00134] As used herein, a "linker" is any chemical moiety that is capable of linking an antibody, antibody fragment (e.g. , antigen binding fragments) or functional equivalent to another moiety, such as a drug moiety. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid based linker.
[00135] In one aspect, the linker used is derived from a crosslinking reagent such as N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), V-succinimidyl-4-(2-pyridyldithio)-2- sulfo-butanoate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl(4- iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfo-SMCC) or 2,5-dioxopyrrolidin-l-yl 17-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-5,8,l l, 14- tetraoxo-4,7, 10, 13-tetraazaheptadecan-l-oate (CXl-1). In another aspect, the linker used is derived from a cross-linking agent such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N- succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-sulfosuccinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (sulfo-SMCC), -succinimidyl-4-(2-pyridyldithio)-2- sulfo-butanoate (sulfo-SPDB) or 2,5-dioxopyrrolidin-l-yl 17-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)- 5,8, 11, 14-tetraoxo-4,7, 10,13-tetraazaheptadecan-l-oate (CXl-1).
[00136] Non-cleavable linkers are any chemical moiety capable of linking a drug moiety, such as a maytansinoid, to an antibody in a stable, covalent manner and does not fall off under the categories listed above for cleavable linkers. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage. Furthermore, non-cleavable refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, photolabile- cleaving agent, a peptidase, an esterase, or a chemical or physiological compound that cleaves a disulfide bond, at conditions under which the drug moiety, such as maytansinoid or the antibody does not lose its activity.
[00137] Acid-labile linkers are linkers cleavable at acidic pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid-labile linkers.
[00138] Photo-labile linkers are linkers that are useful at the body surface and in many body cavities that are accessible to light. Furthermore, infrared light can penetrate tissue. [00139] Some linkers can be cleaved by peptidases, i.e. peptidase cleavable linkers. Only certain peptides are readily cleaved inside or outside cells, see e.g. Trout et al., 79 Proc. Natl.
Acad.Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Furthermore, peptides are composed of a-amino acids and peptidic bonds, which chemically are amide bonds between the carboxylate of one amino acid and the amino group of a second amino acid. Other amide bonds, such as the bond between a carboxylate and the ε-amino group of lysine, are understood not to be peptidic bonds and are considered non-cleavable.
[00140] Some linkers can be cleaved by esterases, i.e. esterase cleavable linkers. Again, only certain esters can be cleaved by esterases present inside or outside of cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters produced with simple alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols.
[00141] Procharged linkers are derived from charged cross-linking reagents that retain their charge after incorporation into an antibody drug conjugate. Examples of procharged linkers can be found in US 2009/0274713.
3. Conjugation and Preparation of ADCs
[00142] The conjugates of the present disclosure can be prepared by any methods known in the art, such as those described in US Patent Nos. 7,811,572, 6,411, 163, 7,368,565, and 8, 163,888, and US application publications 2011/0003969, 2011/0166319, 2012/0253021 and 2012/0259100. The entire teachings of these patents and patent application publications are herein incorporated by reference.
One-Step Process
[00143] In one aspect, the conjugates of the present disclosure can be prepared by a one-step process. The process comprises combining the antibody, drug and cross-linking agent in a substantially aqueous medium, optionally containing one or more co-solvents, at a suitable pH. In one aspect, the process comprises the step of contacting the antibody of the present disclosure with a drug (e.g., DM1 or DM4) to form a first mixture comprising the antibody and the drug, and then contacting the first mixture comprising the antibody and the drug with a cross-linking agent (e.g. , SMCC, Sulfo- SMCC, SPDB, Sulfo-SPDB or CXl-1) in a solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the conjugate (e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Sulfo-SPDB-DM4, or Ab-CXl-l-DMl), (ii) free drug (e.g. , DM1 or DM4), and (iii) reaction by-products.
[00144] In one aspect, the one-step process comprises contacting the antibody with the drug
(e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) in a solution having a pH of about 6 or greater (e.g. , about 6 to about 9, about 6 to about 7, about 7 to about 9, about 7 to about 8.5, about 7.5 to about 8.5, about 7.5 to about 8.0, about 8.0 to about 9.0, or about 8.5 to about 9.0). For example, the process comprises contacting a cell-binding agent with the drug (DM1 or DM4) and then the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) in a solution having a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In another aspect, the process comprises contacting a cell-binding agent with the drug (e.g. , DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) in a solution having a pH of about 7.8 (e.g. , a pH of 7.6 to 8.0 or a pH of 7.7 to 7.9).
[00145] The one-step process (i.e., contacting the antibody with the drug (e.g. , DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) can be carried out at any suitable temperature known in the art. For example, the one-step process can occur at about 20°C or less (e.g., about -10°C (provided that the solution is prevented from freezing, e.g., by the presence of organic solvent used to dissolve the cytotoxic agent and the bifunctional crosslinking reagent) to about 20°C, about 0°C to about 18°C, about 4°C to about 16°C), at room temperature (e.g., about 20°C to about 30°C or about 20°C to about 25°C), or at an elevated temperature (e.g., about 30°C to about 37°C). In one aspect, the one-step process occurs at a temperature of about 16°C to about 24°C (e.g., about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, or about 25°C). In another aspect, the one-step process is carried out at a temperature of about 15°C or less (e.g., about -10°C to about 15°C, or about 0°C to about 15°C). For example, the process comprises contacting the antibody with the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) at a temperature of about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, about 2°C, about 1°C, about 0°C, about -1°C, about -2°C, about -3°C, about -4°C, about -5°C, about -6°C, about -7°C, about -8°C, about -9°C, or about -10°C, provided that the solution is prevented from freezing, e.g., by the presence of organic solvent(s) used to dissolve the cross-linking agent (e.g., SMCC, Sulfo-SMCC, Sulfo-SPDB SPDB, or CXl-1). In one aspect, the process comprises contacting the antibody with the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) at a temperature of about -10°C to about 15°C, about 0°C to about 15°C, about 0°C to about 10°C, about 0°C to about 5°C, about 5°C to about 15°C, about 10°C to about 15°C, or about 5°C to about 10°C. In another aspect, the process comprises contacting the antibody with the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) at a temperature of about 10°C (e.g., a temperature of 8°C to 12°C or a temperature of 9°C to 11°C). [00146] In one aspect, the contacting described above is effected by providing the antibody, then contacting the antibody with the drug (e.g. , DM1 or DM4) to form a first mixture comprising the antibody and the drug (e.g. , DM1 or DM4), and then contacting the first mixture comprising the antibody and the drug (e.g. , DM1 or DM4) with the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1). For example, in one aspect, the antibody is provided in a reaction vessel, the drug (e.g. , DM1 or DM4) is added to the reaction vessel (thereby contacting the antibody), and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) is added to the mixture comprising the antibody and the drug (e.g., DM1 or DM4) (thereby contacting the mixture comprising the antibody and the drug). In one aspect, the antibody is provided in a reaction vessel, and the drug (e.g. , DM1 or DM4) is added to the reaction vessel immediately following providing the antibody to the vessel. In another aspect, the antibody is provided in a reaction vessel, and the drug (e.g., DM1 or DM4) is added to the reaction vessel after a time interval following providing the antibody to the vessel (e.g., about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1 day or longer after providing the cell-binding agent to the space). The drug (e.g. , DM1 or DM4) can be added quickly (i.e., within a short time interval, such as about 5 minutes, about 10 minutes) or slowly (such as by using a pump).
[00147] The mixture comprising the antibody and the drug (e.g., DM1 or DM4) can then be contacted with the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) either immediately after contacting the antibody with the drug (e.g. , DM1 or DM4) or at some later point (e.g., about 5 minutes to about 8 hours or longer) after contacting the antibody with the drug (e.g., DM1 or DM4). For example, in one aspect, the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) is added to the mixture comprising the antibody and the drug (e.g., DM1 or DM4) immediately after the addition of the drug (e.g. , DM1 or DM4) to the reaction vessel comprising the antibody. Alternatively, the mixture comprising the antibody and the drug (e.g., DM1 or DM4) can be contacted with the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo- SPDB or CXl-1) at about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, or longer after contacting the antibody with the drug (e.g. , DM1 or DM4).
[00148] After the mixture comprising the antibody and the drug (e.g., DM1 or DM4) is contacted with the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) the reaction is allowed to proceed for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or longer (e.g., about 30 hours, about 35 hours, about 40 hours, about 45 hours, or about 48 hrs). [00149] In one aspect, the one-step process further comprises a quenching step to quench any unreacted drug (e.g. , DM 1 or DM4) and/or unreacted cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1). The quenching step is typically performed prior to purification of the conjugate. In one aspect, the mixture is quenched by contacting the mixture with a quenching reagent. As used herein, the "quenching reagent" refers to a reagent that reacts with the free drug (e.g., DM1 or DM4) and/or cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1). In one aspect, maleimide or haloacetamide quenching reagents, such as 4-maleimidobutyric acid, 3- maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, or iodoacetamidopropionic acid, can be used to ensure that any unreacted group (such as thiol) in the drug (e.g. , DM1 or DM4) is quenched. The quenching step can help prevent the dimerization of the drug (e.g., DM1). The dimerized DM1 can be difficult to remove. Upon quenching with polar, charged thiol-quenching reagents (such as 4- maleimidobutyric acid or 3-maleimidopropionic acid), the excess, unreacted DM1 is converted into a polar, charged, water-soluble adduct that can be easily separated from the covalently- linked conjugate during the purification step. Quenching with non-polar and neutral thiol-quenching reagents can also be used. In one aspect, the mixture is quenched by contacting the mixture with a quenching reagent that reacts with the unreacted cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1). For example, nucleophiles can be added to the mixture in order to quench any unreacted SMCC. The nucleophile preferably is an amino group containing nucleophile, such as lysine, taurine and hydroxylamine.
[00150] In another aspect, the reaction (i.e., contacting the antibody with the drug (e.g. , DM1 or DM4) and then cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1)) is allowed to proceed to completion prior to contacting the mixture with a quenching reagent. In this regard, the quenching reagent is added to the mixture about 1 hour to about 48 hours (e.g. , about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or about 25 hours to about 48 hours) after the mixture comprising the antibody and the drug (e.g. , DM1 or DM4) is contacted with the cross- linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1).
[00151] Alternatively, the mixture is quenched by lowering the pH of the mixture to about 5.0
(e.g., 4.8, 4.9, 5.0, 5.1 or 5.2). In another aspect, the mixture is quenched by lowering the pH to less than 6.0, less than 5.5, less than 5.0, less than 4.8, less than 4.6, less than 4.4, less than 4.2, less than 4.0. Alternatively, the pH is lowered to about 4.0 (e.g., 3.8, 3.9, 4.0, 4.1 or 4.2) to about 6.0 (e.g., 5.8, 5.9, 6.0, 6.1 or 6.2), about 4.0 to about 5.0, about 4.5 (e.g., 4.3, 4.4, 4.5, 4.6 or 4.7) to about 5.0. In one aspect, the mixture is quenched by lowering the pH of the mixture to 4.8. In another aspect, the mixture is quenched by lowering the pH of the mixture to 5.5. [00152] In one aspect, the one-step process further comprises a holding step to release the unstably bound linkers from the antibody. The holding step comprises holding the mixture prior to purification of the conjugate (e.g. , after the reaction step, between the reaction step and the quenching step, or after the quenching step). For example, the process comprises (a) contacting the antibody with the drug (e.g. , DM1 or DM4) to form a mixture comprising the antibody and the drug (e.g., DM1 or DM4); and then contacting the mixture comprising the antibody and drug (e.g. , DM1 or DM4) with the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1), in a solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the conjugate (e.g., Ab-MCC- DM1, Ab-SPDB-DM4, Sulfo-SPDB-DM4 or Ab-CXl-l-DMl), (ii) free drug (e.g. , DM1 or DM4), and (iii) reaction by-products, (b) holding the mixture prepared in step (a) to release the unstably bound linkers from the cell-binding agent, and (c) purifying the mixture to provide a purified conjugate.
[00153] In another aspect, the process comprises (a) contacting the antibody with the drug
(e.g., DM1 or DM4) to form a mixture comprising the antibody and the drug (e.g., DM1 or DM4); and then contacting the mixture comprising the antibody and the drug (e.g. , DM1 or DM4) with the cross- linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1), in a solution having a pH of about 4 to about 9 to provide a mixture comprising (i) the conjugate, (ii) free drug (e.g., DM1 or DM4), and (iii) reaction by-products, (b) quenching the mixture prepared in step (a) to quench any unreacted drug (e.g. , DM 1 or DM4) and/or unreacted cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1), (c) holding the mixture prepared in step (b) to release the unstably bound linkers from the cell-binding agent, and (d) purifying the mixture to provide a purified conjugate (e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl).
[00154] Alternatively, the holding step can be performed after purification of the conjugate, followed by an additional purification step.
[00155] In another aspect, the reaction is allowed to proceed to completion prior to the holding step. In this regard, the holding step can be performed about 1 hour to about 48 hours (e.g. , about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or about 24 hours to about 48 hours) after the mixture comprising the antibody and the drug (e.g. , DM1 or DM4) is contacted with the cross- linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1).
[00156] The holding step comprises maintaining the solution at a suitable temperature (e.g. , about 0°C to about 37°C) for a suitable period of time (e.g. , about 1 hour to about 1 week, about 1 hour to about 24 hours, about 1 hour to about 8 hours, or about 1 hour to about 4 hours) to release the unstably bound linkers from the antibody while not substantially releasing the stably bound linkers from the antibody. In one aspect, the holding step comprises maintaining the solution at about 20 °C or less (e.g., about 0°C to about 18°C, about 4°C to about 16°C), at room temperature (e.g., about 20°C to about 30°C or about 20°C to about 25°C), or at an elevated temperature (e.g., about 30°C to about 37°C). In one aspect, the holding step comprises maintaining the solution at a temperature of about 16°C to about 24°C (e.g., about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, or about 25°C). In another aspect, the holding step comprises maintaining the solution at a temperature of about 2°C to about 8°C (e.g., about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C). In another aspect, the holding step comprises maintaining the solution at a temperature of about 37°C (e.g., about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C).
[00157] The duration of the holding step depends on the temperature and the pH at which the holding step is performed. For example, the duration of the holding step can be substantially reduced by performing the holding step at elevated temperature, with the maximum temperature limited by the stability of the cell-binding agent-cytotoxic agent conjugate. The holding step can comprise maintaining the solution for about 1 hour to about 1 day (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours), about 10 hours to about 24 hours, about 12 hours to about 24 hours, about 14 hours to about 24 hours, about 16 hours to about 24 hours, about 18 hours to about 24 hours, about 20 hours to about 24 hours, about 5 hours to about 1 week, about 20 hours to about 1 week, about 12 hours to about 1 week (e.g., about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days), or about 1 day to about 1 week.
[00158] In one aspect, the holding step comprises maintaining the solution at a temperature of about 2 °C to about 8 °C for a period of at least about 12 hours for up to a week. In another aspect, the holding step comprises maintaining the solution at a temperature of about 2 °C to about 8 °C overnight (e.g., about 12 to about 24 hours, preferably about 20 hours).
[00159] The pH value for the holding step preferably is about 4 to about 10. In one aspect, the pH value for the holding step is about 4 or more, but less than about 6 (e.g., 4 to 5.9) or about 5 or more, but less than about 6 (e.g. , 5 to 5.9). In another aspect, the pH values for the holding step range from about 6 to about 10 (e.g., about 6.5 to about 9, about 6 to about 8). For example, pH values for the holding step can be about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.
[00160] In other aspects, the holding step can comprise incubating the mixture at 25°C at a pH of about 6-7.5 for about 12 hours to about 1 week, incubating the mixture at 4°C at a pH of about 4.5- 5.9 for about 5 hours to about 5 days, or incubating the mixture at 25°C at a pH of about 4.5-5.9 for about 5 hours to about 1 day.
[00161] The one-step process can optionally include the addition of sucrose to the reaction step to increase solubility and recovery of the conjugates. Desirably, sucrose is added at a concentration of about 0.1% (w/v) to about 20% (w/v) (e.g. , about 0.1% (w/v), 1% (w/v), 5% (w/v), 10% (w/v), 15% (w/v), or 20% (w/v)). Preferably, sucrose is added at a concentration of about 1% (w/v) to about 10% (w/v) (e.g., about 0.5% (w/v), about 1% (w/v), about 1.5% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), or about 11% (w/v)). In addition, the reaction step also can comprise the addition of a buffering agent. Any suitable buffering agent known in the art can be used. Suitable buffering agents include, for example, a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer. In one aspect, the buffering agent is selected from the group consisting of HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), POPSO (piperazine- l,4-bis-(2-hydroxy-propane-sulfonic acid) dehydrate), HEPES (4-(2-hydroxyethyl)piperazine-l- ethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazine-l-propanesulfonic acid), TES (N- [tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and a combination thereof.
[00162] The one-step process can further comprise the step of purifying the mixture to provide purified conjugate (e.g., Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl). Any purification methods known in the art can be used to purify the conjugates of the present disclosure. In one aspect, the conjugates of the present disclosure use tangential flow filtration (TFF), non-adsorptive chromatography, adsorptive chromatography, adsorptive filtration, selective precipitation, or any other suitable purification process, as well as combinations thereof. In another aspect, prior to subjecting the conjugates to purification process described above, the conjugates are first filtered through one or more PVDF membranes. Alternatively, the conjugates are filtered through one or more PVDF membranes after subjecting the conjugates to the purification process described above. For example, in one aspect, the conjugates are filtered through one or more PVDF membranes and then purified using tangential flow filtration. Alternatively, the conjugates are purified using tangential flow filtration and then filtered through one or more PVDF membranes.
[00163] Any suitable TFF systems may be utilized for purification, including a Pellicon® type system (MiUipore, Billerica, MA), a Sartocon® Cassette system (Sartorius AG, Edgewood, NY), and a Centrasette® type system (Pall Corp., East Hills, NY).
[00164] Any suitable adsorptive chromatography resin may be utilized for purification.
Preferred adsorptive chromatography resins include hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (DVIAC), dye ligand chromatography, affinity chromatography, reversed phase chromatography, and combinations thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT Type I and Type II, Bio-Rad Laboratories, Hercules, CA), HA
Ultrogel® hydroxyapatite (Pall Corp., East Hills, NY), and ceramic fluoroapatite (CFT Type I and Type II, Bio-Rad Laboratories, Hercules, CA). An example of a suitable HCIC resin is MEP
Hypercel® resin (Pall Corp., East Hills, NY). Examples of suitable HIC resins include Butyl- Sepharose, Hexyl-Sepaharose, Phenyl-Sepharose, and Octyl Sepharose resins (all from GE Healthcare, Piscataway, NJ), as well as Macro-prep® Methyl and Macro-Prep® t-Butyl resins (Biorad
Laboratories, Hercules, CA). Examples of suitable ion exchange resins include SP-Sepharose®, CM- Sepharose®, and Q-Sepharose® resins (all from GE Healthcare, Piscataway, NJ), and Unosphere® S resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable mixed mode ion exchangers include Bakerbond® ABx resin (JT Baker, Phillipsburg NJ). Examples of suitable IMAC resins include Chelating Sepharose ® resin (GE Healthcare, Piscataway, NJ) and Profinity® IMAC resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable dye ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, NJ) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable affinity resins include Protein A Sepharose resin (e.g. , MabSelect, GE
Healthcare, Piscataway, NJ) and lectin affinity resins, e.g. Lentil Lectin Sepharose® resin (GE Healthcare, Piscataway, NJ), where the antibody bears appropriate lectin binding sites. Examples of suitable reversed phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, CA).
[00165] Any suitable non-adsorptive chromatography resin may be utilized for purification.
Examples of suitable non-adsorptive chromatography resins include, but are not limited to,
SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™ resins (e.g. , S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 and SUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P- 60, and P-100), and others known to those of ordinary skill in the art.
Two-Step Process and One-Pot Process
[00166] In one aspect, the conjugates of the present disclosure can be prepared as described in the U.S. Patent 7,811,572 and U.S. Patent Application Publication No. 2006/0182750. The process comprises the steps of (a) contacting the antibody of the present disclosure with the cross-linking agent (e.g. , SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CXl-1) to covalently attach the linker (i.e., Ab-SMCC, Ab-SPDB or Ab-CXl-1) to the antibody and thereby prepare a first mixture comprising the antibody having the linker bound thereto; (b) optionally subjecting the first mixture to a purification process to prepare a purified first mixture of the antibody having the linker bound thereto; (c) conjugating the drug (e.g. , DM1 or DM4) to the antibody having the linker bound thereto in the first mixture by reacting the antibody having the linker bound thereto with the drug (e.g. , DM1 or DM4) in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) conjugate (e.g. , Ab-MCC-DMl, Ab-SPDB-DM4, Ab-Sulfo-SPDB-DM4 or Ab-CXl-l-DMl), (ii) free drug (e.g. , DM1 or DM4); and (iii) reaction by-products; and (d) subjecting the second mixture to a purification process to purify the conjugate from the other components of the second mixture.
Alternatively, the purification step (b) can be omitted. Any purification methods described herein can be used for steps (b) and (d). In one embodiment, TFF is used for both steps (b) and (d). In another embodiment, TFF is used for step (b) and absorptive chromatography (e.g. , CHT) is used for step (d).
One-Step Reagent and In-situ Process
[00167] In one aspect, the conjugates of the present disclosure can be prepared by conjugating pre-formed drug-linker compound (e.g. , SMCC-DM1, Sulfo-SMCC-DMl, SPDB-DM4, Sulfo-SPDB- DM4 or CXl-l-DMl) to the antibody of the present disclosure, as described in U.S. Patent 6,441, 163 and U.S. Patent Application Publication Nos. 2011/0003969 and 2008/0145374, followed by a purification step. Any purification methods described herein can be used. The drug-linker compound is prepared by reacting the drug (e.g., DM1 or DM4) with the cross-linking agent (e.g., SMCC, Sulfo- SMCC, SPDB, Sulfo-SPDB or CXl-1). The drug-linker compound (e.g. , SMCC-DM1, Sulfo-SMCC- DMl, SPDB-DM4, Sulfo-SPDB-DM4 or CXl-l-DMl) is optionally subjected to purification before being conjugated to the antibody.
4. Characterization and Selection of Desirable Antibodies and Antibody Drug Conjugates
[00168] The antibodies, antibody fragments (e.g. , antigen binding fragments) or antibody drug conjugates of the present disclosure can be characterized and selected for their physical/chemical properties and/or biological activities by various assays known in the art.
[00169] For example, an antibody of the present disclosure can be tested for its antigen binding activity by known methods such as ELISA, FACS, Biacore or Western blot.
[00170] Transgenic animals and cell lines are particularly useful in screening antibody drug conjugates (ADCs) that have potential as prophylactic or therapeutic treatments of cancer overexpression of tumor-associated antigens and cell surface receptors. Screening for a useful ADC may involve administering a candidate ADC over a range of doses to the transgenic animal, and assaying at various time points for the effect(s) of the ADC on the disease or disorder being evaluated. Alternatively, or additionally, the drug can be administered prior to or simultaneously with exposure to an inducer of the disease, if applicable. The candidate ADC may be screened serially and individually, or in parallel under medium or high-throughput screening format.
[00171] One aspect is a screening method comprising (a) transplanting cells from a stable cancer cell line or human patient tumor expressing CDH6 (e.g., an ovarian cell line or tumor fragment, a renal cell line or tumor fragment, a hepatic cell line or tumor fragment, a thyroid cell line or tumor fragment, a CNS cancer cell line or tumor fragment, a cholangiocarcinoma cancer cell line or tumor fragment, ovarian, renal, hepatic, soft tissue, CNS, thyroid, or cholangiocarcinoma primary cells) into a non-human animal, (b) administering an ADC drag candidate to the non-human animal and (c) determining the ability of the candidate to inhibit the growth of tumors from the transplanted cell line. The present disclosure also encompasses a method of screening ADC candidates for the treatment of a disease or disorder characterized by the overexpression of CDH6 comprising (a) contacting cells from a stable cancer cell line expressing CDH6 with a drag candidate, and (b) evaluating the ability of the ADC candidate to inhibit the growth of the stable cell line.
[00172] A further aspect is a screening method comprising (a) contacting cells from a stable cancer cell line expressing CDH6 with an ADC drag candidate and (b) evaluating the ability of the ADC candidate to induce immunogenic cell death. In one aspect the ability of the ADC candidate to induce apoptosis is evaluated.
[00173] Candidate ADC can be screened by being administered to the transgenic animal over a range of doses, and evaluating the animal's physiological response to the compounds over time. In some cases, it can be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound. If cell lines derived from the subject transgenic animals are used to screen for ADCs useful in treating various disorders associated with overexpression of CDH6, the test ADCs are added to the cell culture medium at an appropriate time, and the cellular response to the ADCs is evaluated over time using the appropriate biochemical and/or histological assays.
CDH6 Antibodies
[00174] The present disclosure provides for antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to human CDH6. Antibodies or antibody fragments (e.g., antigen binding fragments) of the present disclosure include, but are not limited to, the human monoclonal antibodies or fragments thereof, isolated as described, in the Examples below.
[00175] The present disclosure in certain aspects provides antibodies or antibody fragments
(e.g., antigen binding fragments) that specifically bind CDH6, said antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VH domain having an amino acid sequence of SEQ ID NO: 20, 34, 48, 62, 76, 90, 104, 118, 132, 146, 160, 174, 188, 202, 216, 230, 244, 258, 272, 286, 300, 314 or 328 (Table 5). The present disclosure also provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 5. In particular aspects, the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to CDH6, said antibodies comprising (or alternatively, consist of) one, two, three, four, five or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 5.
[00176] The present disclosure provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VL domain having an amino acid sequence of SEQ ID NO: 21, 35, 49, 63, 77, 91, 105, 119, 133, 147, 161, 175, 189, 203, 217, 231, 245, 259, 273, 287, 301, 315 or 329 (Table 5). The present disclosure also provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 5, infra. In particular, the disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to CDH6, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise (or alternatively, consist of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 5.
[00177] Other antibodies or antibody fragments (e.g. , antigen binding fragments) of the present disclosure include amino acids that have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity in the CDR regions with the CDR regions depicted in the sequences described in Table 5. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared with the CDR regions depicted in the sequence described in Table 5.
[00178] The present disclosure also provides nucleic acid sequences that encode VH, VL, the full length heavy chain, and the full length light chain of the antibodies that specifically bind to CDH6. Such nucleic acid sequences can be optimized for expression in mammalian cells.
[00179] Other antibodies of the present disclosure include those where the amino acids or nucleic acids encoding the amino acids have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity to the sequences described in Table 5. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared with the variable regions depicted in the sequence described in Table 5, while retaining substantially the same therapeutic activity.
[00180] Since each of these antibodies can bind to CDH6, the VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other CDH6-binding antibodies. Such "mixed and matched" CDH6 -binding antibodies can be tested using the binding assays known in the art (e.g. , ELISAs, and other assays described in the Example section). When these chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence. Likewise a full length heavy chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence. Likewise, a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence. Likewise, a full length light chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length light chain sequence. Accordingly, in one aspect, the disclosure provides for an isolated monoclonal antibody or antigen binding region thereof having: a heavy chain variable region (vH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 34, 48, 62, 76, 90, 104, 1 18, 132, 146, 160, 174, 188, 202, 216, 230, 244, 258, 272, 286, 300, 314 or 328 (Table 5); and a light chain variable region (vL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 35, 49, 63, 77, 91, 105, 119, 133, 147, 161, 175, 189, 203, 217, 231, 245, 259, 273, 287, 301, 315 or 329 (Table 5); wherein the antibody specifically binds to CDH6.
[00181] In another aspect, the disclosure provides (i) an isolated monoclonal antibody having: a full length heavy chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 24, 38, 52, 66, 80, 94, 108, 122, 136, 150, 164, 178, 192, 206, 220, 234, 248, 262, 276, 290, 304, 318 or 332; and a full length light chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 25, 39, 53, 67, 81, 95, 109, 123, 137, 151, 165, 179, 193, 207, 221, 235, 249, 263, 277, 291, 305, 319 or 333 ; or (ii) a functional protein comprising an antigen binding portion thereof.
[00182] In another aspect, the present disclosure provides CDH6 -binding antibodies that comprise the heavy chain and light chain CDRls, CDR2s and CDR3s as described in Table 5, or combinations thereof. The amino acid sequences of the VH CDRls of the antibodies are shown in SEQ ID NOs: 17, 31, 45, 59, 73, 87, 101, 115, 129, 143, 157, 171, 185, 199, 213, 227, 241, 255, 269,
283, 297, 311 and 325. The amino acid sequences of the VH CDR2s of the antibodies and are shown in SEQ ID NOs: 18, 32, 46, 60, 74, 88, 102, 116, 130, 144, 158, 172, 186, 200, 214, 228, 242, 256, 270,
284, 298, 312 and 326. The amino acid sequences of the VH CDR3s of the antibodies are shown in SEQ ID NOs: 19, 33, 47, 61, 75, 89, 103, 117, 131, 145, 159, 173, 187, 201, 215, 229, 243, 257, 271,
285, 299, 313 and 327. The amino acid sequences of the VL CDRls of the antibodies are shown in SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182, 196, 210, 224, 238, 252, 266,
280, 294, 308 and 322. The amino acid sequences of the VL CDR2s of the antibodies are shown in SEQ ID NOs: 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267,
281, 295, 309 and 323. The amino acid sequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268,
282, 296, 310 and 324.
[00183] Given that each of these antibodies can bind to CDH6 and that antigen-binding specificity is provided primarily by the CDR1, 2 and 3 regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and match, although each antibody must contain a VH CDR1, 2 and 3 and a VL CDR1, 2 and 3 to create other CDH6-binding binding molecules. Such "mixed and matched" CDH6-binding antibodies can be tested using the binding assays known in the art and those described in the Examples (e.g., ELISAs). When VH CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular VH sequence should be replaced with a structurally similar CDR sequence(s). Likewise, when VL CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular VL sequence should be replaced with a structurally similar CDR sequence(s). It will be readily apparent to the ordinarily skilled artisan that novel VH and VL sequences can be created by substituting one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences shown herein for monoclonal antibodies of the present disclosure.
[00184] Accordingly, the present disclosure provides an isolated monoclonal antibody or antigen binding region thereof comprising a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 31, 45, 59, 73, 87, 101, 115, 129, 143, 157, 171, 185, 199, 213, 227, 241, 255, 269, 283, 297, 311 and 325; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 32, 46, 60, 74, 88, 102,116, 130, 144, 158, 172, 186, 200, 214, 228, 242, 256, 270, 284, 298, 312 and 326; a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 33, 47, 61, 75, 89, 103, 117, 131, 145, 159, 173, 187, 201, 215, 229, 243, 257, 271, 285, 299, 313 and 327; a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182, 196, 210, 224, 238, 252, 266, 280, 294, 308 and 322; a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309 and 323; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 3 10 and 324; wherein the antibody specifically binds CDH6. [00185] In a specific aspect, an antibody or antibody fragment (e.g. , antigen binding fragments) that specifically binds to CDH6 comprising:
(i) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:224, (b) a LCDR2 of SEQ ID NO:225, (c) a LCDR3 of SEQ ID NO:226; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 227, (e) a HCDR2 of SEQ ID NO: 228, and (f) a HCDR3 of SEQ ID NO:229;
(ii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:210, (b) a LCDR2 of SEQ ID NO:211, (c) a LCDR3 of SEQ ID NO:212; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:213, (e) a HCDR2 of SEQ ID NO: 214, and (f) a HCDR3 of SEQ ID NO:215;
(iii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:266, (b) a LCDR2 of SEQ ID NO:267, (c) a LCDR3 of SEQ ID NO:268; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 269, (e) a HCDR2 of SEQ ID NO:270, and (f) a HCDR3 of SEQ ID NO: 271;
(iv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:308, (b) a LCDR2 of SEQ ID NO:309, (c) a LCDR3 of SEQ ID NO:310; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:311, (e) a HCDR2 of SEQ ID NO:312, and (f) a HCDR3 of SEQ ID NO:313;
(v) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 14, (b) a LCDR2 of SEQ ID NO: 15, (c) a LCDR3 of SEQ ID NO: 16; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 17, (e) a HCDR2 of SEQ ID NO: 18, and (f) a HCDR3 of SEQ ID NO: 19;
(vi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:28, (b) a LCDR2 of SEQ ID NO:29, (c) a LCDR3 of SEQ ID NO:30; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:31, (e) a HCDR2 of SEQ ID NO:32, and (f) a HCDR3 of SEQ ID NO:33;
(vii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:42, (b) a LCDR2 of SEQ ID NO:43, (c) a LCDR3 of SEQ ID NO:44; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:45, (e) a HCDR2 of SEQ ID NO:46, and (f) a HCDR3 of SEQ ID NO:47;
(viii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:56, (b) a LCDR2 of SEQ ID NO:57, (c) a LCDR3 of SEQ ID NO:58; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:59, (e) a HCDR2 of SEQ ID NO:60, and (f) a HCDR3 of SEQ ID NO:61;
(ix) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:70, (b) a LCDR2 of SEQ ID NO:71, (c) a LCDR3 of SEQ ID NO:72; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:73, (e) a HCDR2 of SEQ ID NO:74, and (f) a HCDR3 of SEQ ID NO:75;
(x) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:84, (b) a LCDR2 of SEQ ID NO:85, (c) a LCDR3 of SEQ ID NO:86; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:87, (e) a HCDR2 of SEQ ID NO: 88, and (f) a HCDR3 of SEQ ID NO: 89;
(xi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:98, (b) a LCDR2 of SEQ ID NO:99, (c) a LCDR3 of SEQ ID NO: 100; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 101, (e) a HCDR2 of SEQ ID NO: 102, and (f) a HCDR3 of SEQ ID NO: 103;
(xii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 112, (b) a LCDR2 of SEQ ID NO: 113, (c) a LCDR3 of SEQ ID NO: 114; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 115, (e) a HCDR2 of SEQ ID NO: 116, and (f) a HCDR3 of SEQ ID NO: 117;
(xiii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 126, (b) a LCDR2 of SEQ ID NO: 127, (c) a LCDR3 of SEQ ID NO: 128; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 129, (e) a HCDR2 of SEQ ID NO: 130, and (f) a HCDR3 of SEQ ID NO: 131;
(xiv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 140, (b) a LCDR2 of SEQ ID NO: 141, (c) a LCDR3 of SEQ ID NO: 142; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 143, (e) a HCDR2 of SEQ ID NO: 144, and (f) a HCDR3 of SEQ ID NO: 145;
(xv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 154, (b) a LCDR2 of SEQ ID NO: 155, (c) a LCDR3 of SEQ ID NO: 156; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 157, (e) a HCDR2 of SEQ ID NO: 158, and (f) a HCDR3 of SEQ ID NO: 159;
(xvi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 168, (b) a LCDR2 of SEQ ID NO: 169, (c) a LCDR3 of SEQ ID NO: 170; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 171, (e) a HCDR2 of SEQ ID NO: 172, and (f) a HCDR3 of SEQ ID NO: 173;
(xvii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 182, (b) a LCDR2 of SEQ ID NO: 183, (c) a LCDR3 of SEQ ID NO: 184; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 185, (e) a HCDR2 of SEQ ID NO: 186, and (f) a HCDR3 of SEQ ID NO: 187;
(xviii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 196, (b) a LCDR2 of SEQ ID NO: 197, (c) a LCDR3 of SEQ ID NO: 198; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 199, (e) a HCDR2 of SEQ ID NO:200, and (f) a HCDR3 of SEQ ID NO:201;
(xix) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:238, (b) a LCDR2 of SEQ ID NO:239, (c) a LCDR3 of SEQ ID NO:240; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:241, (e) a HCDR2 of SEQ ID NO:242, and (f) a HCDR3 of SEQ ID NO:243;
(xx) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:252, (b) a LCDR2 of SEQ ID NO:253, (c) a LCDR3 of SEQ ID NO:254; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:255, (e) a HCDR2 of SEQ ID NO:256, and (f) a HCDR3 of SEQ ID NO:257;
(xxi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:280, (b) a LCDR2 of SEQ ID NO:281, (c) a LCDR3 of SEQ ID NO:282; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:283, (e) a HCDR2 of SEQ ID NO:284, and (f) a HCDR3 of SEQ ID NO:285;
(xxii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:294, (b) a LCDR2 of SEQ ID NO:295, (c) a LCDR3 of SEQ ID NO:296; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:297, (e) a HCDR2 of SEQ ID NO:298, and (f) a HCDR3 of SEQ ID NO:299; or
(xxiii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:322, (b) a LCDR2 of SEQ ID NO:323, (c) a LCDR3 of SEQ ID NO:324; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:325, (e) a HCDR2 of SEQ ID NO:326, and (f) a HCDR3 of SEQ ID NO:327.
GITR antibodies
[00186] The present disclosure provides for antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to human GITR. Antibodies or antibody fragments (e.g. , antigen binding fragments) of the present disclosure include, but are not limited to, the human monoclonal antibodies or fragments thereof, isolated as described in the Examples below.
[00187] The present disclosure in certain aspects provides antibodies or antibody fragments
(e.g., antigen binding fragments) that specifically bind GITR, said antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VH domain having an amino acid sequence of SEQ ID NO: 348, 358, 372, 386, 400, 414, 428 or 442 (Table 6). The present disclosure also provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 6. In particular aspects, the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to GITR, said antibodies comprising (or alternatively, consist of) one, two, three, four, five or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 6.
[00188] The present disclosure provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise a VL domain having an amino acid sequence of SEQ ID NO: 349, 359, 373, 387, 401, 415, 429 or 443 (Table 6). The present disclosure also provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g., antigen binding fragments) comprise a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 6, infra. In particular, the disclosure provides antibodies or antibody fragments (e.g. , antigen binding fragments) that specifically bind to GITR, said antibodies or antibody fragments (e.g. , antigen binding fragments) comprise (or alternatively, consist of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 6.
[00189] Other antibodies or antibody fragments (e.g. , antigen binding fragments) of the present disclosure include amino acids that have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity in the CDR regions with the CDR regions depicted in the sequences described in Table 6. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared with the CDR regions depicted in the sequence described in Table 6.
[00190] The present disclosure also provides nucleic acid sequences that encode VH, VL, the full length heavy chain, and the full length light chain of the antibodies that specifically bind to GITR. Such nucleic acid sequences can be optimized for expression in mammalian cells.
[00191] Other antibodies of the present disclosure include those where the amino acids or nucleic acids encoding the amino acids have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity to the sequences described in Table 6. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared with the variable regions depicted in the sequence described in Table 6, while retaining substantially the same therapeutic activity.
[00192] Since each of these antibodies can bind to GITR, the VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other GITR binding antibodies. Such "mixed and matched" GITR binding antibodies can be tested using the binding assays known in the art (e.g., ELISAs, and other assays described in the Example section). When these chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence. Likewise a full length heavy chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence. Likewise, a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence. Likewise, a full length light chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length light chain sequence. Accordingly, in one aspect, the disclosure provides for an isolated monoclonal antibody or antigen binding region thereof having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 348, 358, 372, 386, 400, 414, 428 or 442 (Table 6); and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 349, 359, 373, 387, 401, 415, 429 or 443 (Table 6); wherein the antibody specifically binds to GITR.
[00193] In another aspect, the disclosure provides (i) an isolated monoclonal antibody having: a full length heavy chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 362, 376, 390, 404, 418, 432 or 446; and a full length light chain comprising an amino acid sequence that has been optimized for expression in the cell of a mammalian selected from the group consisting of SEQ ID NO: 363, 377, 391, 405, 419, 433 or 447; or (ii) a functional protein comprising an antigen binding portion thereof .
[00194] In another aspect, the present disclosure provides anti-GITR antibodies that comprise the heavy chain and light chains.
[00195] In another aspect, the present disclosure provides anti-GITR antibodies that comprise the heavy chain and light chain CDRls, CDR2s and CDR3s as described in Table 6, or combinations thereof. In some embodiments, the combination comprises GITR.MAB7.
[00196] In a specific aspect, an antibody or antibody fragment (e.g. , antigen binding fragments) that specifically binds to GITR comprising:
(i) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427;
(ii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:352, (b) a LCDR2 of SEQ ID NO:353, (c) a LCDR3 of SEQ ID NO:354; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 355, (e) a HCDR2 of SEQ ID NO: 356, and (f) a HCDR3 of SEQ ID NO: 357;
(iii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:366, (b) a LCDR2 of SEQ ID NO:367, (c) a LCDR3 of SEQ ID NO:368; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 369, (e) a HCDR2 of SEQ ID NO:370, and (f) a HCDR3 of SEQ ID NO:371;
(iv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:380, (b) a LCDR2 of SEQ ID NO:381, (c) a LCDR3 of SEQ ID NO:382; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 383, (e) a HCDR2 of SEQ ID NO: 384, and (f) a HCDR3 of SEQ ID NO: 385;
(vi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:394, (b) a LCDR2 of SEQ ID NO:395, (c) a LCDR3 of SEQ ID NO:396; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 397, (e) a HCDR2 of SEQ ID NO: 398, and (f) a HCDR3 of SEQ ID NO: 399;
(vii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:408, (b) a LCDR2 of SEQ ID NO:409, (c) a LCDR3 of SEQ ID NO:410; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 411, (e) a HCDR2 of SEQ ID NO: 412, and (f) a HCDR3 of SEQ ID NO: 413; and
(viii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:436, (b) a LCDR2 of SEQ ID NO:437, (c) a LCDR3 of SEQ ID NO:438; and a heavy chain variable region that comprises: (d) a HCDR1 of SEQ ID NO: 439, (e) a HCDR2 of SEQ ID NO: 440, and (f) a HCDR3 of SEQ ID NO: 441.
1. Identification of Epitopes and Antibodies That Bind to the Same Epitope
[00197] The present disclosure provides antibodies and antibody fragments (e.g., antigen binding fragments) that bind to an epitope of the extracellular domain of CDH6 or the extracellular domain of GITR.
[00198] The present disclosure also provides antibodies and antibody fragments (e.g., antigen binding fragments) that bind to the same epitope as do the CDH6 antibodies described in Table 5 and the GITR antibodies described in Table 6. Additional antibodies and antibody fragments (e.g., antigen binding fragments) can therefore be identified based on their ability to cross-compete (e.g. , to competitively inhibit the binding of, in a statistically significant manner) with other antibodies in antibody binding assays. The ability of a test antibody to inhibit the binding of antibodies and antibody fragments (e.g., antigen binding fragments) of the present disclosure to a protein (e.g., human CDH6 or human GITR) demonstrates that the test antibody can compete with that antibody or antibody fragment (e.g. , antigen binding fragments) for binding to CDH6 or GITR; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the respective extracellular domain as the antibody or antibody fragment (e.g., antigen binding fragments) with which it competes. In a certain aspect, the antibody that binds to the same epitope on CDH6 or GITR as the antibodies or antibody fragments (e.g. , antigen binding fragments) of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.
2. Further Alteration of the Framework of Fc Region
[00199] The present disclosure provides site-specific labeled immunoconjugates. These immunoconjugates can comprise modified antibodies or antigen binding fragments thereof that further comprise modifications to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "back-mutated" to the germline sequence by, for example, site-directed mutagenesis. Such "back-mutated" antibodies are also intended to be encompassed.
[00200] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 2003/0153043 by Carr et al.
[00201] In addition or alternative to modifications made within the framework or CDR regions, antibodies can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody can be chemically modified (e.g. , one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below. [00202] In one aspect, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g. , increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
[00203] In another aspect, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc -hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U. S. Patent No. 6, 165,745 by Ward et al.
[00204] In yet other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in, e.g. , U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
[00205] In another aspect, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in, e.g. , U.S. Patent Nos. 6, 194,551 by Idusogie et al.
[00206] In another aspect, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g. , the PCT Publication WO 94/29351 by Bodmer et al. In a specific aspect, one or more amino acids of an antibody or antigen binding fragment thereof of the present disclosure are replaced by one or more allotypic amino acid residues, for the IgGl subclass and the kappa isotype. Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgGl, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al. , MAbs. 1 :332-338 (2009).
[00207] In yet another aspect, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids. This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGl for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).
[00208] In still another aspect, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen." Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.
[00209] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. For example, EP 1, 176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al. , (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g. , beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17: 176-180, 1999).
[00210] In another aspect, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half -life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6, 121,022 by Presta et al.
[00211] In order to minimize the ADCC activity of an antibody, specific mutations in the Fc region result in "Fc silent" antibodies that have minimal interaction with effector cells. In general, the "IgG Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl- terminus of the IgG antibody. The numbering of residues in the Fc region is that of the EU index of Kabat. The C- terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody.
[00212] Silenced effector functions can be obtained by mutation in the Fc region of the antibodies and have been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181 : 6664-69) see also Heusser et al., WO2012065950. Examples of silent Fc lgGl antibodies are the LALA mutant comprising L234A and L235A mutation in the lgGl Fc amino acid sequence. Another example of a silent lgGl antibody is the DAPA (D265A, P329A) mutation (US 6,737,056). Another silent lgGl antibody comprises the N297A mutation, which results in aglycosylated/non-glycosylated antibodies.
[00213] Fc silent antibodies result in no or low ADCC activity, meaning that an Fc silent antibody exhibits an ADCC activity that is below 50% specific cell lysis, No ADCC activity means that the Fc silent antibody exhibits an ADCC activity (specific cell lysis) that is below 1 %.
3. Production of the Antibodies
[00214] The CDH6 or GITR antibodies and antibody fragments (e.g., antigen binding fragments) thereof can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g. , hybridoma or recombinant production. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.
[00215] The disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions as described herein. In some aspects, the polynucleotide encoding the CDH6 antibody heavy chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162,
176, 190, 204, 218, 232, 246, 260, 274, 288, 302, 316, and 330. In some aspects, the polynucleotide encoding the CDH6 antibody light chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 23, 37, 51, 65, 79, 93, 107, 121, 135, 149, 163,
177, 191, 205, 219, 233, 247, 261, 275, 289, 303, 317, and 331.
[00216] In some aspects, the polynucleotide encoding the CDH6 antibody heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 26, 40, 54, 68, 82, 96, 110, 124, 138, 152, 166, 180, 194, 208, 222, 236, 250, 264, 278, 292, 306, 320, and 334. In some aspects, the polynucleotide encoding the CDH6 antibody light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 27, 41, 55, 69, 83, 97, 111, 125, 139, 153, 167, 181, 195, 209, 223, 237, 251, 265, 279, 293, 307, 321, and 335.
[00217] In some aspects, the polynucleotide encoding the GITR antibody heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:364, 378, 392, 406, 420, 434 or 448. In some aspects, the polynucleotide encoding the GITR antibody light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:365, 379, 393, 407, 421, 435 or 449.
[00218] In some aspects, the polynucleotide encoding the GITR antibody heavy chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 350, 360, 374, 388, 402, 416, 430 and 444. In some aspects, the polynucleotide encoding the GITR antibody light chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 351, 361, 375, 389, 403, 417, 431 and 445.
[00219] The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in the Examples below) encoding an anti-CDH6 antibody or its binding fragment. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al. , Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown e/ al, Meth. Enzymol. 68: 109, 1979; the diethylphosphoramidite method of Beaucage et al , Tetra. Lett., 22: 1859, 1981; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al , Nucleic Acids Res. 19:967, 1991; and Eckert et al, PCR Methods and Applications 1 : 17, 1991.
[00220] Also provided in the present disclosure are expression vectors and host cells for producing the anti-CDH6 or anti-GITR antibodies described above. Various expression vectors can be employed to express the polynucleotides encoding the antibody chains or binding fragments. Both viral-based and nonviral expression vectors can be used to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al, Nat Genet 15:345, 1997). For example, nonviral vectors useful for expression of the antibody polynucleotides and polypeptides in mammalian (e.g. , human) cells include pThioHis A, B & C, pcDNA3.1 His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPS V vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adenoassociated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al, supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.
[00221] The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g. , enhancers) that are operably linked to the polynucleotides encoding an antibody chain or fragment. In some aspects, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an antibody chain or fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al, Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al , Meth. Enzymol., 153 :516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.
[00222] The expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted anti-CDH6 or anti-GITR antibody sequences. More often, the inserted anti-CDH6 or anti-GITR antibody sequences are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.
[00223] The host cells for harboring and expressing the anti-CDH6 or anti-GITR antibody chains can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g. , an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express anti-CDH6 or anti-GITR polypeptides. Insect cells in combination with baculovirus vectors can also be used.
[00224] In other aspects, mammalian host cells are used to express and produce the anti-CDH6 or anti-GITR polypeptides of the present disclosure. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., a myeloma hybridoma clone) or a mammalian cell line harboring an exogenous expression vector (e.g. , SP2/0 myeloma cells). These include any normal mortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B- cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g. , Queen et al, Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.
[00225] Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al, supra). Other methods include, e.g. , electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high- yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express anti-CDH6 or anti-GITR antibody chains or binding fragments can be prepared using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.
Therapeutic Uses
[00226] The antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates of the present disclosure are useful in a variety of applications including, but not limited to, treatment of cancer, such as solid cancers. In certain aspects, the antibodies, antibody fragments (e.g., antigen binding fragments), and antibody drug conjugates are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and/or reducing the tumorigenicity of a tumor. The methods of use can be in vitro, ex vivo, or in vivo methods.
[00227] In one aspect, the disclosure provides for a method of treating, preventing or ameliorating a disease comprising administering the antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates to a patient, thereby treating the disease. In certain aspects, the disease treated with the antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates is a cancer. Examples of diseases which can be treated and/or prevented include, but are not limited to, ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma. In certain aspects, the cancer is characterized by CDH6 expressing cells to which the antibodies, antibody fragments (e.g. , antigen binding fragments), and antibody drug conjugates can specifically bind.
[00228] The present disclosure provides for methods of treating cancer comprising administering a therapeutically effective amount of the CDH6 ADC and GITR antibody in combination. In certain aspects, the cancer is a solid cancer. In certain aspects, the subject is a human.
[00229] In certain aspects, the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of the CDH6 ADC and GITR antibody. In certain aspects, the subject is a human. In certain aspects, the subject has a tumor or has had a tumor removed. In another aspect, the subject has a reoccurrence of the tumor.
[00230] In certain aspects, the tumor expresses the CDH6 to which the anti-CDH6 ADC binds.
In certain aspects, the tumor overexpresses the human CDH6. In other aspects, the administration of the anti-CDH6 ADC results in an increase of GITR expression on Treg cells.
[00231] For the treatment of the disease, the appropriate dosage of the CDH6 ADC and GITR antibody combination depends on various factors, such as the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, previous therapy, patient's clinical history, and so on. The CDH6 ADC and GITR antibody combination can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody, antibody fragment (e.g., antigen binding fragment), or antibody drug conjugates. In certain aspects, dosage is from 0.0 lmg to 10 mg (e.g., 0.01 mg, 0.05mg, O.lmg, 0.5mg, lmg, 2mg, 3mg, 4mg, 5mg, 7mg, 8mg, 9mg, or lOmg) per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. In certain aspects, the CDH6 ADC and GITR antibody combination of the present disclosure is given once every two weeks or once every three weeks. In another aspect, the CDH6 ADC is given as a single agent, and the anti-GTIR antibody is administered a certain time afterward. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
Additional Combination Therapy
[00232] In certain instances, the CDH6 ADC/anti-GITR combination of the present disclosure is combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, antinausea agents (or anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.
[00233] General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy- 5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC- Dome®), dactinomycin (Actinomycin D, Cosmegan®), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6- mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine
(Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).
[00234] In one aspect, an antibody, antibody fragment (e.g. , antigen binding fragment), or antibody drug conjugate of the present disclosure is combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen can have complementary activities to the antibody or immunoconjugate of the combination such that they do not adversely affect each other. For example, an antibody, antibody fragment (e.g., antigen binding fragment), or antibody drug conjugate of the present disclosure can be administered in combination with, but not limited to, a chemotherapeutic agent, a tyrosine kinase inhibitor, for example, Imatinib.
[00235] In one aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody drug conjugate in combination with one or more tyrosine kinase inhibitors, including but not limited to, EGFR inhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors.
[00236] For example, tyrosine kinase inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-lH-indazol-4-yl)phenyl]-N'-(2-fluoro-5- methylphenyl)urea, also known as ABT 869, available from Genentech); Sunitinib malate (Sutent®); Bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-l- yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606, and described in US Patent No.
6,780,996); Dasatinib (Sprycel®); Pazopanib (Votrient®); Sorafenib (Nexavar®); Zactima (ZD6474); nilotinib (Tasigna®); Regorafenib (Stivarga®) and Imatinib or Imatinib mesylate (Gilvec® and Gleevec®).
[00237] Epidermal growth factor receptor (EGFR) inhibitors include but are not limited to,
Erlotinib hydrochloride (Tarceva®), Gefitnib (Iressa®); N-[4-[(3-Chloro-4-fluorophenyl)amino]-7- [[(3"S'') etrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®); (3R,4R)-4-Amino-l-((4-((3- methoxyphenyl)amino)pyrrolo[2, 1-f] [l,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514);
Canertinib dihydrochloride (CI-1033); 6-[4-[(4-Ethyl-l-piperazinyl)methyl]phenyl]-N-[(lR)-l- phenylethyl]- 7H-Pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569); Afatinib (BIBW2992); Neratinib (HKI-272); N-[4-[[l-[(3- Fluoropheny l)methyl] - lH-indazol-5-y ljamino] -5-methy lpyrrolo [2, 1 -f] [ 1 ,2,4]triazin-6-y 1] -carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626); N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7- [[(3aa,5 ,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23-8); and 4-[4-[[(lR)-l-Phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrirnidin-6-yl]-phenol (PKI166, CAS 187724-61-4).
[00238] EGFR antibodies include but are not limited to, Cetuximab (Erbitux®); Panitumumab
(Vectibix®); Matuzumab (EMD-72000); Nimotuzumab (hR3); Zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). [00239] Human Epidermal Growth Factor Receptor 2 (HER2 receptor) (also known as Neu,
ErbB-2, CD340, or pl85) inhibitors include but are not limited to, Trastuzumab (Herceptin®);
Pertuzumab (Omnitarg®); Neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridin-2- yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide, and described PCT Publication No. WO 05/028443); Lapatinib or Lapatinib ditosylate (Tykerb®); (3R,4R)-4-amino- 1 -((4-((3 -methoxypheny l)amino)pyrrolo [2, 1-f] [ 1 ,2,4]triazin-5-yl)methyl)piperidin- 3-ol (BMS690514); (2E)-N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-
6- quinazolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[l-[(3- Fluoropheny l)methyl] - lH-indazol-5-y ljamino] -5-methy lpyrrolo [2, 1-f] [1 ,2,4]triazin-6-y 1] -carbamic acid, (38)-3^οφηο1ί^1ηιε%1 ester (BMS 599626, CAS 714971-09-2); Canertinib dihydrochloride (PD 183805 or CI-1033); and N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aa,5 ,6aa)- octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23- 8).
[00240] HER3 inhibitors include but are not limited to, LJM716, MM-121, AMG-888,
RG7116, REGN-1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.
[00241] MET inhibitors include but are not limited to, Cabozantinib (XL184, CAS 849217-68-
1); Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); Tivantinib (ARQ197, CAS 1000873-98-2); l-(2 -Hydroxy -2 -methylpropyl)-^-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5- methyl-3-oxo-2-phenyl-2,3-dihydro-l f-pyrazole-4-carboxamide (AMG 458); Cryzotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-Dihydro-lH-indol-l-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin- l-yl)carbonyl]-lH-pyrrol-2-yl}methylene)-l,3-dihydro-2H-indol-2-one (SU11271); (3Z)-N-(3- Chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-l-yl)carbonyl]-lH-pyrrol-2-yl}methylene)-N- methyl-2-oxoindoline-5-sulfonamide (SU11274); (3Z)-N-(3-Chlorophenyl)-3-{ [3,5-dimethyl-4-(3- moφholin-4-ylpropyl)-lH-pyrrol-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide
(SU11606); 6-[Difluoro[6-(l-methyl-lH-pyrazol-4-yl)-l,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]- quinoline (JNJ38877605, CAS 943540-75-8); 2-[4-[l-(Quinolin-6-ylmethyl)-lH-[l,2,3]triazolo[4,5- b]pyrazin-6-yl]-lH-pyrazol-l-yl]ethanol (PF04217903, CAS 956905-27-4); N-((2R)-l,4-Dioxan-2- ylmethyl)-N-methyl-N'-[3-(l-methyl-lH-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[l,2-b]pyridin-
7- yl]sulfamide (MK2461, CAS 917879-39-1); 6-[[6-(l-Methyl-l /-pyrazol-4-yl)-l,2,4-triazolo[4,3- £]pyridazin-3-yl]thio]-quinoline (SGX523, CAS 1022150-57-7); and (3Z)-5-[[(2,6- Dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2i?)-2-(l-pyrrolidinylmethyl)-l- pyrrolidinyl]carbonyl]-l f-pyrrol-2-yl]methylene]-l,3-d^^ (PHA665752, CAS
477575-56-7).
[00242] IGF1R inhibitors include but are not limited to, BMS-754807, XL-228, OSI-906,
GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. See e.g., Yee, JNCI, 104; 975 (2012) for review.
[00243] In another aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody drug conjugate in combination with one or more FGF downstream signaling pathway inhibitors, including but not limited to, MEK inhibitors, Braf inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTor inhibitors.
[00244] For example, mitogen-activated protein kinase (MEK) inhibitors include but are not limited to, XL-518 (also known as GDC-0973, Cas No. 1029872-29-4, available from ACC Corp.); 2- [(2-Chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as CI- 1040 or PD184352 and described in PCT Publication No. WO2000035436); N-[(2R)-2,3- Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]- benzamide (also known as PD0325901 and described in PCT Publication No. WO2002006213); 2,3-Bis[amino[(2- aminophenyl)thio] methylene] -butanedinitrile (also known as U0126 and described in US Patent No. 2,779,780); N-[3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-l-[(2R)-2,3- dihydroxypropyl]- cyclopropanesulfonamide (also known as RDEAl 19 or BAY869766 and described in PCT Publication No. WO2007014011); (3S,4R,5Z,8S,9S, l lE)-14-(Ethylamino)-8,9, 16-trihydroxy- 3,4-dimethyl-3,4,9, 19-tetrahydro-lH-2-benzoxacyclotetradecine-l,7(8H)-dione] (also known as E6201 and described in PCT Publication No. WO2003076424); 2'-Amino-3 '-methoxyflavone (also known as PD98059 available from Biaffin GmbH & Co., KG, Germany); Vemurafenib (PLX-4032, CAS 918504-65-1); (R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8- methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (TAK-733, CAS 1035555-63-5);
Pimasertib (AS-703026, CAS 1204531-26-9); and Trametinib dimethyl sulfoxide (GSK-1120212, CAS 1204531-25-80).
[00245] Phosphoinositide 3 -kinase (PI3K) inhibitors include but are not limited to, 4-[2-(lH-
Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-l-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]moφholine (also known as GDC 0941 and described in PCT Publication Nos. WO 09/036082 and WO
09/055730); 2-Methyl-2-[4-[3-methyl-2-oxo-8-(qmnolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-l- yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ 235, and described in PCT Publication No. WO 06/122806); 4-(trifluoromethyl)-5-(2,6-dimoφholinopyrimidin-4-yl)pyridin-2 -amine (also known as BKM120 or NVP-BKM120, and described in PCT Publication No. WO2007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-Pyridinyl)-6- quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 958852-01-2);
(lE,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-l-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a- octahydro- 11 -hydroxy -4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho [ 1 ,2-c]pyran- 2,7, 10(lH)-trione (PX866, CAS 502632-66-8); and 8-Phenyl-2-(moφholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).
[00246] mTor inhibitors include but are not limited to, Temsirolimus (Torisel®);
Ridaforolimus (formally known as deferolimus, (lR,2R,4S)-4-[(2R)-2
[(1R,9S,12S, 15R, 16E, 18R,19R,21R, 23S,24£,26£,28Z,30S,32S,35i?)-l,18-dihydroxy-19,30- dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10, 14,20-pentaoxo-l l,36-dioxa-4- azatricyclo[30.3.1.04'9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); Everolimus (Afinitor® or RADOOl); Rapamycin (AY22989, Sirolimus®);
Simapimod (CAS 164301-51-3); (5-{2,4-Bis[(3S)-3-methylmoφholin-4-yl]pyrido[2,3-d]pyrimidin-7- yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[/ra«5-4-(2-hydroxyethoxy)cyclohexyl]-6- (6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-^pyrirnidin-7(8 f)-one (PF04691502, CAS 1013101- 36-4); and Λί2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4 ί-l-benzopyran-2-yl)moφholinium-4- yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartylL-serine-, inner salt (SF1126, CAS 936487-67-1).
[00247] In yet another aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof a CDH6 ADC and anti-GITR combination with one or more pro-apoptotics, including but not limited to, IAP inhibitors, Bcl2 inhibitors, MCll inhibitors, Trail agents, Chk inhibitors.
[00248] For examples, IAP inhibitors include but are not limited to, NVP-LCL161, GDC-
0917, AEG-35156, AT406, and TL32711. Other examples of IAP inhibitors include but are not limited to those disclosed in WO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118, WO 06/017295, and WO08/134679, all of which are incoφorated herein by reference.
[00249] BCL-2 inhibitors include but are not limited to, 4-[4-[[2-(4-Chlorophenyl)-5,5- dimethyl-l-cyclohexen-l-yl]methyl]-l-piperazinyl]-N-[[4-[[(lR)-3-(4-moφholinyl)-l- [(phenyltWo)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide (also known as ABT-263 and described in PCT Publication No. WO 09/155386); Tetrocarcin A; Antimycin; Gossypol ((-)BL-193); Obatoclax; Ethyl-2-amino-6-cyclopentyl-4-(l-cyano-2-ethoxy-2- oxoethyl)-4Hchromone-3-carboxylate (HA14 - 1); Oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-Gossypol acetic acid (AT-101); 4-[4-[(4'-CWoro[l,l'-biphenyl]-2-yl)methyl]-l- piperazinyl]-N-[[4-[[(lR)-3-(dimethylami∞
nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9); and Navitoclax (ABT-263, CAS 923564-51-6).
[00250] Proapoptotic receptor agonists (PARAs) including DR4 (TRAILR1) and DR5
(TRAILR2), including but are not limited to, Dulanermin (AMG-951, RhApo2L/TRAIL);
Mapatumumab (HRS-ETR1, CAS 658052-09-6); Lexatumumab (HGS-ETR2, CAS 845816-02-6); Apomab (Apomab®); Conatumumab (AMG655, CAS 896731-82-1); and Tigatuzumab (CS1008, CAS 946415-34-5, available from Daiichi Sankyo).
[00251] Checkpoint Kinase (CHK) inhibitors include but are not limited to, 7-
Hydroxystaurosporine (UCN-01); 6-Bromo-3-(l-methyl-l i-pyrazol-4-yl)-5-(3i?)-3-piperidinyl- pyrazolo[l,5-a]pyrimidin-7-amine (SCH900776, CAS 891494-63-6); 5-(3-Fluorophenyl)-3- ureidothiophene-2-carboxylic acid N-[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4- [((3S)-l-Azabicyclo[2.2.2]oct-3-yl)amino]-3-(lH-benzimidazol-2-yl)-6-cUoroquinolin-2(lH)-one (CHIR 124, CAS 405168-58-3); 7-Aminodactinomycin (7-AAD), Isogranulatimide,
debromohymenialdisine; N-[5-Bromo-4-methyl-2-[(2S)-2-moφholinylmethoxy]-phenyl]-N'-(5- methyl-2-pyrazinyl)urea (LY2603618, CAS 911222-45-2); Sulforaphane (CAS 4478-93-7, 4- Methylsulfinylbutyl isothiocyanate); 9, 10, 11, 12-Tetrahydro- 9, 12-epoxy-l i-diindolo[l,2,3- J¾:3',2',l'- :/]pyrrolo[3,4-/] [l,6]benzodiazocine-l,3(2 f)-dione (SB-218078, CAS 135897-06-2); and TAT-S216A (Sha et al., Mol. Cancer. Ther 2007; 6(1): 147-153), and CBP501.
[00252] In one aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody drug conjugate in combination with one or more
FGFR inhibitors. For example, FGFR inhibitors include but are not limited to, Brivanib alaninate
(BMS-582664, (5)-((i?)-l-(4-(4-Fluoro-2-methyl-l i-indol-5-yloxy)-5-methylpyrrolo[2,l- ] [l,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); Vargatef (BIBF1120, CAS 928326-83-4);
Dovitinib dilactic acid (TKI258, CAS 852433-84-2); 3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-l-{6-[4-
(4-ethyl-piperazin-l-yl)-phenylamino]-pyrimidin-4-yl}-l-methyl-urea (BGJ398, CAS 872511-34-7);
Danusertib (PHA-739358); and (PD173074, CAS 219580-11-7). In a specific aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof a CDH6
ADC and anti-GITR antibody combination with an FGFR2 inhibitor, such as 3-(2,6-dichloro-3,5- dimethoxyphenyl)- 1 -(6((4-(4-ethy lpiperazin- 1 -yl)pheny l)amino)pyrimidin-4-yl)- 1-methy lurea (also known as BGJ-398); or 4-amino-5-fluoro-3-(5-(4-methylpiperazinl-yl)-l /-benzo[if|imidazole-2- yl)quinolin-2(l /)-one (also known as dovitinib or TKI-258). AZD4547 (Gavine et al, 2012, Cancer Research 72, 2045-56, N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3R,5S)- diemthylpiperazin-l-yl)benzamide), Ponatinib (AP24534; Gozgit e/ a/. , 2012, Mol Cancer Ther., 11; 690-99; 3-[2-(imidazo[l,2-b]pyridazin-3-yl)emynyl]-4-methyl-N-{4-[(4-methylpiperazin-l- yl)methyl]-3-(trifluoromethyl)phenyl}benzamide, CAS 943319-70-8)
[00253] In one embodiment, a CDH6 ADC and anti-GITR antibody combination is used with a PD-1 inhibitor, e.g., as described in WO2015/026684 or WO2016/057846. WO2016/057846 in particular discloses that treatment with an anti-GITR antagonist antibody causes an increase in PD-1 expression on CD8+ T cells in a mouse colon tumor model. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab.
[00254] In some embodiments, the anti-PD-1 antibody is Nivolumab. Alternative names for
Nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD- 1 antibody is Nivolumab (CAS Registry Number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody which specifically blocks PD 1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US 8,008,449 and WO2006/121168. In one embodiment, the inhibitor of PD-1 is Nivolumab, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).
[00255] In some embodiments, the anti-PD-1 antibody is Pembrolizumab. Pembrolizumab
(also referred to as Lambrolizumab, MK-3475, MK03475, SCH-900475 or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, US 8,354,509 and WO2009/114335.
[00256] In one embodiment, the inhibitor of PD-1 is Pembrolizumab disclosed in, e.g., US
8,354,509 and WO 2009/114335, and having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).
[00257] In some embodiments, the anti-PD-1 antibody is Pidilizumab. Pidilizumab (CT-011;
Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009/101611. Other anti-PDl antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD l antibodies disclosed in US 8,609,089, US 2010028330, and/or US 20120114649.
[00258] In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 and
WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and
B7-H1.
[00259] In certain embodiments the CDH6 ADC is in combination with an anti-GITR antibody, wherein the anti-GITR antibody is BMS-986156, INCAGN01876, AMG 228, TRX518, MEDI1873, MK-4166, MK-1248 or FPA-154. In some embodiments, the combination comprises an anti-GITR antibody or agonist described in any of the following publications: WO2015187835, WO2015031667, WO2015026684, WO2015184099, WO2016196792, WO2016126781,
WO17015623, WO2017025610, WO2013039954, WO2011028683, WO2009009116,
WO2006105021, WO2005007190, US20070098719, US20050014224, or US20140072566.
[00260] In other embodiments, the anti-GITR antibody is in combination with an ADC. For example, the ADC NOV169N31Q-MCC-DM1 is a P-cadherin antibody conjugated to DM1, and is disclosed in US 2016/0137730. DM1 as well as DM4 can induce immunogenic cell death, and the combination of NOV169N31Q-MCC-DM1 and anti-GITR antibody can be used for the treatment of P-cadherin positive cancers.
Pharmaceutical Compositions
[00261] To prepare pharmaceutical or sterile compositions the CDH6 ADC and GITR antibody of the present disclosure are mixed with a pharmaceutically acceptable carrier or excipient. The CDH6 ADC and GITR antibody can be prepared separately or as an admixture. The compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing cancer, for example, ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma.
[00262] Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g. , lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g. , Hardman et al. , Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y., 2000).
[00263] Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain aspects, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y., 1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y., 1993; Baert et al , New Engl. J. Med. 348:601-608, 2003; Milgrom et al, New Engl. J. Med. 341 : 1966-1973, 1999; Slamon et al, New Engl. J. Med. 344:783-792, 2001; Beniaminovitz et al, New Engl. J. Med.
342:613-619, 2000; Ghosh et al , New Engl. J. Med. 348:24-32, 2003; Lipsky et al , New Engl. J. Med. 343 : 1594-1602, 2000).
[00264] Actual dosage levels of the CDH6 ADC and GITR antibody in pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts. [00265] Compositions comprising the CDH6 ADC and GITR antibody combination can be provided by continuous infusion, or by doses at intervals of, e.g. , one day, one week, or 1-7 times per week. Doses can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.
[00266] The dosage of each respective CDH6 ADC and GITR antibody administered to a patient can be 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dosage can be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight. The dosage of each CDH6 ADC and GITR antibody of the combination can thereof be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
[00267] Doses of the CDH6 ADC and GITR antibody combination can be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In a specific aspect, doses of the CDH6 ADC and GITR antibody combination of the present disclosure are repeated every 3 weeks.
[00268] An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route and dose of administration and the severity of side effects (see, e.g., Maynard et al, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).
[00269] The route of administration may be by, e.g. , topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g. , Sidman et al, Biopolymers 22:547-556, 1983; Langer e/ a/., J. Biomed. Mater. Res. 15: 167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al. , Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al , Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent or a local anesthetic such as lidocaine to ease pain at the site of the injection, or both. In addition, pulmonary administration can also be employed, e.g. , by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g. , U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO
98/31346, and WO 99/66903, each of which is incorporated herein by reference their entirety.
[00270] A composition of the present disclosure can also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Selected routes of administration for the immunoconjugates include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, a composition of the present disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one aspect, the CDH6 ADC and GITR antibody combination of the present disclosure is administered by infusion. In another aspect, the CDH6 ADC and GITR antibody combination is administered subcutaneously.
[00271] If the CDH6 ADC and anti-GITR combination of the present disclosure are administered in a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald e/ a/., Surgery 88:507, 1980; Saudek e/ a/., N. Engl. J. Med. 321 :574, 1989). Polymeric materials can be used to achieve controlled or sustained release of the therapies of the
immunoconjugates (see e.g. , Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23 :61, 1983; see also Levy et al , Science 228: 190, 1985; During et al, Ann. Neurol. 25:351, 1989; Howard et al , J. Neurosurg. 7 1 : 105, 1989; U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5, 128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly (methacry lie acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),
polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one aspect, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).
[00272] Controlled release systems are discussed in the review by Langer, Science 249: 1527-
1533, 1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising the CDH6 ADC and anti-GITR combination of the present disclosure. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al , Radiotherapy & Oncology 39: 179-189, 1996; Song et al , PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek e/ a/., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853- 854, 1997; and Lam et al , Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, 1997, each of which is incorpo rated herein by reference in their entirety.
[00273] If the CDH6 ADC and anti-GITR combination of the disclosure is administered topically, they can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.
[00274] If the compositions comprising the immunoconjugates are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[00275] The CDH6 ADC and GITR antibody combination as disclosed herein can be administered together or separately. The CDH6 ADC and GITR antibody can be administered separately less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. The CDH6 ADC and GITR antibody combination can be administered within one same patient visit.
[00276] In certain aspects, the CDH6 ADC and GITR antibody combination can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g. , U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannosides (Umezawa e/ a/., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (Bloeman e/ al, (1995) FEBS Lett. 357: 140; Owais et al , (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al, (1995) Am. J. Physiol. 1233 : 134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
[00277] The present disclosure provides protocols for the administration of a CDH6 ADC and
GITR antibody combination to a subject in need thereof. The CDH6 ADC and GITR antibody combination can be administered concomitantly or sequentially to a subject. The CDH6 ADC and GITR antibody combination can also be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g. , CDH6 ADC) for a period of time, followed by the
administration of a second therapy (e.g. , anti-GITR antibody) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies to avoid or reduce the side effects of one of the therapies, and/or to improve the efficacy of the therapies.
[00278] The CDH6 ADC and GITR antibody combination of the disclosure can be administered to a subject concurrently. The term "concurrently" is not limited to the administration of the combination at exactly the same time, but rather it is meant that a pharmaceutical composition comprising the combination is administered to a subject in a sequence and within a time interval such that the combination can act together to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route.
[00279] The CDH6 ADC and GITR antibody combination can be administered to a subject in the same pharmaceutical composition. Alternatively, the CDH6 ADC and GITR antibody combination can be administered concurrently to a subject in separate pharmaceutical compositions.
EXAMPLES
Example 1: Identification of CDH6 antibodies by phase-display technology
HuCAL PLATINUM® Pannings [00280] Antibodies were identified by the selection of clones that bound to CDH6-ECD. As a source of antibody variants a commercially available phage display library, the Morphosys HuCAL PLATINUM® library, was used. The phagemid library is based on the HuCAL® concept (Knappik et al., . J Mol Biol. 2000: 296(l):57-86; Prassler et al., J Mol Biol. 2011 : 413(l):261-78 2011) and employs the CysDisplayTM technology for displaying the Fab on the phage surface (Rothe et al., J Mol Biol. 2008: 376(4): 1182-200). For isolation of anti-CDH6 antibodies, standard panning strategies were performed using solid phase and solution panning approaches.
[00281] Pannings were done on various forms of CDH6 antigens. An overview of recombinant
CDH6 proteins and CDH6-expressing cell lines used as antigens for pannings and subsequent screening and characterization is given in Table 1.
[00282] Human, mouse and rat CDH6 extracellular domains were gene synthesized based on amino acid sequences from the GenBank or Uniprot databases (see Table 1). Cynomolgus CDH6 cDNA template was gene synthesized based on amino acid sequences and homology information generated using mRNA from various cyno tissues (e.g. Zyagen Laboratories, San Diego, CA). All synthesized DNA fragments were cloned into appropriate expression vectors listed affinity tags to allow for purification or expression vectors Zyagen Laboratories for mammalian cell surface expression.
[00283] For the generation of a stable CHO cell line exogenously expressing human CDH6 the
TREX expression system (Invitrogen, Carlsbad, CA) was used according to the manufacturer's instructions. Briefly, CHO-TREx cells (Invitrogen, R718-07, Carlsbad, CA) were grown in DMEM media (Invitrogen 11995-085) withl0% FBS (Invitrogen, 10082-147, Carlsbad, CA), and 10 μg/ml Blasticidin (Invitrogen Al 1139-02, Carlsbad, CA). Transfection was performed in 6 well plates when cell reach 90% confluence. 4 μg of linearized CDH6 plasmid DNA was mixed with 100 μΐ DMEM media (no FBS) in a sterile Eppendorf tube, 12 μΐ of Lipofectamine® 2000 (Invitrogen 11668-019, Carlsbad, CA) were mixed with 100 μΐ DMEM media in another sterile Eppendorf tube. DNA and Lipofectamine® were mixed together, and incubated in room temperature for 15 min. The culture media of CHO-TREx cells was changed to 1 ml DMEM media without FBS for each well; half above DNA Lipofectamine® mix were added into each well, incubated for 2 hours. Then 2 ml growth media (with FBS) were added into each well, and the cells were incubated overnight. The transfected cells were split from 6 well plate to T175 flask next day and grew in selection DMEM media with 10% FBS, 10 μg/ml Blasticidin, 800-1000 μg/ml Geneticin (Invitrogen 10131-027). CDH6 expression was induced with final 1 μg/ml Tetracycline in selection media for 20-24 hours. Positive cells were labeled with final 5 μg/ml anti-CDH6 primary monoclonal antibody (MAB2715 from R&D Systems, Minneapolis, MN), and then labeled with PE conjugated anti-mouse secondary antibody (cat #12- 4010-87, eBioscience, San Diego, CA) and sorted by FACS. [00284] Stable CHO cell lines featuring exogenous expression of CDH6 from mouse, rat and cynomolgus origin were generated by transfection of CHO-K1 cells (Invitrogen, Carlsbad, CA) with the respective cDNAs cloned into a mammalian expression vector (pcDNA6.1, Invitrogen, Carlsbad, CA). Transfection was performed in 6 well plates when cell reach 90% confluence. 4 μg of linearized CDH6 plasmid DNA were mixed with 100 μΐ DMEM media without FBS in a sterile Eppendorf tube, 12 μΐ of Lipofectamine® 2000 (Invitrogen 11668-019, Carlsbad, CA) were mixed with 100 μΐ DMEM media in another sterile Eppendorf tube. The DNA and Lipofectamine® solution were mixed together, and incubated at room temperature for 15 min. The culture media of CHO cells was changed to 1 ml DMEM media without FBS for each well; half above DNA/Lipofectamine® mix were added into each well, incubated for 2 hours. Then 2 ml growth media (with FBS) were added into each well, and the cells were incubated overnight. The transfected cells were split from 6 well plate to T175 flask next day and grew in selection DMEM media with 10% FBS, 10 μg/ml Blasticidin, 800-1000 μg/ml Geneticin (Invitrogen 10131-027 Carlsbad, CA). CDH6 expression was induced with final 1 μg/ml Tetracycline in selection media for 20-24 hours. Positive cells were labeled with final 5 μg/ml anti- CDH6 primary monoclonal antibody (MAB2715 from R&D Systems, Minneapolis, MN), and then labeled with PE conjugated anti-mouse secondary antibody (cat #12-4010-87, eBioscience, San Diego, CA) and sorted by FACS. An alternative commercially available anti-CDH6 antibody is the 2B6 antibody (#GWB-E8FDF3 - Genway, San Diego, CA). This antibody was also used to confirm CDH6 expression in cells.
Table 1
Overview of antigen, antibody and cell line reagents for pannings and screenings
Antigen/ Antibody Accession Description Sequence
Human CDH6 AK291290 Full length MRTYRYFL-LLFWVGQPYPTLSTPLSKRTS
GFPAKKRALELSGNSKNELNRSKRSWM
human cDNA
W QFFLLEEYTGSDYQYVGKLHSDQDR
(SEQ ID NO:2) GD G SLKYTL S GD G A GDLFII ENTGD IQ A
TKRLDREEKPV n..RAQAINRRTGRPVEP
ESEFIIKIHDINDNEPIFTKEV TATWEMS
DVGTFWQVTATDADDPTYGNSAKWY
SILQGQPYFSVESETGIIKTALLNMDRENR
EQYOVViQA DMGGQMGGLSGTTTVNI
TLTDVNDNPPRFPQSTYQFKTPESSPPGTP
IGRIKASD AD VGENAEIEY SITDGEGLDM
FDVITDQETQEGilTVKKLLDFEK KVYT
L VEASNPYVEPRFLYLGPF DSATVRIV
VEDVDEPPVFSKLAYILQ1REDAQINTTIG
S VTAQDPD AARNPVK Y S VDRHTDMDRIF
NIDSGNGSIFTSKLLDRETLLWHMTVIAT
E1NNPK Q S SR VPL Y IK VLD VNDN APEF AE
FYETFVCEKAKADQLIQTLHAVDKDDPY
SGHQFSFSLAPEAASGSNFTIQDNKDNTA
GILTR NGYNRHEMSTYLLPVVTSDNDY
PVQSSTGTVTVRVCACDHHGNMQSCHA E ALIHPTGL STG AL VAILLCI VILL VT WL
FAALRRQRKKEPLIISKEDIRDNIVSYNDE GGGEEDTQAFDIGTLRNPEAIEDNKLRRD I VPE ALFLPRRTPT ARDNTD VRDF1N QRL KENDTDPTAPPYDSLATYAYEGTGSVAD SLSSLESVTTD ADQDYD YLSD WGPRFKK L ADMYGG VD SDKD S
His6-SUMO- NM_004932.3 human EC 1/2 SWMWNQFFLLEEYTGSDYQYVGKLHSD
QDRGDGSLKYILSGDGAGDLFIINENTGD CDH6 aa54-260- domain variant
IQATKRLDREEKPVYILRAQAINRRTGRP APP-Avi (SEQ ID NO:3) VEPESEFIIKIHDINDNEPIFTKEVYTATVP
EMSDVGTFWQVTATDADDPTYGNSAK
WYSILQGQPYFSVESETGIIKTALLNMD
RENREQYQWIQAKDMGGQMGGLSGTT
TVNITLTDGGGGSEFRHDSGLNDIFEAQK
IEWHE
hCDH6aa54-615 NM_004932.3 human full- SWMWNQFFLLEEYTGSDYQYVGKLHSD
QDRGDGSLKYILSGDGAGDLFIINENTGD APPavi length ECD
IQATKRLDREEKPVYILRAQAINRRTGRP
(SEQ ID NO:4) VEPESEFIIKIHDINDNEPIFTKEVYTATVP
EMSDVGTFWQVTATDADDPTYGNSAK
WYSILQGQPYFSVESETGIIKTALLNMD
RENREQYQWIQAKDMGGQMGGLSGTT
TVNITLTDVNDNPPRFPQSTYQFKTPESSP
PGTPIGRIKASDADVGENAEIEYSITDGEG
LDMFDVITDQETQEGIITVKKLLDFEKKK
WTLKVE ASNPYVEPRFLYLGPFKD SAT
VRIWEDVDEPPVFSKLAYILQIREDAQIN
TTIGSVTAQDPDAARNPVKYSVDRHTDM
DRIFNIDSGNGSIFTSKLLDRETLLWHNIT
VIATEINNPKQSSRVPLYIKVLDVNDNAP
EFAEFYETFVCEKAKADQLIQTLHAVDK
DDPYSGHQFSFSLAPEAASGSNFTIQDNK
DNTAGILTRKNGYNRHEMSTYLLPVVIS
DNDYPVQSSTGTVTVRVCACDHHGNMQ
SCHAE ALIHPTGL STGA
hCDH6aa54-615 NM_004932.3 human full- SWMWNQFFLLEEYTGSDYQYVGKLHSD
QDRGDGSLKYILSGDGAGDLFIINENTGD APPavi-biotin length ECD,
IQATKRLDREEKPVYILRAQAINRRTGRP
biotinylated VEPESEFIIKIHDINDNEPIFTKEVYTATVP
EMSDVGTFWQVTATDADDPTYGNSAK
(SEQ ID NO:5)
WYSILQGQPYFSVESETGIIKTALLNMD
RENREQYQWIQAKDMGGQMGGLSGTT
TVNITLTDVNDNPPRFPQSTYQFKTPESSP
PGTPIGRIKASDADVGENAEIEYSITDGEG
LDMFDVITDQETQEGIITVKKLLDFEKKK
WTLKVE ASNPYVEPRFLYLGPFKD SAT
VRIWEDVDEPPVFSKLAYILQIREDAQIN
TTIGSVTAQDPDAARNPVKYSVDRHTDM
DRIFNIDSGNGSIFTSKLLDRETLLWHNIT
VIATEINNPKQSSRVPLYIKVLDVNDNAP
EFAEFYETFVCEKAKADQLIQTLHAVDK DDPYSGHQFSFSLAPEAASGSNFTIQDNK
DNTAGILTRKNGYNRHEMSTYLLPVVIS DNDYPVQSSTGTVTVRVCACDHHGNMQ SCH AEALIHPTGLSTGAGSEFRHD SGLND IFEAQK(BIOTIN)IEWHE
hCDH6aa54-615 NM_004932.3 human full- SWMWNQFFLLEEYTGSDYQYVGKLHSD
QDRGDGSLKYILSGDGAGDLFIINENTGD
Fc length ECD,
IQATKRLDREEKPVYILRAQAINRRTGRP
dimerized VEPESEFIIKIHDINDNEPIFTKEVYTATVP
EMSDVGTFWQVTATDADDPTYGNSAK
(SEQ ID NO:6)
WYSILQGQPYFSVESETGIIKTALLNMD
RENREQYQWIQAKDMGGQMGGLSGTT
TVNITLTDVNDNPPRFPQSTYQFKTPESSP
PGTPIGRIKASDADVGENAEIEYSITDGEG
LDMFDVITDQETQEGIITVKKLLDFEKKK
VYTLKVE ASNPYVEPRFLYLGPFKD SAT
VRIWEDVDEPPVFSKLAYILQIREDAQIN
TTIGSVTAQDPDAARNPVKYSVDRHTDM
DRIFNIDSGNGSIFTSKLLDRETLLWHNIT
VIATEINNPKQSSRVPLYIKVLDVNDNAP
EFAEFYETFVCEKAKADQLIQTLHAVDK
DDPYSGHQFSFSLAPEAASGSNFTIQDNK
DNTAGILTRKNGYNRHEMSTYLLPVVIS
DNDYPVQSSTGTVTVRVCACDHHGNMQ
SCHAEALIHPTGLSTGAGSDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVT
CVWDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVM
HE ALHNH YTQKSL SL SP GK
cynoCDH6 FL See above Cynomolgus SWMWNQFFLLEEYTGSDYQYVGKLHSD
QDRGDGSLKYILSGDGAGDLFIINENTGD
description on (Macaca
IQATKRLDREEKPVYILRAQAINRRTGRP
cynomolgus fascicularis) VEPESEFIIKIHDINDNEPIFTKEVYTATVP
EMSDVGTFWQVTATDADDPTYGNSAK
sequence full-length
WYSILQGQPYFSVESETGIIKTALLNMD
derivation CDH6 RENREQYQWIQAKDMGGQMGGLSGTT
TVNITLTDVNDNPPRFPQSTYQFKTPESSP
(SEQ ID NO:7)
PGTPIGRIKASDADVGENAEIEYSITDGEG
LDMFDVITDQETQEGIITVKKLLDFEKKK
WTLKVE ASNPH VEPRFLYLGPFKD SAT
VRIWEDVDEPPVFSKLAYILQIREDAQIN
TTIGSVTAQDPDAARNPVKYSVDRHTDM
DRIFNIDSGNGSIFTSKLLDRETLLWHNIT
VIATEINNPKQSSRVPLYIKVLDVNDNAP
EFAEFYETFVCEKAKADQLIQTLRAVDK
DDPYSGHQFSFSLAPEAASGSNFTIQDNK
DNTAGILTRKNGYNRHEMSTYLLPVVIS
DNDYPVQSSTGTVTVRVCACDHHGNMQ
SCH AEALIHPTGLSTGAGSEFRHD SGLND
IFEAQKIEWHE moCDH6 FL NM_007666.3 Mouse (Mus SWMWNQFFLLEEYTGSDYQYVGKLHSD
QDRGDGSLKYILSGDGAGDLFIINENTGD
musculus) full-
IQATKRLDREEKPVYILRAQAVNRRTGRP
length CDH6 VEPESEFIIKIHDINDNEPIFTKDVYTATVP
EMADVGTFWQVTATDADDPTYGNSAK
(SEQ ID NO:8)
WYSILQGQPYFSVESETGIIKTALLNMD
RENREQYQWIQAKDMGGQMGGLSGTT
TVNITLTDVNDNPPRFPQSTYQFKTPESSP
PGTPIGRIKASDADVGENAEIEYSITDGEG
HEMFDVITDQETQEGIITVKKLLDFEKKK
VYTLKVE ASNPH VEPRFLYLGPFKD SAT
VRIWDDVDEPPVFSKLAYILQIREDARIN
TTIGSVAAQDPDAARNPVKYSVDRHTD
MDRIFNIDSGNGSIFTSKLLDRETLLWHNI
T VI ATEINNPKQ S SRVPL YIKVLD VNDNA
PEFAEFYETFVCEKAKADQLIQTLRAVD
KDDPYSGHQFSFSLAPEAASSSNFTIQDN
KDNTAGILTRKNGYNRHEMSTYLLPVVI
SDNDYPVQSSTGTVTVRVCACDHHGNM
QSCHAEALIHPTGLSTGAGSEFRHDSGLN
DIFEAQKIEWHE
ratCDH6 FL NM_012927.1 Rat (Rattus SWMWNQFFLLEEYTGSDYQYVGKLHSD
QDRGDGSLKYILSGDGAGDLFIINENTGD
norvegicus) full-
IQATKRLDREEKPVYILRAQAINRRTGRP
length CDH6 VEPESEFIIKIHDINDNEPIFTKDVYTATVP
EMADVGTFWQVTATDADDPTYGNSAK
(SEQ ID NO:9)
WYSILQGQPYFSVESETGIIKTALLNMD
RENREQYQWIQAKDMGGQMGGLSGTT
TVNITLTDVNDNPPRFPQSTYQFKTPESSP
PGTPIGRIKASDADVGENAEIEYSITDGEG
HDMFDVITDQETQEGIITVKKLLDFEKKR
VYTLKVEASNPHIEPRFLYLGPFKDSATV
RIWDDVDEPPVFSKLAYILQIREDAQINT
TIGSVAAQDPDAARNPVKYSVDRHTDM
DRIFNIDSGNGSIFTSKLLDRETLLWHNIT
VIATEINNPKQSSRVPLYIKVLDVNDNAP
EFAEFYETFVCEKAKADQLIQTLHAVDK
DDPYSGHQFSFSLAPEAASGSNFTIQDNK
DNTAGILTRKNGYNRHEMSTYLLPVVIS
DNDYPVQSSTGTVTVRVCACDHHGNMQ
SCH AEALIHPTGLSTGAGSEFRHD SGLND
IFEAQKIEWHE
Recombinant protein expression and purification:
His6-SUMO-CDH6 aa54-260-APP-Avi
[00285] Protein was expressed in Rosetta® 2 De3 pLysS cells (Millipore, Billerica, MA) in pET vector using 2xYT + 50 μg/ml Kanamycin and 30 μg/ml chloramphenicol. Cells were induced with 0.1 mM IPTG at 30°C. The pellet resuspended in 400 ml 20mM Tris-Cl, pH 8.0, 150 mM NaCl, 3mM CaC12, 20 mM Imidazole, + PI tabs (1 per 50 ml) + lmg/ml lysozyme added fresh, homogenized to break up clumps and passed through microfluidizer 3 times. Following centrifugation at 20K x g for 30 min the soluble fraction of supernatant was used for purification over a 2 ml Ni-NTA (Qiagen superflow resin, Qiagen, Venlo, Netherlands). The lysate was passed over the column at 2.0 ml/min, washed with 10 CV (column volume) 20mM Tris-Cl, pH 8.0, 150 mM NaCl, 3mM CaC12, 20 mM Imidazole and protein was eluted with 4 x 3.0 ml elution fractions using 20mM Tris-Cl, pH 8.0, 500 mM NaCl, 3mM CaC12, 250 mM Imidazole. Fractions were pooled. 200 units of SUMO Protease (Invitrogen) were added with ~15ml protein (~20mgs) and dialyzed into 5L of 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 3 mM CaC12 overnight at 4°C using Snakeskin® pleated tubing with 10K cutoff (Thermo Scientific, Rockford, IL). Dialyzed material was collected and re-purified over 2.0 ml Ni- NTA resin to remove uncleaved protein and SUMO Protease. Cleaved protein was collected from flow through and wash fractions. Concentrated flow through and wash from reverse Ni-NTA to 5 ml volume, added NaCl to final concentration of 500mM and injected onto S200 column, buffer = 20mM Tris-Cl, pH 8.0, 150 mM NaCl, 3mM CaC12.
hCDH6aa54-615 APPavi, hCDH6aa54-615 Fc, cynoCDH6 FL, moCDH6 FL, ratCDH6 FL
[00286] The protein was expressed in HEK293 (ATCC CRL-1573) derived cell lines previously adapted to suspension culture and grown in a Novartis proprietary serum-free medium. Small scale expression verification was undertaken in transient 6-well-plate transfection assays on the basis of lipofection. Large-scale protein production via transient transfection and was performed at the 10-20 L scale in the Wave™ bioreactor system (GE Healthcare, Pittsburgh, PA). DNA Polyethylenimine (Polysciences, Warrington, PA) was used as a plasmid carrier at a ratio of 1 :3 (w:w). The cell culture supernatants were harvested 7-10 days post transfection and concentrated by cross- flow filtration and diafiltration prior to purification.
[00287] For purification, clarified and filtered conditioned supernatant from the transient transfection was passed over a 20 ml affinity column at 5ml/min. Wash 20 CV PBS, 1% TX100, 0.3% t-n-butylphospate buffer, 20CV PBS. Elute using 100 mM sodium citrate, 150 mM NaCl pH3.0, protein collecting 2 ml fractions. Pooled fractions were neutralized and then dialyzed overnight at 4°C with 5L 50 mM Tris, 150 mM NaCl pH 7.4, 3 mM CaC12. Sample was then subjected to size exclusion chromatography on a Superdex® S200 16/60 column (GE Healthcare, Pittsburgh, PA) equilibrated with dialysis buffer.
[00288] An overview of the various panning strategies is indicated in Table 2
Table 2
Overview of panning strategies
Pancode Phage subpool Target and conditions
1038.1 VHlA/B/5 λ+κ
SP on hCDH6aa54-615 APPavi, without CaCl2
1038.2 VH3 λ+κ Pancode Phage subpool Target and conditions
1038.3 VH2/6 λ+κ
1038.4 VH6, K
1038.5 VH1A/B/5 λ+κ SP on hCDH6aa54-615 APPavi, with CaCl2
1038.6 VH3 λ+κ
1038.7 VH2/6 λ+κ
1038.8 VH6, κ
1038.9 VH1A/B/5 λ+κ SP on His6-SUMO-CDH6 aa54-260-APP-Avi, with lmM CaCl2
1038.10 VH3 λ+κ
1038.11 VH2/6 λ+κ
1038.12 VH6, κ
1038.13 VH1A/B/5 λ+κ SP on His6-SUMO-CDH6 aa54-260-APP-Avi, with lmM CaCl2
1038.14 VH3 λ+κ Binding competition using tool antibody MAB2715
1038.15 VH2/6 λ+κ
1038.16 VH6, κ
1038.17 VH1A/B/5 λ+κ LP on biotinylated hCDH6aa54-615 APPavi
1038.18 VH3 λ+κ
1038.19 VH2/6 λ+κ
1038.20 VH1A/B/5 λ+κ LP on hCDH6aa54-615 APPavi
1038.21 VH3 λ+κ Pull-down panning using biotinylated tool antibody 2B6
1038.22 VH2/6 λ+κ
1038.23 VH1A/B/5 λ+κ LP on Biotinylated hCDH6aa54-615-Fc
1038.24 VH3 λ+κ
1038.25 VH2/6 λ+κ
1038.26 VH1A/B/5 λ+κ SP on hCDH6aa54-615-Fc
1038.27 VH3 λ+κ
1038.28 VH2/6 λ+κ
Biotinylation of proteins
[00289] Avi-tagged proteins were biotinylated using BirA enzyme following manufacture
(Avidity, Aurora, CO) standard protocol for protein biotinylation on an Avi-tagged protein. After biotinylation the protein was subjected to size exclusion chromatography on a Superdex® S200 16/60 column into 50 mM Tris, 150 mM NaCl pH 7.4, 3 niM CaC12.
[00290] Non- Avi-tagged proteins were dialyzed using Slide-A-Lyzer Mini Dialysis units,
10000 MWCO (#69576 Thermo Scientific, Rockford, IL) twice for 2h against 5L of lx PBS under stirring at room temperature prior to biotinylation. EZ-link Sulfo-NHS-LC-biotin (#21327 Thermo Scientific, Rockford, IL) was reconstituted according to the manufacturer's protocol. The proteins were incubated with a 20-fold molar excess of biotin reagent for 30 min at room temperature. To remove non-reacted biotin reagent the sample was again dialyzed twice for 2h against 5L of lx PBS under stirring at room temperature. Protein concentrations were determined by means of the
Nanodrop® device (Thermo Scientific, Rockford, IL). Samples were stored at 4°C until usage.
Solid phase panning
[00291] In solid phase pannings the Fab fragment displaying phages are incubated with antigen that has been bound to a surface support by direct immobilization.
[00292] Prior to the antigen selection process, a coating check ELISA was performed to determine the optimal antigen coating concentration. For this purpose a 2 fold dilution series of hCDH6aa54-615 APPavi (see Table 1) covering 24 to 0.19 μg/mL was generated. The individual dilutions were used to coat wells on a 96-well Maxisorp™ plate (#442404 Nunc, Rochester, NY) via direct immobilization. After coating the plate was washed thrice with 300 μΐ, PBS and subsequently blocked with lx blocking buffer (2.5% milk, 2.5% BSA, 0.05% Tween 20) for 2h at room temperature.
[00293] To detect bound antigen, an anti-APP antibody (generated in-house) was added to 1 μΜ, 0.18 μΜ or 0.04 μΜ. Secondary detection was done using AP -labeled anti-mouse IgG-F(ab)2 specific antibody in a 1 :5000 dilution Ref. 115-056-006 Jackson-Immunoresearch, West Grove, PA). The antigen concentration at which signal saturation was observed was chosen as coating concentrations for SP pannings.
Pancodes 1038.1-8
[00294] For SP pannings 1038.1-8 (see Table 2) hCDH6aa54-615 APPavi (see Table 1, SEQ
ID NO:4) was coated on a 96-well Maxisorp™ plate (Nunc) via direct immobilization. Two wells were coated with 300 μΐ, antigen solution (10 μg/mL in PBS) per phage subpool combination. Coating was done at 4°C overnight with (pannings 1038.1-4) or without (pannings 1038.4-8) the presence of 1 niM CaC12. The wells were subsequently washed twice with 400 μΐ, PBS and blocked with 400 μΐ, blocking buffer for 2h at room temperature at 400 rpm. After blocking the cells were washed twice with 400 uL PBS.
[00295] Prior to binding phage subpools were blocked and depleted with unrelated FLT2-
APPavi at 10 μg/mL for lh at room temperature to eliminate unspecific and tag binders. 300 μΐ, of blocked and depleted phage pools were transferred per coated well and incubated for 2h at room temperature and 400 rpm. Non-specific bound phages were removed by the washing steps listed below in Table 3. Table 3
First Round Second Round Third round (only pancodes 1038.9-16)
3x PBST (quick) lx PBST (quick) lOx PBST (quick)
2x PBST / 5min 4x PBST / 5min 5x PBST / 5min
3x PBS (quick) lx PBS (quick) lOx PBS (quick)
2x PBS / 5min 4x PBS / 5min 5x PBS / 5min
[00296] The specifically bound phages were eluted with 300 μΐ^ elution buffer for 10 min at room temperature. Eluted phages were used to reinfect 14 mL of E. coli TG1F+ (Stratagene/ Agilent, Santa Clara, CA),at an optical density of OD600= 0.6-0.8. The mix of E. coli TG1F+ and phage eluate was incubated for 45 min in a water bath at 37°C for phage infection. The bacterial pellets were re- suspended in 2xYT medium, plated on LB agar plates supplemented with 34 μg/mL chloramphenicol and incubated overnight at 37°C. Colonies were scraped off the plates and were used for polyclonal amplification of enriched clones and phage production. With purified phage the next panning round was started. The second round of pannings was performed according to the protocol of the first round except for a more stringent washing condition. A third panning round was omitted due to the low second round output titers of -104 cfu/mL.
Pancodes 1038.9-16
[00297] For SP pannings 1038.9-16 (Table 2) His6-SUMO-CDH6 aa54-260-APP-Avi (see
Table 1) was coated on a 96-well Maxisorp™ plate (Nunc) via direct immobilization as described for pancodes 1038.1-8. For parmings 1038.13-16 300 μΐ. of a 100 nM solution of tool antibody MAB2715 was added per well and incubated for 1 h at room temperature to avoid selection of binders recognizing the same epitope as MAB2715. Besides the antigen coated and a third panning round the pannings were done as described for pancodes 1038.1-8.
Pancodes 1038.26-28
[00298] For SP pannings 1038.26-28 (see Table 2) hCDH6aa54-615-Fc (see Table 1, SEQ ID
NO:6 ) was coated on a 96-well Maxisorp™ plate (Nunc) via direct immobilization as described for pancodes 1038.1-8. Besides the antigen coated and the washing stringencies the pannings were done as described for pancodes 1038.1-8.
Liquid phase panning
[00299] During solution panning, the Fab fragment displaying phage and biotinylated antigens are incubated in solution which is expected to increase accessibility of the antigen by the phage. Antigen biotinylation was done as described above.
Pancodes 1038.17-22 [00300] Each phage library subpool was blocked with an equal volume of 2x Chemiblocker for 2h at room temperature on a turning wheel. To avoid selection of antibodies against the APPavi and biotin tag unrelated FLT3-APPAvi and biotinylated anti-Cyclosporin msIgG was added to 7.5 and 18 μg/mL in the blocking step, respectively. For removal of phage particles binding to Streptavidin- beads, pre-adsorption of blocked phage particles was performed using blocked Streptavidin beads (# 112.06D Invitrogen, Carlsbad, CA). Blocked and pre-adsorbed phages were incubated with biotinylated hCDH6aa54-615 APPavi (see Table 1, SEQ ID NO:4) for lh at room temperature on a turning wheel. Phage-antigen complexes were captured either with washed beads (Pancodes 1038.17- 19) or with beads precoated with biotinylated anti-CDH6 antibody 2B6 (Genway, San Diego, CA) to enrich binders for non-2B6 epitopes (Pancodes 1038.20-22). Non-specific phage particles were removed by several washing steps (see below). Elution of specifically bound phage particles, infection, and amplification were done as described for pancodes 1038.1-8.
Table 4
Washing conditions Pancodes 1038.17-25
First Round Second Round Third round (only pancodes
1038.9-16)
5x PBST (quick) lOx PBST (quick) lOx PBST (quick)
2x PBST / 5min 3x PBST / 5min 5x PBST / 5min
3x PBS (quick) 5x PBS (quick) 5x PBS (quick)
Pancodes 1038.23-25
[00301] Pannings were done as described for pancodes 1038.1-8 but using biotinylated hCDH6aa54-615-Fc (see Table 1, SEQ ID NO:6) as antigen.
Subcloning
Conversion for FAB-FH expression
[00302] To facilitate rapid expression of soluble Fab fragments, the Fab encoding inserts of the selected HuCAL® PLATINUM phage particles were subcloned from the pMORPH®30 display vector into the pMORPH® 11 FH expression vector. Glycerol stocks containing E. coli that were infected with the final panning round output phages were used to inoculate cultures for pMORPH®30 DNA purification using the Nucleobond® Xtra Midi Plus kit according to the manufacturers manual (#740.412.50 Machery Nagel, Bethlehem, PA). 5 μg of each pMORPH®30 DNA output pool was triple digested via EcoRI/Xbal/Bmtl (all restriction enzymes were purchased from New England Biolabs, Ipswich, MA). The resulting EcoRI/Xbal Fab encoding 1485 bp fragment was gel purified using the Wizard SV Gel/PCR clean-up kit according to the manufacturers manual (A9282, Promega, Madison, WI) and ligated into the EcoRI/Xbal cut pMORPH® 11 FH vector backbone.
Conversion for IgG expression
[00303] In order to express full length IgG, variable domain fragments of VH and VL were subcloned from Fab expression vectors into pMORPH®4_hIgGlf vectors following the RapCLONE® protocol (Morphosys, Martinsried/Planegg, Germany). RapCLONE® is a two-step cloning method for the batch conversion of a large amount of Fab expression vectors into IgG expression vectors. In a first cloning step, a eukaryotic expression cassette was introduced into the pMORPH® 11 expression vectors via BsiWI/Mfel (for pools) or Hpal Mfel (for λ pools) digestion and subsequent ligation. This was followed by a second cloning step, in which the Fab pools containing the expression cassette were digested using EcoRV BlpI (both and λ pools) and subsequently cloned into the
pMORPH®4_IgGlf acceptor vector for expression in mammalian cells.
Expression and Purification of Fab Fragments
Transformation of E. coli TG1F
[00304] Electroporation-competent E. coli TG1F- aliquots of 50 were thawed on ice, mixed with 50 pg/μΐ pMORPH® 11 FH plasmid DNA and transferred into pre-cooled electroporation cuvettes. The cells were electroporated (Bio-Rad Gene Pulser; settings: 1.75 kV, 200 Ω, and 25 μΡ (Bio-Rad, Hercules, CA), transferred into 950 μΐ pre-warmed SOB medium and incubated for 1 h at 37 °C shaking at 220 rpm. An appropriate volume of the transformation samples were plated onto LB agar plates supplemented with 34 μg/mL Chloramphenicol and incubated overnight at 37°C to obtain single colonies.
Generation of Master plates
[00305] Chloramphenicol resistant single clones were picked into the wells of a sterile 384- well microtiter plate (Nunc) pre-filled with 60 μΐ 2xYT medium supplemented with 34 μg/ml of Chloramphenicol and 1 % glucose and grown overnight at 37°C. Next morning, 20 μΐ sterile 2xYT media containing 60 % glycerol and 1 % glucose were added into each well of the master plates. Plates were sealed with aluminum foil and stored at -8 °C.
Generation of Fab -Containing Bacterial Lysates for ELISA Screening
[00306] 5 μΐ of each well of a Master plate was transferred to a sterile 384-well microtiter plate pre-filled with 40 μΐ 2xYT medium per well supplemented with 34 μg/ml of Chloramphenicol and 0.1 % glucose. Plates were incubated at 37°C at 480 rpm and 80% humidity until the cultures were slightly turbid. 10 μΐ of 2xYT medium supplemented with 34 μg/ml Chloramphenicol and 5 niM IPTG were added per well for induction of Fab fragment expression. Plates were sealed with a gas- permeable tape and incubated overnight at 22°C at 500 rpm and 80% humidity. The next day 15 μΐ BEL lysis buffer (2.5 mg/ml lysozyme (#10837059001, Roche, Nutley, NJ ), 4 mM EDTA, 10 U/μΙ Benzonase (# 1.01654.0001 Merck, White House Station, NJ) was added to each well and plates were incubated for 1 h at 500 rpm and 80% humidity. Resulting Fab lysates were blocked by adding 15 μΐ. of 2x Chemiblocker per well followed by incubation for 30 min at 22°C, 500 rpm and 80% humidity.
Generation of Fab -Containing Bacterial Lysates for FACS Screening
[00307] 5 μΐ of each well of a compression plate was transferred to a sterile 96-well round bottom microtiter plate pre-filled with 100 μΐ 2xYT medium per well supplemented with 34 μg/ml of Chloramphenicol. Plates were incubated at 37°C at 500 rpm and 80% humidity until the cultures were slightly turbid. 10 μΐ of 2xYT medium supplemented with 34 μg/ml Chloramphenicol and 5 mM IPTG were added per well for induction of Fab fragment expression. Plates were sealed with a gas- permeable tape and incubated overnight at 22°C at 500 rpm and 80% humidity. The next day the plate was centrifuged for 10 min at 1200g and the supernatant discarded. The bacterial pellets were frozen overnight at -20°C to facilitate the lysis step. The thawed pellets were re-suspended in 200 μΐ. of BEL lysis buffer (see above) and incubated for 1 h at 22°C and 250 rpm. Resulting Fab lysates were centrifuged to remove cellular debris for 10 min at 1200 g. Fab containing supernatants were used for screening purposes.
Expression and Purification of His6-tagged Fab Fragments in E. coli
[00308] E. coli TG1F- transformants containing pMORPH®l 1 Fab FH DNA were singled out on 2xYT supplemented with 34 μg/mL Chloramphenicol and 1% glucose. Individual clones were picked and transferred into 3 mL 2xYT seed cultures supplemented with 34 μg/mL Chloramphenicol and 1% glucose and incubated at 37°C, 220 rpm for 4h. The seed culture was used to inoculate 50 mL main cultures with 2xYT supplemented with 34 μg/mL Chloramphenicol and 1% glucose and incubated in 250 mL shake flasks at 30°C until the OD600 reached a value of 0.6. Fab expression was induced by addition of IPTG to a final concentration of 0.75 mM and cultures were further incubated overnight at 25°C and 220 rpm. The next day cells were harvested and the cell pellets frozen overnight at -20°C. Cells pellets were disrupted by re-suspending and incubating in lysis buffer ((25 mM TRIS/pH=8, 500 mM NaCl, 2 mM MgCl2, 10 U/μΙ Benzonase (# 1.01654.0001 Merck), 0.1% Lysozyme (# 10837059001, Roche), Protease Inhibitor Complete w/o EDTA 1 tablet/ 50 mL of buffer (#11873580001 Roche)) for lh at room temperature on a rocking table. Lysates were clarified by centrifugation for 30 min at 15000 g and filtration of the supernatant through a 200 nm pore sized filter. His6-tagged Fab fragments were isolated via immobilized metal ion affinity chromatography (Ni-NTA Superflow® beads, #30430 Qiagen, Venlo, Netherlands) and eluted using imidazole. Buffer exchange to lx PBS was performed using PD-10 columns (# 17-0851-01 GE Healthcare, Pittsburgh, PA). Samples were sterile-filtered and the protein concentrations were determined by UV- spectrophotometry. The purity of the samples was analyzed in denaturing, reducing 15% SDS-PAGE. The identity of the samples was confirmed by MS.
Expression and purification of IgGs
Expression of IgGs at screening scale
[00309] Eukaryotic HEK293cl8 cells (ATCC CRL-10852) were used in a 96-well expression system for the generation of conditioned cell culture supematants containing full-length IgG for the subsequent use in specificity and/or functional assays. Eukaryotic HEK293 cl8 cells were re- suspended in transfection medium (D-MEM supplemented with 2% L-glutamine (#25030-024 Gibco, Grand Island, NY) 10 % FCS (#3302 PAN Biotech, Aidenbach, Germany) and 1 %
penicillin/streptomycin (#15140-122 Gibco, Grand Island, NY)) and seeded in 96-well F-bottom plates to a density of approximately 4xl04 cells in 50 μΐ per well the day before. A transfection master mix was prepared by mixing 0.6 μΐ Lipofectamine® 2000 (#11668 Invitrogen, Carlsbad, CA) per well with 25 μΐ. Opti-MEM® I medium. (#31985-047 Invitrogen, Carlsbad, CA). The master mix was incubated for 15 min at room temperature. On a new plate 20 μΐ. of Opti- MEM® I medium was added to wells containing 300 ng DNA and gently mixed. After that, the DNA was combined with the pre-incubated Lipofectamine® 2000, mixed gently and incubated for 20 min at room temperature. ΙΟΟμΙ of the pre-incubated Lipofectamine® 2000 DNA complexes were then transferred to each well of the plates with the cells and gently mixed. Plates were incubated for 40 h at 37°C and 6 % C02 for transient expression. The culture supematants were transferred to 96-well V-bottom plates and cleared by centrifugation. The resulting IgG-containing supematants were tested by an anti-Fd capture ELISA for assessment of IgG protein concentration in reference to a known standard and stored at -80°C for later use.
IgG expression check by anti-Fd ELISA
[00310] To assess IgG levels in screening scale derived supematants Maxisorp ® 96-well plates (#437111 Nunc) were coated with 50 μΐ/well Fd-fragment-specific sheep anti-human IgG (#PC075 The Binding Site, San Diego, CA) diluted 1 : 1000 in PBS. After coating, the plates were washed twice with TBST and blocked for lh with 5 % skim milk powder in TBST. In the meantime the IgG containing supematants were diluted 1 :50 in 2.5% skim milk powder in TBST. After washing the ELISA plates three times with TBST, 100 iL of the diluted supematants were transferred per well and plates were incubated for 1 h at room temperature. Subsequently, the plates were washed five times with TBST and the captured IgGs were detected by incubation with 50 μΐ F(ab')2-specific goat anti-human IgG (#109-055-097 Dianova, Hamburg, Germany) (diluted 1 :5000) in 0.5 % skim milk powder in TBST for lh. After washing the plates five times with TBST the AttoPhos fluorescence substrate (#1484281 Roche, Nutley, NJ) was added according to the manufacturer's instructions and the fluorescence emission at 535 nm was recorded with excitation at 430 nm with an ELISA reader. Expression and Purification of IgGs in Microscale
[00311] CAP-T® cells (CEVEC Pharmaceuticals, Cologne, Germany) were transiently transfected with pMORPH®4 IgG expression plasmid in FreeStyle293® expression medium (#12338 Invitrogen, Carlsbad, CA) using 40 kDa linear PEI ((PEI Max (#24765-2 Polysciences Warrington, PA)) as gene delivery vehicle.
[00312] One day prior to the transfection cells were diluted to -0.8E+06 cells/ml to ensure exponential growth. At the day of transfection cells were diluted in 9 mL pre-warmed Freestyle® (Invitrogen, Carlsbad, CA) medium to ~0.5xl07 cells/ml and transferred into a 125ml shake flask. 30 μg DNA was diluted in 500 μΐ OptiMEM. 1200 μg PEI Max (#24765-2 Polysciences Warrington, PA) was diluted in 8.80 mL OptiMEM medium. The DNA solution was added drop wise to the cells and gently mixed. After that, 500 μΐ. PEI Max solution was added to the cells and gently mixed.
[00313] The cells were incubated at 37°C, 6 % C02, 85 % humidity with agitation at 100 rpm.
After 4 h 10 mL pre-warmed PEM supplemented with 4 mM L-Glutamine and 5μg/ml blasticidin (#R21001 Invitrogen, Carlsbad, CA) was added. Simultaneously, 400 μΐ valproic acid (#P4543-25G Sigma Aldrich, St. Louis, MO) was added to a final concentration of 4 mM. Cells were incubated for 6 days at 37°C, 6 % C02, 85 % humidity with agitation at 100 rpm.
[00314] After centrifugation for 5 min at 1200 g at 4°C the supernatant was sterile-filtered (0.2 μπι pore size) and subjected to a Protein A affinity chromatography (MabSelect® SURE, GE Healthcare, Pittsburgh, PA) using a liquid handling station. Buffer exchange was performed to lx PBS and samples were sterile filtered (0.2 μπι pore size). Protein concentrations were determined by UV spectrophotometry and purity of IgGs was analyzed under denaturing, reducing conditions in SDS- PAGE.
Screening of Fab-containing raw bacterial lysates
ELISA screening
[00315] Using ELISA screening, single Fab clones were identified from panning outputs for binding to the target antigen. Fab fragments were tested using Fab containing crude E. coli lysates.
ELISA screening on directly coated antigen
[00316] Maxisorp® (#442404 Nunc, Rochester, NY) 384-well plates were coated overnight at
4°C with huCDH6 proteins at a concentration of 3 μg/ml in PBS. After washing plates were blocked for 2 h with 5% skimmed milk in lxPBST. Fab-containing E. coli lysates were added and binding allowed for 1 h at room temperature.
[00317] To detect bound Fab fragments plates were washed 5x with TBST and AP-anti human
IgG F(ab')2 (#109-055-097 Jackson Immunoresearch, West Grove, PA)was added in a 1/2500 dilution. After lh at room temperature plates were washed 5x with TBST and AttoPhos substrate was added according to the manufacturer's specifications. Plates were read in an ELISA reader 5 minutes after adding the substrate.
ELISA Screening on Biotinylated Antigen using NeutrAvidin® Plates
[00318] Maxisorp® (#442404 Nunc, Rochester, NY) 384-well plates were coated overnight at
4°C with NeutrAvidin® (#31000 Thermo Scientific, Rockford, IL) at a concentration of 10 μg/ml in PBS. After washing, plates were blocked for 2 h with lx Chemiblocker. Fab-containing E. coli lysates were dispensed into a fresh 384-well microtiter plate and biotinylated CDH6 antigens added at a concentration of 2.5 μg/mL. Fab-antigen complexes were then transferred to the NeutrAvidin® coated plates and capturing allowed for lh at room temperature and detected as described above.
FACS screening
[00319] In FACS screening, Fab fragments binding to cell surface expressed CDH6 antigen were identified from the panning output. lxlO5 cells/well were transferred into U bottom 96 well plates and mixed with 40 μΐ/well of the Fab-containing bacterial lysates. Plates were incubated shaking at 4°C for 1 h. After the incubation, 100 μΐ/well ice-cold FACS buffer (3% FCS, 0.02% NaN3, 2 mM EDTA in PBS) was added, cells were spun down at 4°C for 5 min at 250 g and washed twice with 180 μΐ/well ice-cold FACS buffer. After each washing step, cells were centrifuged and carefully re-suspended. After the last washing step cells were re-suspended in 50 μΐ, of 1/200 diluted secondary detection antibody (PE-conjugated goat anti-human IgG (#109-116-088, Jackson Immuno research, West Grove, PA). After lh incubation at 4°C cells were again washed twice in 180 μΕΛνεΙΙ ice-cold FACS buffer. Finally, cell pellets were re-suspended in 120 μΐ/well FACS buffer with 0.4 % paraformaldehyde and analyzed in a FACSCalibur® (BD Biosciences, San Jose, CA) equipped with an HTS plate reader.
Clone sequencing
Fab clone sequencing
[00320] Confirmed cell binding Fab hits were subjected to VL and VH sequencing. 1.2 mL
2xYT supplemented with 34 μg/mL Chloramphenicol and 1% glucose were inoculated with 5 μΐ, of bacterial glycerol stock deriving from the compression plates and grown in 96-well deep well microtiter plates overnight at 37°C and 500 rpm. The next day the bacteria were harvested and the pMORPH® 11 FH plasmids purified using a 96-well DNA purification kit (Nucleospin® 96 Plasmid Purification Kit #740625.24, Machery Nagel, Bethlehem, PA) according to the manufacturers protocol. To sequence the VL and VH primers M13rev (5' CAGGAAACAGCTATGAC 3' (SEQ ID NO: 10) and HuCAL VH for ((5' GATAAGCATGCGTAGGAGAAA 3 (SEQ ID NO: 11)) were used, respectively. IgG clone sequencing
[00321] Unique cell binding Fab clones were converted in the IgGl format in a polyclonal manner and afterwards retrieved by VL and VH sequencing. 1.5 mL 2xYT medium supplemented with 100 μg/mL Amp and 1% glucose were inoculated with single clones and grown in 96-well deep well microtiter plates overnight at 37°C, 450 rpm and 80% humidity. The next day the bacteria were harvested and the IgG encoding pMORPH®4 plasmids purified using a 96-well DNA purification kit according to the manufacturer's protocol (Nucleospin® 96 Plasmid Purification Kit #740625.24, Machery Nagel, Bethlehem, PA). To sequence the VL and VH primers ((T7 5'
TAATACGACTCACTATAGGG 3 ' (SEQ ID NO: 12) and CMV HC for (5'
CTCTAGCGCCACCATGAAACA 3 ' (SEQ ID NO: 13)) were used, respectively.
[00322] In vitro assays
Affinity assessment using Octet® QK
[00323] Affinity assessments by determining kinetic parameters were performed via Bio-Layer
Interferometry technology. All Fab samples were measured using Streptavidin Dip and Read biosensors (#18-0009 ForteBio, Menlo Park, CA). The plate was placed in an Octet® QK instrument (ForteBio, Menlo Park, CA) and allowed to equilibrate to 27°C in the chamber. The run was initiated by placing the sensors in the wells containing 150 nM of biotinylated CDH6 protein for 300s. After that the sensors were placed in the wells containing 250 nM Fab sample. Fab association and dissociation were each recorded for 600s by measuring the change in layer thickness (in nanometers, nm) with time, all under computer control. Data were processed automatically using the Octet® User Software version 3.0.
Assessment of antibody cellular internalization propensity
[00324] Cell Internalization of IgGs by target mediated endocytosis was assessed by microscopy using a VTI Array Scan® HC reader (ThermoFischer, Waltham, MA) as described below. The underlying analysis protocol was the Spot Detector V4 algorithm (Thermo Scientific Cellomics®, Thermo Scientific, Rockford, IL). In brief, this analysis protocol provides a fast and generic spot analysis that identifies intracellular punctuate objects, such as IgG containing lysosomal vesicles after immunofluorescent staining. The total punctuate fluorescent intensity can then be averaged over the number of cells analyzed.
[00325] OVCAR3 cells (ATCC HTB-161) were grown in T150 tissue culture flasks to approximately 90% confluency. Media was discarded and cells flushed with 10 mL PBS. Cells were detached by incubation with 4 mL cell dissociation buffer for 5 min at 37°C. Detached cells were re- suspended thoroughly in growth medium and transferred to a 50 mL tube. After counting the cells on a Vi-Cell® analyzer (Beckman Coulter, Brea, CA) the suspension was adjusted to 105 cells/mL in growth medium. 100 μΐ^ of the cell suspension was seeded per well of a 96-well microtiter plate with transparent bottom. Plates were incubated for 24 h at 37°C in 5% C02 to allow the cells to adhere and to expand.
[00326] IgG containing cell culture supernatants were generated as described and diluted in
PBS in a fourfold dilution series covering dilutions ranging from 1:4 to 1 : 128. 100 μΐ of each sample dilution was dispensed per well of the cell containing microtiter plate and incubated for 2h at 37°C to permit IgG internalization. Subsequently, cells were fixed by adding 100 μΐ lx CellFix® reagent (#340181 BD Biosciences, San Jose, CA) per well. After 10 minutes plates were washed twice with PBS and cells then permeabilized by adding 100 μΐ 0.1% Triton X-100 per well. After 10 min plates were washed again twice with PBS and blocked with 100 μΐ lx Odyssey blocking buffer (#927-40000 LiCor, Lincoln, NE) for 2 hours at room temperature. To detect internalized IgGs 100 μΐ of a 1 :000 dilution of secondary antibody Alexa Fluor® 488 goat anti-human IgG (#11001 Molecular Probes, Grand Island, NY) supplemented with a 1 : 10000 dilution of Hoechst nucleus staining reagent (#B2261 Sigma, St. Louis, Mo) were added per well and incubated for 1 hours at room temperature in the dark. Cells were then washed twice with PBS without final aspiration. Plates were then loaded into the Cellomics® VTI Array Scan HC reader (ThermoFischer, Waltham, MA) and analyzed. The extent of IgG internalization was assessed by the mean average spot intensity (MS AI) per cell.
Surrogate ADC assay using anti-human FAB-DM1 conjugate
[00327] To test the ability of CDH6 antibodies to internalize after receptor binding and deliver cytotoxic pay load, a surrogate ADC assay was performed mixing anti-human Fab-DMl reagent (AffiniPure® Fab Fragment Goat Anti-Human IgG H+L conjugated with SMCC-DM1) with purified IgGs at a fixed 1:2 ratio. Cytotoxic potential was tested on the cancer cell line OVCAR3 (ovarian serous carcinoma, cultured in McCoys + 20% FCS) as these cells show high expression of CDH6.
[00328] Cells in culture were counted and diluted in medium to a concentration of lxlO5 cells/ml. 1000 cells/well were transferred to 384-well plates (Corning Costar#3707, Corning, Tewksbury, MA). Primary antibody Fab-DMl solution was prepared in 1.4ml Matrix tubes (Thermo, # 3790, Rockford, IL) by combining 666nM Fab-DMl with 333nM primary human IgG diluted in cell culture media and incubated at 37°C for 30 minutes. A 10-point, 1 :3 serial dilution was prepared in a 384-well deep-well plate (Brandtech Scientific Inc #701355, Essex, CT ) and 25μ1 were transferred per assay plate (triplicates) to yield a highest starting concentration of FAB-DMl/human IgG of 66nM and 33nM, respectively. For controls, wells with cells only (=100% viability control) and cells only incubated with Fab-DMl (to check for unspecific killing of the secondary reagent) were prepared. Plates were incubated for 120 h at 37°C and 5% C02. Cellular activity of the primary antibody /Fab- DM1 complexes was determined using CellTiter-Glo® reagent (#G7571 Promega, Madison, WI) according to the manufacturer's instructions. Viability was normalized to the cells only control.
Antibody screening summary
[00329] The HuCAL® PLATINUM phagemid library was used to select specific Fab fragments against human CDH6-ECD antigens. Recombinant human APPAvi and Fc fusion as well as truncated domain APPAvi fusion proteins were used for the pannings. HuCAL® PLATINUM antibody -phage particles were subjected in different subpool combinations to a total of 8 different panning strategies resulting in 28 panning output pools. In summary, six out of eight strategies have been productive and resulted in 771 ELISA positive screening hits.
[00330] FACS screening on CHO cells expressing human CDH6 as well as OVCAR3 cells resulted in 271 confirmed cell binding hits. To consolidate these cell binding hits VL and VH sequencing was performed leading to the identification of 53 unique Fab antibody clones.
[00331] To allow for the functional characterization, the Fab antibody clones were converted into the IgG format, yielding 47 unique IgG clones of which 44 were successfully expressed at screening scale. The purified, unique IgGs were subjected to a series of characterization assays including human/cy no/rat/mouse cross-reactivity by FACS, affinity ranking by Octet, cellular internalization assays as well as surrogate ADC assays using an anti-human IgG Fab-SMCC-DMl secondary reagent. Based on these assays, human IgGs were selected for scaled up production, subsequent direct conjugation to ADC linker/pay loads and testing as ADCs in in vitro and in vivo experiments.
[00332] The sequence information for the anti-CDH6 IgGs selected for further in-depth characterization are shown in Table 5.
Table 5
Sequence information of the IgGs selected for in-depth characterization.
Figure imgf000099_0001
(SEQ ID
NO: 16)
HCDR1
(Kabat)
(SEQ ID
SYAIS NO: 17)
HCDR2
(Kabat)
(SEQ ID
GIIPIFGTANYAQKFQG NO: 18)
HCDR3
(Kabat)
(SEQ ID
KFPGRGPFAY NO: 19)
vH full
sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPG (SEQ ID QGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSL NO:20) RSEDT AVYYC ARKFPGRGPF AYWGQGTL VTVS S
vL full
sequence DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSQYVYWYQQLPGTA (SEQ ID PKLLIYYNSERPSGMPDRFSGSKSGTSASLAITGLQAEDEADYY NO:21) CQTWDASSQSFVFGGGTKLTVL
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAAC
CGGGCAGCAGCGTGAAAGTTAGCTGCAAAGCATCCGGAGG
GACGTTTTCTTCTTACGCTATCTCTTGGGTGCGCCAGGCCCC
GGGCCAGGGCCTCGAGTGGATGGGCGGTATCATCCCGATCT
TCGGCACTGCGAACTACGCCCAGAAATTTCAGGGCCGGGTG
ACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGA
vH DNA ACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTATTATT sequence GCGCGCGTAAATTCCCGGGTCGTGGTCCGTTCGCTTACTGGG (SEQ ID GCCAAGGCACCCTGGTGACTGTTAGCTCA
NO:22)
GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA ACATTGGTTCTCAGTACGTGTACTGGTACCAGCAGCTGCCG GGCACGGCGCCGAAACTGCTGATCTACTACAACTCTGAACG
vL DNA CCCGAGCGGCATGCCGGATCGCTTTAGCGGATCCAAAAGCG sequence GCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAA (SEQ ID GACGAAGCGGATTATTACTGCCAGACTTGGGACGCTTCTTCT NO:23) CAGTCTTTCGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPG QGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSL RSEDTAVYYCARKFPGRGPF AYWGQGTL VTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
HC full RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ sequence PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG (SEQ ID QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV NO:24) MHE ALHNHYTQKSL SL SPGK
LC full DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSQYVYWYQQLPGTA sequence PKLLIYYNSERPSGMPDRFSGSKSGTSASLAITGLQAEDEADYY (SEQ ID CQTWDASSQSFVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA NO:25) NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAAC
CGGGCAGCAGCGTGAAAGTTAGCTGCAAAGCATCCGGAGG
GACGTTTTCTTCTTACGCTATCTCTTGGGTGCGCCAGGCCCC
GGGCCAGGGCCTCGAGTGGATGGGCGGTATCATCCCGATCT
TCGGCACTGCGAACTACGCCCAGAAATTTCAGGGCCGGGTG
ACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGA
ACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTATTATT
GCGCGCGTAAATTCCCGGGTCGTGGTCCGTTCGCTTACTGGG
GCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAG
GGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC
TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC
ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG
TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
HC DNA CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC
sequence AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG (SEQ ID TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT NO:26) ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC
GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA
ACATTGGTTCTCAGTACGTGTACTGGTACCAGCAGCTGCCG
GGCACGGCGCCGAAACTGCTGATCTACTACAACTCTGAACG
CCCGAGCGGCATGCCGGATCGCTTTAGCGGATCCAAAAGCG
GCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAA
GACGAAGCGGATTATTACTGCCAGACTTGGGACGCTTCTTCT
CAGTCTTTCGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA
GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCC
TCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTG
TCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCT
GGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGAC
LC DNA CACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCA
sequence GCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCAC (SEQ ID AGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGT NO:27) GGAGAAGACAGTGGCCCCTACAGAATGTTCA
NOV0672 LCDR1
(Kabat)
TGTSSDVGAYNYVS
(SEQ ID
Figure imgf000102_0001
TCWVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHE ALHNHYTQKSL SL SPGK
DIALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGK
LC full APKLMIYGVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADY sequence YCQSYDHLLHWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA (SEQ ID NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN NO:39) KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTAACACTTACGGTATCCATTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCTACATCCATTACTCTGGTT
CTTCTACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACCA
TCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATG
AACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGC
GCGTCATGCTTACGGTTACATGGATTTCTGGGGCCAAGGCA
CCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA
ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG
GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT
ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG
GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTG
ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTC
CTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG
TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC
AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG
CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT
HC DNA GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
sequence GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT (SEQ ID CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG NO:40) AGCCTCTCCCTGTCTCCGGGTAAA
GATATCGCGCTGACCCAGCCGGCGAGCGTGAGCGGTAGCCC
GGGCCAGAGCATTACCATTAGCTGCACCGGCACCAGCAGCG
ATGTGGGCGCTTACAACTACGTGTCTTGGTACCAGCAGCAT
CCGGGCAAGGCGCCGAAACTGATGATCTACGGTGTTTCTAA
ACGTCCGAGCGGCGTGAGCAACCGTTTTAGCGGATCCAAAA
GCGGCAACACCGCGAGCCTGACCATTAGCGGCCTGCAAGCG
GAAGACGAAGCGGATTATTACTGCCAGTCTTACGACCATCT
LC DNA GCTGCATGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCT
sequence AGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCC (SEQ ID CTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGT N0:41) GTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCC
Figure imgf000104_0001
A
QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPG
KGLEWVSGISGGGSNTYYADSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARGGGQYFDYWGQGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCWVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQYNSTY
HC full RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ sequence PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG (SEQ ID QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV NO:52) MHE ALHNHYTQKSL SL SPGK
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGYNYVSWYQQLPGTA
LC full PKLLIYRDNQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYY sequence CAAWTSGSIGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA (SEQ ID NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN NO:53) KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTTCTTACGCTATGACTTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCGGTATCTCTGGTGGTGGTT
CTAACACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG
CGCGTGGTGGTGGTCAGTACTTCGATTACTGGGGCCAAGGC
ACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATC
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG
CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCG
AACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG
GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTG
ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTC
CTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG
TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC
AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG
CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT
HC DNA GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
sequence GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT (SEQ ID CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG NO:54) AGCCTCTCCCTGTCTCCGGGTAAA
LC DNA GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC
sequence GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA (SEQ ID ACATTGGTTACAACTACGTGTCTTGGTACCAGCAGCTGCCG
Figure imgf000106_0001
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAG
CGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGA
CTATTAACTCTTACCTGAACTGGTACCAGCAGAAACCGGGC
AAAGCGCCGAAACTATTAATCTACCGTGCTTCTAACCTGCA
AAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCA
vL DNA CCGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT sequence TTGCGACCTATTATTGCCAGCAGGGTGACTCTTCTTGGACCT (SEQ ID TTGGCCAGGGCACGAAAGTTGAAATTAAA
NO:65)
QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAISWVRQAPGK
GLEW VGFIKSNAD GYTTNYAAP VKGRFTISRDD SKNTL YLQM
NSLKTEDTAVYYCARIRYFRNWDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFP A VLQ SSGLYSLSS WT VP S S SL GTQTYICN VNHKP SNTK V
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
HC full NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA sequence KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE (SEQ ID SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC NO:66) SVMHEALHNHYTQKSLSLSPGK
DIQMTQSPSSLSASVGDRVTITCRASQTINSYLNWYQQKPGKA
LC full PKLLIYRASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC sequence QQGDSSWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO:67) S STLTL SKAD YEKHKVYACE VTHQGL S SP VTKSFNRGEC
CAGGTGCAATTGGTGGAAAGCGGCGGTGGCCTGGTGAAACC
AGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCTCCGGATTCA
CCTTTTCTTCTTACGCTATCTCTTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTGGGCTTCATCAAATCTAACGCT
GACGGTTACACTACTAACTATGCCGCCCCAGTGAAAGGCCG
CTTTACCATTAGCCGCGATGATTCGAAAAACACCCTGTATCT
GCAAATGAACAGCCTGAAAACCGAAGATACGGCCGTGTATT
ATTGCGCGCGTATCCGTTACTTCCGTAACTGGGATTACTGGG
GCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAG
GGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC
TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC
ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG
TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
HC DNA AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
sequence CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC (SEQ ID CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG NO:68) GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
Figure imgf000108_0001
(SEQ ID GCGTGAGCGGTAACTCTGCTGCTTGGAACTGGATTCGTCAG NO:78) AGCCCGAGCCGTGGCCTCGAGTGGCTGGGCATCATCTACTA
CCGTAGCAAATGGTACAACGACTATGCCGTGAGCGTGAAAA
GCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTT
AGCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGT
GTATTATTGCGCGCGTTCTTCTTACTCTGGTGGTTTCGATTAC
TGGGGCCAAGGCACCCTGGTGACTGTTAGCTCA
GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC
GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA
ACATTGGTTCTTACTACGTGTCTTGGTACCAGCAGCTGCCGG
GCACGGCGCCGAAACTGCTGATCTACTACAACACTAAACGC
CCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGG
CACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAG
vL DNA ACGAAGCGGATTATTACTGCCAGTCTTGGGACAAACTGGGT sequence AAAGGTTACGTGTTTGGCGGCGGCACGAAGTTAACCGTCCT (SEQ ID A
NO:79)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSGNSAAWNWIRQSP
SRGLEWLGIIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLN
SVTPEDTAVYYCARSSYSGGFDYWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQS SGLYSLS S WTVPS SSLGTQTYICNVNHKPSNTKVDK
RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQYNST
HC full YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG sequence QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN (SEQ ID GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV NO:80) MHE ALHNHYTQKSL SL SPGK
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSYYVSWYQQLPGTA
LC full PKLLIYYNTKRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYY sequence CQSWDKLGKGYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA (SEQ ID NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN N0:81) KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACC
GAGCCAGACCCTGAGCCTGACCTGCGCGATTTCCGGAGATA
GCGTGAGCGGTAACTCTGCTGCTTGGAACTGGATTCGTCAG
AGCCCGAGCCGTGGCCTCGAGTGGCTGGGCATCATCTACTA
CCGTAGCAAATGGTACAACGACTATGCCGTGAGCGTGAAAA
GCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTT
AGCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGT
GTATTATTGCGCGCGTTCTTCTTACTCTGGTGGTTTCGATTAC
TGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCAC
CAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAG
CACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG
ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA
GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA
ATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA
HC DNA GCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCC
sequence CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC (SEQ ID CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT NO:82) GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
Figure imgf000110_0001
sequence KGLEWVSVISSSGSNTNYADSVKGRFTISRDNSKNTLYLQMNS (SEQ ID LRAEDTAVYYCARPSYFQAMDYWGQGTLVTVSS
NO:90)
vL full DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNFVSWYQQLPGTAP sequence KLLIYDNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC (SEQ ID SSYDSFDHSWVFGGGTKLTVL
N0:91)
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTTCTTTCGCTATGAACTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCGTTATCTCTTCTTCTGGTT
CTAACACCAACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
vH DNA GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG sequence CGCGTCCGTCTTACTTCCAGGCTATGGATTACTGGGGCCAAG (SEQ ID GCACCCTGGTGACTGTTAGCTCA
NO:92)
GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC
GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA
ACATTGGTTCTAACTTCGTGTCTTGGTACCAGCAGCTGCCGG
GCACGGCGCCGAAACTGCTGATCTACGACAACTCTAACCGC
CCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGG
vL DNA CACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAG sequence ACGAAGCGGATTATTACTGCTCTTCTTACGACTCTTTCGACC (SEQ ID ATTCTTGGGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA NO:93)
QVQLLESGGGLVQPGGSLRLSCAASGFTFSSFAMNWVRQAPG
KGLEWVSVISSSGSNTNYADSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARPSYFQAMDYWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQS SGLYSLS S WTVPS SSLGTQTYICNVNHKPSNTKVDK
RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQYNST
HC full YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG sequence QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN (SEQ ID GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV NO:94) MHE ALHNHYTQKSL SL SPGK
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNFVSWYQQLPGTAP
LC full KLLIYDNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC sequence S S YD SFDH SWVFGGGTKLT VLGQPKAAP S VTLFPPS SEELQ AN (SEQ ID KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNK NO:95) YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTTCTTTCGCTATGAACTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCGTTATCTCTTCTTCTGGTT
CTAACACCAACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG
CGCGTCCGTCTTACTTCCAGGCTATGGATTACTGGGGCCAAG
HC DNA GCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCA
sequence TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG (SEQ ID GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC NO:96) CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA
CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC
TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCC
CAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA
CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGG
TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC
AGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG
GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA
AGAGCCTCTCCCTGTCTCCGGGTAAA
GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC
GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA
ACATTGGTTCTAACTTCGTGTCTTGGTACCAGCAGCTGCCGG
GCACGGCGCCGAAACTGCTGATCTACGACAACTCTAACCGC
CCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGG
CACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAG
ACGAAGCGGATTATTACTGCTCTTCTTACGACTCTTTCGACC
ATTCTTGGGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA
GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCC
TCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTG
TCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCT
GGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGAC
LC DNA CACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCA
sequence GCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCAC (SEQ ID AGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGT NO:97) GGAGAAGACAGTGGCCCCTACAGAATGTTCA
NOV0690 LCDR1
(Kabat)
(SEQ ID
SGDAIGTKFAH NO:98)
LCDR2
(Kabat)
(SEQ ID
YDHERPS NO:99)
LCDR3
(Kabat)
(SEQ ID
YSRASSNLV NO: 100)
HCDRl
(Kabat)
(SEQ ID
DHAID NO: 101)
VIAGDGSITYYADSVKG
HCDR2 (Kabat)
(SEQ ID
NO: 102)
HCDR3
(Kabat)
(SEQ ID
DTGVYREYMDV NO: 103)
vH full QVQLLESGGGLVQPGGSLRLSCAASGFTFSDHAIDWVRQAPGK sequence GLEWVSVIAGDGSITYYADSVKGRFTISRDNSKNTLYLQMNSL (SEQ ID RAEDTAVYYCARDTGVYREYMDVWGQGTLVTVSS
NO: 104)
vL full DIELTQPPSVSVSPGQTASITCSGDAIGTKFAHWYQQKPGQAPV sequence LVIYYDHERPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCY (SEQ ID SRAS SNL VFGGGTKLT VL
NO: 105)
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTGACCATGCTATCGACTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCGTTATCGCTGGTGACGGTT
CTATCACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
vH DNA GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG sequence CGCGTGACACTGGTGTTTACCGTGAATACATGGATGTTTGG (SEQ ID GGCCAAGGCACCCTGGTGACTGTTAGCTCA
NO: 106)
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATGCTATCG
GTACTAAATTCGCTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACTACGACCATGAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
vL DNA CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA sequence GCGGATTATTACTGCTACTCTCGTGCTTCTTCTAACCTGGTG (SEQ ID TTTGGCGGCGGCACGAAGTTAACCGTCCTA
NO: 107)
QVQLLESGGGLVQPGGSLRLSCAASGFTFSDHAIDWVRQAPGK
GLEWVSVIAGDGSITYYADSVKGRFTISRDNSKNTLYLQMNSL
RAEDTAVYYCARDTGVYREYMDVWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFP A VLQ SSGLYSLSS WT VP S S SL GTQTYICN VNHKP SNTK V
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
HC full NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA sequence KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE (SEQ ID SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC NO: 108) SVMHEALHNHYTQKSLSLSPGK
DIELTQPPSVSVSPGQTASITCSGDAIGTKFAHWYQQKPGQAPV
LC full LVIYYDHERPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCY sequence SRASSNLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATL (SEQ ID VCLISDFYPGAVT VAWKAD S SP VKAGVETTTP SKQ SNNKYAA NO: 109) S S YL SLTPEQ WKSHRS YS CQ VTHEGST VEKT VAPTECS
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA CCTTTTCTGACCATGCTATCGACTGGGTGCGCCAGGCCCCGG GCAAAGGTCTCGAGTGGGTTTCCGTTATCGCTGGTGACGGTT CTATCACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG
CGCGTGACACTGGTGTTTACCGTGAATACATGGATGTTTGG
GGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAA
GGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT
ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC
TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC
ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG
TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC
AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG
TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATGCTATCG
GTACTAAATTCGCTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACTACGACCATGAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA
GCGGATTATTACTGCTACTCTCGTGCTTCTTCTAACCTGGTG
TTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCAGCCCAA
GGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGA
GCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTG
ACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGAT
AGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTC
LC DNA CAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTG
sequence AGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAG (SEQ ID CTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACA NO: 111) GTGGCCCCTACAGAATGTTCA
NOV0691 LCDR1
(Kabat)
(SEQ ID
TGTSSDVGRYNFVS NO: 112)
LCDR2
(Kabat)
(SEQ ID
RVSNRPS NO: 113)
QSWTTYSNW
LCDR3
Figure imgf000115_0001
sequence APKLMIYRVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADY (SEQ ID YCQSWTTYSNWFGGGT LTVLGQPKAAPSVTLFPPSSEELQA NO: 123) NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN
KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGGTGGAAAGCGGCGGTGGCCTGGTGAAACC
AGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCTCCGGATTCA
CCTTTTCTTCTTACGCTCTGAACTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTGGGCCGTATCAAATCTAAAACT
TACGGTGGTTCTACTGACTATGCCGCCCCAGTGAAAGGCCG
CTTTACCATTAGCCGCGATGATTCGAAAAACACCCTGTATCT
GCAAATGAACAGCCTGAAAACCGAAGATACGGCCGTGTATT
ATTGCGCGCGTGACCGTGGTGGTTACGTTGGTTTCGATTCTT
GGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACC
AAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCG
CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT
CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT
CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC
CCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA
GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG
AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA
TCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG
CCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC
HC DNA GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG
sequence CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC (SEQ ID GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC NO: 124) TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
GATATCGCGCTGACCCAGCCGGCGAGCGTGAGCGGTAGCCC
GGGCCAGAGCATTACCATTAGCTGCACCGGCACCAGCAGCG
ATGTGGGCCGTTACAACTTCGTGTCTTGGTACCAGCAGCATC
CGGGCAAGGCGCCGAAACTGATGATCTACCGTGTTTCTAAC
CGTCCGAGCGGCGTGAGCAACCGTTTTAGCGGATCCAAAAG
CGGCAACACCGCGAGCCTGACCATTAGCGGCCTGCAAGCGG
AAGACGAAGCGGATTATTACTGCCAGTCTTGGACTACTTAC
TCTAACGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA
GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCC
TCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTG
TCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCT
GGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGAC
LC DNA CACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCA
sequence GCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCAC (SEQ ID AGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGT NO: 125) GGAGAAGACAGTGGCCCCTACAGAATGTTCA
NOV0692 SGDSIGSKYAQ
LCDR1
Figure imgf000117_0001
NO: 136) FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP AVLQS S GL YSL S S VVT VPS S SLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EWCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQWTLPPSREEMT NQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHE ALHNH YTQKSL SL SP GK
DIELTQPPSVSVSPGQTASITCSGDSIGSKYAQWYQQKPGQAPV
LC full LVIYYNSERPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQS sequence WDGQSTIRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKA (SEQ ID TLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA NO: 137) ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGGTGGAAAGCGGCGGTGGCCTGGTGAAACC
AGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCTCCGGATTCA
CCTTTTCTCGTTACTGGATGGACTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTGGGCCGTATCAAATCTAAAGCT
AACGGTGGTATCACTGACTATGCCGCCCCAGTGAAAGGCCG
CTTTACCATTAGCCGCGATGATTCGAAAAACACCCTGTATCT
GCAAATGAACAGCCTGAAAACCGAAGATACGGCCGTGTATT
ATTGCGCGCGTGGTATGACTTTCCTGGGTATCTGGGGCCAA
GGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC
CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC
AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGG
ACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA
GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG
CCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAAT
CTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC
ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCA
AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC
GGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT
CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG
GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT
CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
HC DNA GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC
sequence ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC (SEQ ID ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC NO: 138) AGAAGAGCCTCTCCCTGTCTCCGGGTAAA
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATTCTATCG
GTTCTAAATACGCTCAGTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACTACAACTCTGAACGTCCGAG
LC DNA CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
sequence CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA (SEQ ID GCGGATTATTACTGCCAGTCTTGGGACGGTCAGTCTACTATC NO: 139) CGTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCA
Figure imgf000119_0001
AGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCAC CGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTT TGCGACCTATTATTGCCATCAGTACTCTTACTGGCTGCGTAC CTTTGGCCAGGGCACGAAAGTTGAAATTAAA
Q VQLLES GGGL VQPGGSLRL SC AAS GFTFS S YALHW VRQ APG
KGLEWVSYIFYDSSSTYYADSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARFLYSAYGVANWGQGTLVTVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFP AVLQS S GL YSL S S VVT VPS S SLGTQTYICNVNHKPSNTKVD
KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EWCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
HC full TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES (SEQ ID NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS NO: 150) VMHE ALHNH YTQKSL SL SP GK
DIQMTQSPSSLSASVGDRVTITCRASQSISFYLAWYQQKPGKAP
LC full KLLIYGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCH sequence QYSYWLRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 151) S STLTL SKAD YEKHKVYACE VTHQGL S SP VTKSFNRGEC
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTTCTTACGCTCTGCATTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCTACATCTTCTACGACTCTT
CTTCTACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACCA
TCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATG
AACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGC
GCGTTTCCTGTACTCTGCTTACGGTGTTGCTAACTGGGGCCA
AGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTC
CATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG
GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC
CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG
GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC
AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA
GCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAA
TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACC
TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA
CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC
CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCC
GGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
HC DNA CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAG
sequence CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT (SEQ ID CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA NO: 152) CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
Figure imgf000121_0001
GCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGTG TATTATTGCGCGCGTGAACGTTCTTACCGTGACTACTTCGAT TACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCA
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAG
CGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGG
GTATTTTCACTTACCTGAACTGGTACCAGCAGAAACCGGGC
AAAGCGCCGAAACTATTAATCTCTGCTGCTTCTACTCTGCAA
AGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCAC
vL DNA CGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTT sequence TGCGACCTATTATTGCCAGCAGTACTACTCTACTTCTCTGAC (SEQ ID CTTTGGCCAGGGCACGAAAGTTGAAATTAAA
NO: 163)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSP
SRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQL
NSVTPEDTAVYYCARERSYRDYFDYWGQGTLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 164) CSVMHEALHNHYTQKSLSLSPGK
DIQMTQSPSSLSASVGDRVTITCRASQGIFTYLNWYQQKPGKA
LC full PKLLISAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC sequence QQYYSTSLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV (SEQ ID VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS NO: 165) LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACC
GAGCCAGACCCTGAGCCTGACCTGCGCGATTTCCGGAGATA
GCGTGAGCTCTAACTCTGCTGCTTGGAACTGGATTCGTCAGA
GCCCGAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTAC
CGTAGCAAATGGTACAACGACTATGCCGTGAGCGTGAAAAG
CCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTTA
GCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGTG
TATTATTGCGCGCGTGAACGTTCTTACCGTGACTACTTCGAT
TACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTC
CACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA
GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT
ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC
GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAG
TTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG
TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC
TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAG
ACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
HC DNA GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA
sequence ACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCAC (SEQ ID CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC NO: 166) CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
Figure imgf000123_0001
sequence VLVIYRDNNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYC (SEQ ID QSWDSFLAWFGGGTKLTVL
NO: 175)
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTTCTTACGCTATGCATTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCTTCATCTCTTCTCTGGGTT
CTTACACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
vH DNA GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG sequence CGCGTGAAACTGCTGGTTACGGTTACGCTTTCGATCCGTGGG (SEQ ID GCCAAGGCACCCTGGTGACTGTTAGCTCA
NO: 176)
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACATCC
GTAAATACGTTGTTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACCGTGACAACAACCGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
vL DNA CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA sequence GCGGATTATTACTGCCAGTCTTGGGACTCTTTCCTGGCTGTT (SEQ ID GTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA
NO: 177)
QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPG
KGLEWVSFISSLGSYTYYADSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARETAGYGYAFDPWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFP A VLQ SSGLYSLSS WT VP S S SL GTQTYICN VNHKP SNTK V
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
HC full NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA sequence KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE (SEQ ID SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC NO: 178) SVMHEALHNHYTQKSLSLSPGK
DIELTQPPSVSVSPGQTASITCSGDNIRKYWHWYQQKPGQAP
LC full VLVIYRDNNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYC sequence QSWDSFLAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANK (SEQ ID ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY NO: 179) AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTTCTTACGCTATGCATTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCTTCATCTCTTCTCTGGGTT
CTTACACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG
CGCGTGAAACTGCTGGTTACGGTTACGCTTTCGATCCGTGGG
GCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAG
GGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC
TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
HC DNA TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
sequence CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC (SEQ ID AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA NO: 180) CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC
Figure imgf000125_0001
(Kabat)
(SEQ ID
NO: 187)
vH full QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPG sequence KGLEWMGFIDPGVSYTRYSPSFQGQVTISADKSISTAYLQWSSL (SEQ ID KASDTAMYYCARVLAHSTEYNWP AF WGQGTLVTVS S NO: 188)
vL full DIVLTQPPSVSGAPGQRVTISCSGSSSNIGLDYVNWYQQLPGTA sequence PKLLIYRNKQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYY (SEQ ID CQAWAGRTNYWFGGGTKLTVL
NO: 189)
CAGGTGCAATTGGTGCAGAGCGGTGCGGAAGTGAAAAAAC
CGGGCGAAAGCCTGAAAATTAGCTGCAAAGGCTCCGGATAT
AGCTTCACTAACTACTGGATCGGTTGGGTGCGCCAGATGCC
GGGCAAAGGTCTCGAGTGGATGGGCTTCATCGACCCGGGTG
TTAGCTACACCCGTTATAGCCCGAGCTTTCAGGGCCAGGTG
ACCATTAGCGCGGATAAAAGCATCAGCACCGCGTATCTGCA
vH DNA ATGGAGCAGCCTGAAAGCGAGCGATACCGCGATGTATTATT sequence GCGCGCGTGTTCTGGCTCATTCTACTGAATACAACTGGCCGG (SEQ ID CTTTCTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCA NO: 190)
GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC
GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA
ACATTGGTCTGGACTACGTGAACTGGTACCAGCAGCTGCCG
GGCACGGCGCCGAAACTGCTGATCTACCGTAACAAACAGCG
CCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCG
vL DNA GCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAA sequence GACGAAGCGGATTATTACTGCCAGGCTTGGGCTGGTCGTAC (SEQ ID TAACTACGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCT NO: 191) A
QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPG
KGLEWMGFIDPGVSYTRYSPSFQGQVTISADKSISTAYLQWSSL
KASDTAMYYCARVLAHSTEYNWP AFWGQGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 192) CSVMHEALHNHYTQKSLSLSPGK
DIVLTQPPSVSGAPGQRVTISCSGSSSNIGLDYVNWYQQLPGTA
LC full PKLLIYRNKQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYY sequence CQAWAGRTNYWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ (SEQ ID ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NO: 193) NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGGTGCAGAGCGGTGCGGAAGTGAAAAAAC
CGGGCGAAAGCCTGAAAATTAGCTGCAAAGGCTCCGGATAT
AGCTTCACTAACTACTGGATCGGTTGGGTGCGCCAGATGCC
GGGCAAAGGTCTCGAGTGGATGGGCTTCATCGACCCGGGTG
HC DNA TTAGCTACACCCGTTATAGCCCGAGCTTTCAGGGCCAGGTG
sequence ACCATTAGCGCGGATAAAAGCATCAGCACCGCGTATCTGCA (SEQ ID ATGGAGCAGCCTGAAAGCGAGCGATACCGCGATGTATTATT NO: 194) GCGCGCGTGTTCTGGCTCATTCTACTGAATACAACTGGCCGG CTTTCTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCT
CCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTC
AAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTC
AGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC
TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA
CGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGA
GTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACC
GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC
TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA
AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG
AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT
CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC
CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAA
GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACC
GGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCA
ACATTGGTCTGGACTACGTGAACTGGTACCAGCAGCTGCCG
GGCACGGCGCCGAAACTGCTGATCTACCGTAACAAACAGCG
CCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCG
GCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAA
GACGAAGCGGATTATTACTGCCAGGCTTGGGCTGGTCGTAC
TAACTACGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCT
AGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCC
CTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGT
GTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCC
TGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGA
LC DNA CCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCC
sequence AGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCA (SEQ ID CAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACC NO: 195) GTGGAGAAGACAGTGGCCCCTACAGAATGTTCA
NOV0709 LCDR1
(Kabat)
(SEQ ID
TGTSSDVGSYNYVS NO: 196)
LCDR2
(Kabat)
(SEQ ID
YVSNRPS NO: 197)
LCDR3
(Kabat)
(SEQ ID
ASYTHQGSWV NO: 198)
Figure imgf000128_0001
NO:207) NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTACTTACTACATGCATTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCGTTATCTCTTCTGACGGTT
CTTTCACCTTCTATGCGGATAGCGTGAAAGGCCGCTTTACCA
TCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATG
AACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGC
GCGTCATGGTTACGGTGCTTTCGATTACTGGGGCCAAGGCA
CCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA
ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG
GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT
ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG
GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTG
ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTC
CTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG
TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC
AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG
CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT
HC DNA GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
sequence GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT (SEQ ID CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG NO:208) AGCCTCTCCCTGTCTCCGGGTAAA
GATATCGCGCTGACCCAGCCGGCGAGCGTGAGCGGTAGCCC
GGGCCAGAGCATTACCATTAGCTGCACCGGCACCAGCAGCG
ATGTGGGCTCTTACAACTACGTGTCTTGGTACCAGCAGCATC
CGGGCAAGGCGCCGAAACTGATGATCTACTACGTTTCTAAC
CGTCCGAGCGGCGTGAGCAACCGTTTTAGCGGATCCAAAAG
CGGCAACACCGCGAGCCTGACCATTAGCGGCCTGCAAGCGG
AAGACGAAGCGGATTATTACTGCGCTTCTTACACTCATCAG
GGTTCTTGGGTGTTTGGCGGCGGCACGAAGTTAACCGTCCT
AGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCC
CTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGT
GTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCC
TGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGA
LC DNA CCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCC
sequence AGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCA (SEQ ID CAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACC NO:209) GTGGAGAAGACAGTGGCCCCTACAGAATGTTCA
NOV0710 LCDR1
(Kabat)
RASQSISLWLN
(SEQ ID
Figure imgf000130_0001
TCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHE ALHNHYTQKSL SL SPGK
DIQMTQSPSSLSASVGDRVTITCRASQSISLWLNWYQQKPGKA
LC full PKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC sequence QQYYTSPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV (SEQ ID VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS NO:221) LSSTLTLSKADYEKHKVYACEVTHQGLSSPVT SFNRGEC
CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAACC
TGGCGGATCCCTGAGGCTGAGCTGCGCTGCTAGTGGCTTCA
CCTTCTCTAGCTACGCTATGAGCTGGGTCCGCCAGGCCCCTG
GTAAAGGCCTCGAGTGGGTGTCAGTGATTAGATCTAGCGGC
TCTAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCAC
TATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGA
TGAACTCCCTGAGGGCCGAGGACACCGCCGTCTACTACTGC
GCTAGAGGCGGAGGCTACTTCGACTACTGGGGTCAAGGCAC
CCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCAAGTG
TGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAA
CTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGC
CCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGC
GTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTA
CAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGG
GAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGC
AACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCG
ACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTG
CTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG
GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGT
GGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACC
AAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGG
TGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGC
AAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGC
CCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCA
CGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGA
GATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGG
GCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAAC
GGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCT
HC DNA GGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCG
sequence TGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGC (SEQ ID AGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA NO:222) GTCCCTGAGCCTGAGCCCCGGCAAG
GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCTAG
TGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGT
CTATTAGCCTGTGGCTGAACTGGTATCAGCAGAAGCCCGGT
AAAGCCCCTAAGCTGCTGATCTACGCCGCCTCTACCCTGCA
GTCAGGCGTGCCCTCTAGGTTTAGCGGTAGCGGTAGTGGCA
CCGACTTCACCCTGACTATCTCTAGCCTGCAGCCCGAGGACT
TCGCTACCTACTACTGTCAGCAGTACTACACTAGCCCCTACA
LC DNA CCTTCGGTCAGGGCACTAAGGTCGAGATTAAGCGTACGGTG
sequence GCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCA (SEQ ID GCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACA NO:223) ACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGAC
Figure imgf000132_0001
ACTACCTTCGGTCAGGGCACTAAGGTCGAGATTAAG
QVQLLESGGGLVQPGGSLRLSCAASGFTFSSHGMHWVRQAPG
KGLEWVSVISGSGSNTGYADSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARQWGSYAFDSWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQS SGLYSLS S WTVPS SSLGTQTYICNVNHKPSNTKVDK
RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQYNST
HC full YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG sequence QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN (SEQ ID GQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF SCS V NO:234) MHE ALHNHYTQKSL SL SPGK
DIQMTQSP S SL S AS VGDRVTITCRASQ SIS S YLNWYQQKPGKAP
LC full KLLIYAVSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ sequence QSGTFPPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO:235) S STLTL SKAD YEKHKVYACE VTHQGL S SP VTKSFNRGEC
CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCC
TGGCGGATCCCTGAGGCTGAGCTGCGCTGCTAGTGGCTTCA
CCTTTAGCTCTCACGGAATGCACTGGGTCCGCCAGGCCCCTG
GTAAAGGCCTCGAGTGGGTGTCAGTGATTAGCGGTAGCGGC
TCTAACACCGGCTACGCCGATAGCGTGAAGGGCCGGTTCAC
TATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGA
TGAACTCCCTGAGGGCCGAGGACACCGCCGTCTACTACTGC
GCTAGACAGTGGGGCTCCTACGCCTTCGATAGCTGGGGTCA
AGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCC
CAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCG
GCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTC
CCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGAC
TTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCG
GCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGC
TCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAA
GCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAG
AGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCC
AGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAA
GCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGA
CCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTG
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGC
CAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC
AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCT
GAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCC
CTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGG
GCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGC
CGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT
GGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGG
AGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
HC DNA CCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCA
sequence AGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGT (SEQ ID GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACT NO:236) ACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG
LC DNA GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCTAG
sequence TGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGT (SEQ ID CTATCTCTAGCTACCTGAACTGGTATCAGCAGAAGCCCGGT
Figure imgf000134_0001
AGCTCA
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACCTGC
GTTCTTACTACGTTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACGGTAACAACAAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
vL DNA CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA sequence GCGGATTATTACTGCGGTGTTTACACTCTGTCTTCTGTTGTG (SEQ ID TTTGGCGGCGGCACGAAGTTAACCGTCCTA
NO:247)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSP
SRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQL
NSVTPEDTAVYYCARGLVGRYGQPYHFDVWGQGTLVTVSSAS
T GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKP
SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
HC full EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK sequence TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA (SEQ ID VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NO:248) NVFSCSVMHEALHNHYTQKSLSLSPGK
DIELTQPPSVSVSPGQTASITCSGDNLRSYYVHWYQQKPGQAP
LC full VLVIYGNNKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYC sequence GVYTLS S WFGGGT LTVLGQPKAAPS VTLFPPS SEELQANKA (SEQ ID TLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA NO:249) ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACC
GAGCCAGACCCTGAGCCTGACCTGCGCGATTTCCGGAGATA
GCGTGAGCTCTAACTCTGCTGCTTGGAACTGGATTCGTCAGA
GCCCGAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTAC
CGTAGCAAATGGTACAACGACTATGCCGTGAGCGTGAAAAG
CCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTTA
GCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGTG
TATTATTGCGCGCGTGGTCTGGTTGGTCGTTACGGTCAGCCG
TACCATTTCGATGTTTGGGGCCAAGGCACCCTGGTGACTGTT
AGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCA
CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG
CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTA
CATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC
AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT
CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA
GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACC
HC DNA GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG
sequence CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA (SEQ ID CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG NO:250) TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCA
Figure imgf000136_0001
(SEQ ID STASGTWFGGGTKLTVL
NO:259)
CAGGTGCAATTGGTGGAAAGCGGCGGTGGCCTGGTGAAACC
AGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCTCCGGATTCA
CCTTTAACTCTTACGCTCTGCATTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTGGGCCGTATCAAATCTAAAACT
AACGGTGGTACTACTGACTATGCCGCCCCAGTGAAAGGCCG
CTTTACCATTAGCCGCGATGATTCGAAAAACACCCTGTATCT
GCAAATGAACAGCCTGAAAACCGAAGATACGGCCGTGTATT
vH DNA ATTGCGCGCGTGTTGACGCTACTTACTCTTACTCTGGTTACT sequence ACTACCCGATGGATTACTGGGGCCAAGGCACCCTGGTGACT (SEQ ID GTTAGCTCA
NO:260)
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAAAATCC
CGACTTACACTGTTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACGACGACAACAAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
vL DNA CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA sequence GCGGATTATTACTGCCAGTCTACTGCTTCTGGTACTGTTGTG (SEQ ID TTTGGCGGCGGCACGAAGTTAACCGTCCTA
NO:261)
QVQLVESGGGLVKPGGSLRLSCAASGFTFNSYALHWVRQAPG
KGLEWVGRIKSKTNGGTTDYAAPVKGRFTISRDDSKNTLYLQ
MNSLKTEDTAVYYCARVDATYSYSGYYYPMDYWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK
HC full T PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA sequence PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP (SEQ ID SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW NO:262) QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DIELTQPPSVSVSPGQTASITCSGDKIPTYTVHWYQQKPGQAPV
LC full LVIYDDNKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQ sequence STASGTWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKAT (SEQ ID LVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA NO:263) ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGGTGGAAAGCGGCGGTGGCCTGGTGAAACC
AGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCTCCGGATTCA
CCTTTAACTCTTACGCTCTGCATTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTGGGCCGTATCAAATCTAAAACT
AACGGTGGTACTACTGACTATGCCGCCCCAGTGAAAGGCCG
CTTTACCATTAGCCGCGATGATTCGAAAAACACCCTGTATCT
GCAAATGAACAGCCTGAAAACCGAAGATACGGCCGTGTATT
ATTGCGCGCGTGTTGACGCTACTTACTCTTACTCTGGTTACT
ACTACCCGATGGATTACTGGGGCCAAGGCACCCTGGTGACT
GTTAGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTG
GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCT
GGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
HC DNA TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
sequence TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC (SEQ ID AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGAC NO:264) CTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG
Figure imgf000138_0001
HCDR3
(Kabat)
(SEQ ID
GSYDMAFDV NO:271)
vH full QVQLLESGGGLVQPGGSLRLSCAASGFTFSSHWVHWVRQAPG sequence KGLEWVSVISYMGSSTYYADSVKGRFTISRDNSKNTLYLQMNS (SEQ ID LRAEDT AVYYC ARGS YDM AFD VWGQGTL VTVS S
NO:272)
vL full DIQMTQSP S SL S AS VGDRVTITCRASQ SI VS YLNWYQQKPGKAP sequence KLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ (SEQ ID QSGSHSITFGQGTKVEIK
NO:273)
CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCC
TGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCA
CCTTTAGCTCTCACTGGGTGCACTGGGTCAGACAGGCCCCTG
GTAAAGGCCTGGAGTGGGTGTCAGTGATTAGCTATATGGGC
TCTAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCAC
TATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGA
vH DNA TGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGC sequence GCTAGAGGCTCCTACGATATGGCCTTCGACGTGTGGGGTCA (SEQ ID GGGCACCCTGGTCACCGTGTCTAGC
NO:274)
GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCTAG TGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGT CTATCGTCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGT AAAGCCCCTAAGCTGCTGATCTACGACGCCTCTAGCCTGCA
vL DNA GTCAGGCGTGCCCTCTAGGTTTAGCGGTAGCGGTAGTGGCA sequence CCGACTTCACCCTGACTATTAGTAGCCTGCAGCCCGAGGAC (SEQ ID TTCGCTACCTACTACTGTCAGCAGTCAGGCTCTCACTCTATC NO:275) ACCTTCGGTCAGGGCACTAAGGTCGAGATTAAG
QVQLLESGGGLVQPGGSLRLSCAASGFTFSSHWVHWVRQAPG
KGLEWVSVISYMGSSTYYADSVKGRFTISRDNSKNTLYLQMNS
LRAEDTA VYYCARGSYDMAFD VWGQGTL VTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQS SGLYSLS S WTVPS SSLGTQTYICNVNHKPSNTKVDK
RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
WCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
HC full YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG sequence QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN (SEQ ID GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV NO:276) MHE ALHNHYTQKSL SL SPGK
DIQMTQSP S SL S AS VGDRVTITCRASQ SI VS YLNWYQQKPGKAP
LC full KLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ sequence QSGSHSITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC (SEQ ID LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS NO:277) STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCC
TGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCA
CCTTTAGCTCTCACTGGGTGCACTGGGTCAGACAGGCCCCTG
GTAAAGGCCTGGAGTGGGTGTCAGTGATTAGCTATATGGGC
HC DNA TCTAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCAC
sequence TATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGA (SEQ ID TGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGC NO:278) GCTAGAGGCTCCTACGATATGGCCTTCGACGTGTGGGGTCA GGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCC
CAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCG
GCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTC
CCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGAC
TTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCG
GCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGC
TCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAA
GCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAG
AGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCC
AGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAA
GCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGA
CCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTG
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGC
CAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC
AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCT
GAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCC
CTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGG
GCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGC
CGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT
GGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGG
AGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCA
AGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGT
GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACT
ACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG
GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCTAG
TGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGT
CTATCGTCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGT
AAAGCCCCTAAGCTGCTGATCTACGACGCCTCTAGCCTGCA
GTCAGGCGTGCCCTCTAGGTTTAGCGGTAGCGGTAGTGGCA
CCGACTTCACCCTGACTATTAGTAGCCTGCAGCCCGAGGAC
TTCGCTACCTACTACTGTCAGCAGTCAGGCTCTCACTCTATC
ACCTTCGGTCAGGGCACTAAGGTCGAGATTAAGCGTACGGT
GGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGC
AGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAAC
AACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGA
CAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACC
LC DNA GAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCA
sequence CCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTG (SEQ ID TACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGT NO:279) GACCAAGAGCTTCAACAGGGGCGAGTGC
NOV0720 LCDR1
(Kabat)
(SEQ ID
SGDNIGSMTAH NO:280)
LCDR2
(Kabat)
(SEQ ID
DKNERPS NO:281)
LCDR3
(Kabat)
(SEQ ID
QSWDDSYNSVV NO:282)
SNSAGWN
HCDR1 (Kabat)
(SEQ ID
NO:283)
HCDR2
(Kabat)
(SEQ ID
RIYYRSKWYNDYAVSVKS NO:284)
HCDR3
(Kabat)
(SEQ ID
EKYTVSFYDFFDY NO:285)
vH full QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAGWNWIRQSP sequence SRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQL (SEQ ID NSVTPEDTAVYYCAREKYTVSFYDFFDYWGQGTLVTVSS NO:286)
vL full DIELTQPPSVSVSPGQTASITCSGDNIGSMTAHWYQQKPGQAPV sequence LVIYDKNERPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQ (SEQ ID SWDDSYNSWFGGGTKLTVL
NO:287)
CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACC
GAGCCAGACCCTGAGCCTGACCTGCGCGATTTCCGGAGATA
GCGTGAGCTCTAACTCTGCTGGTTGGAACTGGATTCGTCAG
AGCCCGAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTA
CCGTAGCAAATGGTACAACGACTATGCCGTGAGCGTGAAAA
GCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTT
AGCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGT
vH DNA GTATTATTGCGCGCGTGAAAAATACACTGTTTCTTTCTACGA sequence CTTCTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAG (SEQ ID CTCA
NO:288)
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACATCG
GTTCTATGACTGCTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACGACAAAAACGAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
vL DNA CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA sequence GCGGATTATTACTGCCAGTCTTGGGACGACTCTTACAACTCT (SEQ ID GTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA
NO:289)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAGWNWIRQSP
SRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQL
NSVTPEDTAVYYCAREKYTVSFYDFFDYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFP A VLQ S SGL YSL S S WTVP S S SLGTQTYICN VNHKPS
NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
HC full EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI sequence SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV (SEQ ID EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN NO:290) VFSCSVMHEALHNHYTQKSLSLSPGK
LC full DIELTQPPSVSVSPGQTASITCSGDNIGSMTAHWYQQKPGQAPV sequence LVIYDKNERPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQ (SEQ ID SWDDSYNSVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANK NO:291) ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACC
GAGCCAGACCCTGAGCCTGACCTGCGCGATTTCCGGAGATA
GCGTGAGCTCTAACTCTGCTGGTTGGAACTGGATTCGTCAG
AGCCCGAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTA
CCGTAGCAAATGGTACAACGACTATGCCGTGAGCGTGAAAA
GCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTT
AGCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGT
GTATTATTGCGCGCGTGAAAAATACACTGTTTCTTTCTACGA
CTTCTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAG
CTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACC
CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT
GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA
TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG
TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT
CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA
ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
HC DNA TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG
sequence GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG (SEQ ID CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC NO:292) CGGGTAAA
GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCC
GGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACATCG
GTTCTATGACTGCTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACGACAAAAACGAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
CCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAA
GCGGATTATTACTGCCAGTCTTGGGACGACTCTTACAACTCT
GTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCA
GCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTC
TGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCA
TAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAG
GCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCA
LC DNA CACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAG
sequence CTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAA (SEQ ID GCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAG NO:293) AAGACAGTGGCCCCTACAGAATGTTCA
NOV1126 SGDAIGT FAH
LCDR1
Figure imgf000143_0001
HTFP A VLQ SSGLYSLSS WT VP S S SL GTQTYICN VNHKP SNTK V
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
SYELTQPLSVSVALGQTARITCSGDAIGTKFAHWYQQKPGQAP
LC full VLVIYYDHERPSGIPERFSGSNSGNTATLTISRAQAGDEADYYC sequence YSRASSNLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKA (SEQ ID TLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA NO:305) ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
GAAGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCC
GGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCA
CCTTTTCTGACCATGCTATCGACTGGGTGCGCCAGGCCCCGG
GCAAAGGTCTCGAGTGGGTTTCCGTTATCGCTGGTAGCGGTT
CTATCACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACC
ATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT
GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCG
CGCGTGACACTGGTGTTTACCGTGAATACATGGATGTTTGG
GGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAA
GGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT
ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC
TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC
ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG
TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
HC DNA CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC
sequence AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG (SEQ ID TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT NO:306) ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
AGCTATGAACTGACCCAGCCGCTGAGCGTGAGCGTGGCGCT
GGGCCAGACCGCGCGCATTACCTGTAGCGGCGATGCTATCG
GTACTAAATTCGCTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACTACGACCATGAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
LC DNA CCGCGACCCTGACCATTAGCCGCGCGCAGGCGGGCGACGAA
sequence GCGGATTATTACTGCTACTCTCGTGCTTCTTCTAACCTGGTG (SEQ ID TTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCAGCCCAA NO:307) GGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGA
Figure imgf000145_0001
CCGACTTCACCCTGACTATCTCTAGCCTGCAGCCCGAGGACT TCGCTACCTACTACTGTCAGCAGTACTACTCTACTAGCCTGA CCTTCGGTCAGGGCACTAAGGTCGAGATTAAG
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSP
SRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQL
NSVTPEDTAVYYCARERSYRDYFDYWGQGTLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO:318) CSVMHEALHNHYTQKSLSLSPGK
DIQMTQSPSSLSASVGDRVTITCRASQGIFTYLNWYQQKPGKA
LC full PKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC sequence QQYYSTSLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV (SEQ ID VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS NO:319) LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
CAGGTGCAGCTGCAGCAGTCAGGCCCTGGCCTGGTCAAGCC
TAGTCAGACCCTGAGCCTGACCTGCGCTATTAGCGGCGATA
GTGTGTCTAGTCAGTCAGCCGCCTGGAACTGGATTAGACAG
TCACCCTCTAGGGGCCTGGAGTGGCTGGGTAGAATCTACTA
TAGGTCTAAGTGGTATAACGACTACGCCGTCAGCGTGAAGT
CTAGGATCACTATTAACCCCGACACCTCTAAGAATCAGTTTA
GCCTGCAGCTGAATAGCGTGACCCCCGAGGACACCGCCGTC
TACTACTGCGCTAGAGAGCGGTCCTATAGAGACTACTTCGA
CTACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCGCTA
GCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGC
AAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTG
AAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTC
TGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCT
GCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAG
TGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACG
TGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
GGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCT
GCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGT
TCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACC
CCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGA
CCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG
TGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAA
CAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACC
AGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCC
AACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCA
AGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTG
CCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCT
GACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCG
TGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAA
GACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCC
HC DNA TGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAG
sequence GGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCA (SEQ ID CAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCA NO:320) AG
Figure imgf000147_0001
AGCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGT GTATTATTGCGCGCGTGAAAAATACACTGTTTCTTTCTACGA CTTCTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAG CTCA
AGCTATGAACTGACCCAGCCGCTGAGCGTGAGCGTGGCGCT
GGGCCAGACCGCGCGCATTACCTGTAGCGGCGATAACATCG
GTTCTATGACTGCTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACGACAAAAACGAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
vL DNA CCGCGACCCTGACCATTAGCCGCGCGCAGGCGGGCGACGAA sequence GCGGATTATTACTGCCAGTCTTGGGACGACTCTTACACCTCT (SEQ ID GTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA
NO:331)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAGWNWIRQSP
SRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQL
NSVTPEDTAVYYCAREKYTVSFYDFFDYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFP A VLQ S SGL YSL S S WTVP S S SLGTQTYICN VNHKPS
NT VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
HC full EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI sequence SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV (SEQ ID EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN NO:332) VFSCSVMHEALHNHYTQKSLSLSPGK
SYELTQPLSVSVALGQTARITCSGDNIGSMTAHWYQQKPGQAP
LC full VLVIYDKNERPSGIPERFSGSNSGNTATLTISRAQAGDEADYYC sequence QSWDDSYTSWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQAN (SEQ ID KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNK NO:333) YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
CAGGTGCAATTGCAGCAGAGCGGTCCGGGCCTGGTGAAACC
GAGCCAGACCCTGAGCCTGACCTGCGCGATTTCCGGAGATA
GCGTGAGCTCTCAGTCTGCTGGTTGGAACTGGATTCGTCAG
AGCCCGAGCCGTGGCCTCGAGTGGCTGGGCCGTATCTACTA
CCGTAGCAAATGGTACAACGACTATGCCGTGAGCGTGAAAA
GCCGCATTACCATTAACCCGGATACTTCGAAAAACCAGTTT
AGCCTGCAACTGAACAGCGTGACCCCGGAAGATACGGCCGT
GTATTATTGCGCGCGTGAAAAATACACTGTTTCTTTCTACGA
CTTCTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAG
CTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACC
CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT
GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA
TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG
TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT
CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
HC DNA CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG
sequence CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG (SEQ ID CAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGT NO:334) CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA
ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG
GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC
CGGGTAAA
AGCTATGAACTGACCCAGCCGCTGAGCGTGAGCGTGGCGCT
GGGCCAGACCGCGCGCATTACCTGTAGCGGCGATAACATCG
GTTCTATGACTGCTCATTGGTACCAGCAGAAACCGGGCCAG
GCGCCGGTGCTGGTGATCTACGACAAAAACGAACGTCCGAG
CGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACA
CCGCGACCCTGACCATTAGCCGCGCGCAGGCGGGCGACGAA
GCGGATTATTACTGCCAGTCTTGGGACGACTCTTACACCTCT
GTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCA
GCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTC
TGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCA
TAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAG
GCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCA
LC DNA CACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAG
sequence CTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAA (SEQ ID GCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAG NO:335) AAGACAGTGGCCCCTACAGAATGTTCA
Example 2: GITR antibody isolation
[00333] Human antibodies GITR.MAB2, GITR.MAB3, GITR.MAB4, GITR.MAB5,
GITR.MAB6, GITR.MAB7 and GITR.MAB8 were generated by engineering a murine monoclonal GITR agonist antibody GITR.MAB 1 to have greater sequence homology to a human germline antibody. GITR.MAB2, GITR.MAB3, GITR.MAB4, GITR.MAB5, GITR.MAB6, GITR.MAB7 and GITR.MAB8 retain the epitope specificity, affinity, and cynomolgus macaque GITR cross-reactivity of the parental murine antibody, GITR.MAB 1. GITR.MAB2, GITR.MAB3, GITR.MAB4, GITR.MAB 5, GITR.MAB6, GITR.MAB7 and GITR.MAB8 have much higher homology to the human germline sequence than the original murine antibody and should therefore be better tolerated by the human immune system.
[00334] Mouse monoclonal MAB 1 was engineered to bring its protein sequence closer to a human germline sequence and decrease its immunogenicity using the Humaneered® technology platform available through KaloBios (South San Francisco, CA). Humaneered® antibodies are very close to human antibodies with V-region sequences that have high homology to a human germline sequence while still retaining the specificity and affinity of the parent or reference antibody (U.S. Patent Publ. 2005/0255552 and 2006/0134098). The process first identifies the minimum antigen binding specificity determinants (BSDs) in heavy and light chain variable regions of a reference Fab (typically sequences within the heavy chain CDR3 and the light chain CDR3). As these heavy and light chain BSDs are maintained in all libraries constructed during the process, each library is epitope- focused, and the resulting Humaneered® antibodies retain the epitope specificity of the original mouse antibody.
[00335] Next, full chain libraries (in which an entire light or heavy chain variable region is replaced with a library of human sequences) and/or cassette libraries (in which a portion of the heavy or light chain variable region of the mouse Fab is replaced with a library of human sequences) are generated. A bacterial secretion system is used to express members of the library as antibody Fab fragments, and the library is screened for Fabs that bind antigen using a colony lift binding assay (CLBA). Positive clones are further characterized to identify those with the highest affinity.
Identified human cassettes supporting binding in the context of residual murine sequences are the combined in a final library screen to generate completely human V-regions.
[00336] Resulting Humaneered® antibody Fabs have V-segment sequences derived from human libraries, retain the short BSD sequences identified within the CDR3 regions, and have human germline Framework 4 regions. These Fabs are converted to full IgGs by cloning variable regions of the heavy and light chains into IgG expression vectors. Humaneered® antibodies generated in this process retain the binding specificity of the parent, murine antibody, typically having equivalent or higher affinity for antigen that the parent antibody, and have V-regions with a high degree of sequence identity compared with human germline antibody genes at the protein level.
[00337] Bcl-2 transgenic mice (C57BL/6-Tgn (bcl-2) 22 wehi strain) were immunized with the
N-terminal region of human GITR (aa 26-161) using a procedure that calls for Repetitive
Immunization at Multiple Sites (RIMMS) (Mclntyre GD. Hybridoma 1997) followed by hybridoma generation from high titer mice. A hybridoma secreting MAB 1 was identified and selected using a sandwich ELISA against hGITR and an NF B Reporter Gene Assay to confirm hGITR binding and agonist activity.
Human GITR (SEQ ID NO:336) aa 26-161 is bolded and underlined
MAOHGAMGAFRALCGLALLCALSLGORPTGGPGCGPGRLLLGTGTDARCCRVHTTRCC RDYPGEECCSEWDCMCVOPEFHCGDPCCTTCRHHPCPPGOGVOSOGKFSFGFOCIDCA SGTFSGGHEGHCKPWTDCTOFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTWLLAVAA
CVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGD LWV
[00338] Variable region DNA from murine monoclonal MAB 1 was amplified by RT-PCR from RNA obtained from the hybridoma cell line using standard methods. Heavy chain variable region was amplified from MAB 1 cDNA with
HV3 (5'- GGGTCTAGACACCATGGCTGTCTTGGGGCTGCTCTTC-3' (SEQ ID NO: 337)) and HCconstant (5 ' -GCGTCT AGA AYCTCC AC AC AC AGGRRCC AGTGGAT AGAC-3 ' (SEQ ID NO: 338)). Light chain variable region was amplified from the same cDNA with LV3 (5 ' -GGGTCT AG AC ACC ATGGAGWC AC AKWCTC AGGTCTTTRTA-3 ' (SEQ ID NO: 339)) and LCconstant (5'- GCGTCTAGAACTGGATGGTGGGAAGATGG-3 ' (SEQ ID NO: 340)).
Variable heavy and light chain products were inserted into a pcDNA3.1 vector and sequence verified. The heavy and light vectors were used as templates for PCR incorporating restriction enzyme sites for cloning into KaloBios vectors: Vh into KB 1292-His (modified version of KB 1292 that encodes a C- terminal flexible linker and 6-His tag of amino acid sequence AAGASHHHHHH (SEQ ID NO:341) on CHI) at Ncol (5') and el (3 '); Vk into KB 1296 at Ncol (5') and BsiWl (3 '). These separate heavy and light chain vectors were then combined into a single dicistronic KaloBios Fab expression vector by restriction digest with BssHll and CM and ligation. Fab fragments were expressed in E. coli from this vector. This Fab was tested for hGITR-antigen binding and is referred to as MAB IrFab.
[00339] Fab fragments were expressed by secretion from E. coli using KaloBios expression vectors. Cells were grown in 2 x YT medium to an OD500 of -0.6. Expression was induced by adding IPTG to 100 μΜ and shaking for 4 hours at 33°C. Assembled Fab was obtained from periplasmic fractions by osmotic lysis and purification by affinity chromatography using Ni-NTA columns HisTrap HP columns; GE Healthcare catalog # 17- 5247-01) according to standard methods. Fabs were eluted in buffer containing 500 mM imidazole and thoroughly dialyzed against PBS pH7.4 without calcium and magnesium.
[00340] To limit the complexity to identify complimentary human CDRs that support
BSD-FR4 in human GITR binding, a cassette library approach, in which only part of the parent murine V-segment is replaced by a library of human sequences, was taken. The original murine MAB 1 Vk is closest to human germline Vklll, so a mixture of two KaloBios libraries that contains Vklll germlines (KB 1423 and KB 1424) was used in making the Vk cassette libraries. KaloBios libraries that contains VH3 germlines (KB 1413, KB 1414) were used to construct Vh cassette libraries. Two types of cassettes were constructed by overlap PCR: front-end cassettes (8C 1VK3FE-01, and MAB 1VH3FE-01) containing human sequences in FR1, CDR1, and FR2, and FR3 cassettes (MAB 1 VK3FR3 -01 , and
MAB 1VH3FR3-01) containing human sequences in the FR3 were amplified using the above mentioned germline restricted KaloBios libraries. Each Vh cassette library was cloned into vector KB 1292-His at Ncol (5 ') and Kpnl (3 '); each Vk cassette library was cloned into vector KB 1296-B (modified version of KaloBios vector KB 1296 which has a silent Hindlll site added in FR4) at Ncol (5 ') and Hindlll (3 '). Resultant Vh or Vk plasmid libraries were then combined with the complementary chain from the optimized reference Fab (MAB lopVK or MAB l opVH (e.g., the Vh front-end library was combined with the optimized reference Vk vector) by digestion with ZfasHII and CM and subsequent ligation to create libraries of dicistronic vectors expressing full Fabs.
Table 6
Sequence information of the IgGs selected for in-depth characterization.
Figure imgf000152_0001
Figure imgf000153_0001
TTGCAGTGTACTACTGCGGCCAGAGCTATAGCTATCCATTTA CCTTTGGCCAGGGCACCAAGCTTGAAATTAAG
QVQLVESGGGLVQPGGSLRLSCAASGFSLSSYGVDWVRQAPG
KGLEWVGVIWGGGGTYYASSVMARFTISRDNSKNTLYLQMNS
LRAEDTAVYYCAKHAYGHDGGFAMDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEWCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 362) CSVMHEALHNHYTQKSLSLSPGK
EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA
LC full PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC sequence GQSYSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 363) S STLTL SKAD YEKHKVYACE VTHQGLS SP VTKSFNRGEC
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTC
CCTCAGCAGCTATGGTGTGGACTGGGTTCGCCAGGCTCCAG
GAAAGGGTCTGGAGTGGGTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATGCTTCTTCTGTCATGGCCAGATTCACCATC
TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA
CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA
AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT
GGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACC
AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG
CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGG
ACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGA
GCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCC
CAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGA
ACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGA
GCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCC
CAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCC
CCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCC
AGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAG
CACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGG
ACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAAC
AAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGC
CAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCC
CCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACC
TGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACC
HC DNA ACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTA
sequence CAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGC (SEQ ID AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAA NO: 364) CCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG
LC DNA GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCT
sequence CCAGGAGAAAGAGCCACCCTCTCCTGCAGGGCCAGTGAGAG
Figure imgf000155_0001
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCT
CCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAG
TGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCC
AGGCTCCCAGGCTCCTCATCTACGGGGCATCCAACCGGGCC
ACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC
vL DNA AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT sequence TTGCAGTTTACTACTGCGGCCAGAGCTATAGCTATCCATTTA (SEQ ID CCTTTGGCCAGGGCACCAAGCTTGAAATTAAA
NO: 375)
QVQLVESGGGLVQPGGSLRLSCAASGFSLSSYGVDWVRQAPG
KGLEWLGVIWGGGGTYYTASLMGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCAKHAYGHDGGFAMDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEWCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 376) CSVMHEALHNHYTQKSLSLSPGK
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQA
LC full PRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC sequence GQSYSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 377) S STLTL SKAD YEKHKVYACE VTHQGLS SP VTKSFNRGEC
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTC
CCTCAGCAGCTATGGTGTGGACTGGGTTCGCCAGGCTCCAG
GAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATACTGCTTCTCTCATGGGCAGATTCACCATC
TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA
CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA
AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT
GGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACC
AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG
CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGG
ACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGA
GCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCC
CAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGA
ACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGA
GCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCC
CAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCC
CCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCC
AGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAG
CACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGG
ACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAAC
AAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGC
HC DNA CAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCC
sequence CCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACC (SEQ ID TGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA NO: 378) GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACC
Figure imgf000157_0001
NO: 388) GAAAGGGTCTGGAGTGGGTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATGCTTCTTCTCTCATGGGCAGATTCACCATC
TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA
CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA
AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT
GGGGCCAGGGTACCCTTGTGACCGTGAGCTCA
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCT
CCAGGAGAAAGAGCCACCCTCTCCTGCAGGGCCAGTGAGAG
TGTTAGCAGTAATGTAGCCTGGTACCAGCAGAGACCTGGCC
AGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGGGCC
vL DNA ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGAC sequence AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT (SEQ ID TTGCAGTGTACTACTGCGGCCAGAGCTATAGCTATCCATTTA NO: 389) CCTTTGGCCAGGGCACCAAGCTTGAAATTAAG
EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG
KGLEWVGVIWGGGGTYYASSLMGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCAKHAYGHDGGFAMDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEWCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 390) CSVMHEALHNHYTQKSLSLSPGK
EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA
LC full PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC sequence GQSYSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 391) S STLTL SKAD YEKHKVYACE VTHQGLS SP VTKSFNRGEC
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTC
CCTCAGCAGCTATGGTGTGGACTGGGTTCGCCAGGCTCCAG
GAAAGGGTCTGGAGTGGGTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATGCTTCTTCTCTCATGGGCAGATTCACCATC
TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA
CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA
AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT
GGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACC
AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG
CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGG
ACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGA
GCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCC
CAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGA
ACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGA
GCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCC
CAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCC
CCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCC
HC DNA AGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
sequence ACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAG (SEQ ID CACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGG NO: 392) ACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAAC
Figure imgf000159_0001
sequence PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID GQSYSYPFTFGQGTKLEIK
NO: 401)
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTC
CCTCAGCAGCTATGGTGTGGACTGGGTTCGCCAGGCTCCAG
GAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATACTTCTTCTCTCATGGGCAGATTCACCATC
vH DNA TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA sequence CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA (SEQ ID AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT NO: 402) GGGGCCAGGGTACCCTTGTGACCGTGAGCTCA
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCT
CCAGGAGAAAGAGCCACCCTCTCCTGCAGGGCCAGTGAGAG
TGTTAGCAGTAATGTAGCCTGGTACCAGCAGAGACCTGGCC
AGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGGGCC
vL DNA ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGAC sequence AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT (SEQ ID TTGCAGTGTACTACTGCGGCCAGAGCTATAGCTATCCATTTA NO: 403) CCTTTGGCCAGGGCACCAAGCTTGAAATTAAG
EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG
KGLEWLGVIWGGGGTYYTSSLMGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCAKHAYGHDGGFAMDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEWCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 404) CSVMHEALHNHYTQKSLSLSPGK
EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA
LC full PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC sequence GQSYSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 405) S STLTL SKAD YEKHKVYACE VTHQGLS SP VTKSFNRGEC
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTC
CCTCAGCAGCTATGGTGTGGACTGGGTTCGCCAGGCTCCAG
GAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATACTTCTTCTCTCATGGGCAGATTCACCATC
TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA
CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA
AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT
GGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACC
AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG
CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGG
ACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGA
GCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAG
AGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCC
HC DNA CAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGA
sequence ACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGA (SEQ ID GCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCC NO: 406) CAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCC
Figure imgf000161_0001
NO: 413)
vH full
sequence EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG (SEQ ID KGLEWLGVIWGGGGTYYTSSLMARFTISRDNSKNTLYLQMNS NO: 414) LRAEDTAVYYCAKHAYGHDGGFAMDYWGQGTLVTVSS vL full
sequence EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA (SEQ ID PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC NO: 415) GQSYSYPFTFGQGTKLEIK
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTC
CCTCAGCAGCTATGGTGTGGACTGGGTTCGCCAGGCTCCAG
GAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATACTTCTTCTCTCATGGCCAGATTCACCATC
vH DNA TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA sequence CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA (SEQ ID AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT NO: 416) GGGGCCAGGGTACCCTTGTGACCGTGAGCTCA
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTTTCT
CCAGGAGAAAGAGCCACCCTCTCCTGCAGGGCCAGTGAGAG
TGTTAGCAGTAATGTAGCCTGGTACCAGCAGAGACCTGGCC
AGGCACCCAGGCTCCTCATCTACGGGGCATCCAACCGGGCC
vL DNA ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGAC sequence AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT (SEQ ID TTGCAGTGTACTACTGCGGCCAGAGCTATAGCTATCCATTTA NO: 417) CCTTTGGCCAGGGCACCAAGCTTGAAATTAAG
EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG
KGLEWLGVIWGGGGTYYTSSLMARFTISRDNSKNTLYLQMNS
LRAEDTAVYYCAKHAYGHDGGFAMDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEWCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 418) CSVMHEALHNHYTQKSLSLSPGK
EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA
LC full PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC sequence GQSYSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 419) S STLTL SKAD YEKHKVYACE VTHQGLS SP VTKSFNRGEC
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGTC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTC
CCTCAGCAGCTATGGTGTGGACTGGGTTCGCCAGGCTCCAG
GAAAGGGTCTGGAGTGGCTGGGAGTTATATGGGGTGGTGGA
GGCACATATTATACTTCTTCTCTCATGGCCAGATTCACCATC
TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA
CAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCGCCA
AACATGCCTATGGCCATGATGGCGGCTTTGCTATGGATTATT
HC DNA GGGGCCAGGGTACCCTTGTGACCGTGAGCTCAGCTAGCACC
sequence AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG (SEQ ID CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGG NO: 420) ACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGA
Figure imgf000163_0001
(Kabat)
(SEQ ID
NO: 426)
HCDR3
(Kabat)
(SEQ ID
HAYGHDGGFAMDY NO: 427)
vH full
sequence EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG (SEQ ID KGLEWVGVIWGGGGTYYASSLMGRFTISRDNSKNTLYLQMNS NO: 428) LRAEDTAVYYCARHAYGHDGGF AMDYWGQGTLVTVS S vL full EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA sequence PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID GQSYSYPFTFGQGTKLEIK
NO: 429)
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGTC
CGGCGGCTCTCTGAGACTGTCTTGCGCTGCCTCCGGCTTCTC
CCTGTCCTCTTACGGCGTGGACTGGGTGCGACAGGCCCCTG
GCAAGGGCCTGGAATGGGTGGGAGTGATCTGGGGCGGAGG
CGGCACCTACTACGCCTCTTCCCTGATGGGCCGGTTCACCAT
vH DNA CTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA sequence ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCC (SEQ ID AGACACGCCTACGGCCACGACGGCGGCTTCGCCATGGATTA NO: 430) TTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC
GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCT
CCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCCTCCGAGTC
CGTGTCCTCCAACGTGGCCTGGTATCAGCAGAGACCTGGTC
AGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGGGCC
vL DNA ACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCAC sequence CGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACT (SEQ ID TCGCCGTGTACTACTGCGGCCAGTCCTACTCATACCCCTTCA NO: 431) CCTTCGGCCAGGGCACCAAGCTGGAAATCAAG
EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG
KGLEWVGVIWGGGGTYYASSLMGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARHAYGHDGGF AMD YWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEWCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 432) CSVMHEALHNHYTQKSLSLSPGK
EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA
LC full PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC sequence GQSYSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 433) S STLTL SKAD YEKHKVYACE VTHQGLS SP VTKSFNRGEC
GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGTC CGGCGGCTCTCTGAGACTGTCTTGCGCTGCCTCCGGCTTCTC
HC DNA CCTGTCCTCTTACGGCGTGGACTGGGTGCGACAGGCCCCTG
sequence GCAAGGGCCTGGAATGGGTGGGAGTGATCTGGGGCGGAGG (SEQ ID CGGCACCTACTACGCCTCTTCCCTGATGGGCCGGTTCACCAT NO: 434) CTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCC
AGACACGCCTACGGCCACGACGGCGGCTTCGCCATGGATTA
TTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCA
CCAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAG
TCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAG
GACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG
GGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCA
GAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGC
CCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGG
AGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGC
CCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTC
CCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCC
CGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACC
CAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACA
GCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAG
GACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAA
CAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG
GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCC
CCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGA
CCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTG
GAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGA
CCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGT
ACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGG
CAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACA
ACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG
GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCT
CCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCCTCCGAGTC
CGTGTCCTCCAACGTGGCCTGGTATCAGCAGAGACCTGGTC
AGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGGGCC
ACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCAC
CGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACT
TCGCCGTGTACTACTGCGGCCAGTCCTACTCATACCCCTTCA
CCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTG
GCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCA
GCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACA
ACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGAC
AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCG
LC DNA AGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACC
sequence CTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTA (SEQ ID CGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGA NO: 435) CCAAGAGCTTCAACAGGGGCGAGTGC
GITR.MAB8 LCDR1
(Kabat)
(SEQ ID
RASES VSSNVA NO: 436)
LCDR2
(Kabat)
(SEQ ID
GASNRAT NO: 437)
LCDR3
(Kabat)
GQSYSYPFT
(SEQ ID NO: 438)
HCDR1
(Kabat)
(SEQ ID
SYGVD NO: 439)
HCDR2
(Kabat)
(SEQ ID
VIWGGGGTYY AS SLMG NO: 440)
HCDR3
(Kabat)
(SEQ ID
NAYGHDGGFAMDY NO: 441)
vH full
sequence EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG (SEQ ID KGLEWVGVIWGGGGTYYASSLMGRFTISRDNSKNTLYLQMNS NO: 442) LRAEDTAVYYCARNAYGHDGGF AMDYWGQGTLVTVS S vL full
sequence EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA (SEQ ID PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC NO: 443) GQSYSYPFTFGQGTKLEIK
GAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTGCAGTC
AGGCGGTAGCCTGAGACTGAGCTGCGCCGCCTCCGGCTTTA
GCCTGTCTAGCTACGGCGTGGACTGGGTCCGACAGGCCCCT
GGCAAAGGCCTGGAGTGGGTCGGAGTGATCTGGGGCGGAG
GCGGAACCTACTACGCCTCTAGCCTGATGGGCCGGTTCACT
vH DNA ATCTCTAGGGACAACTCTAAGAACACCCTGTACCTGCAGAT sequence GAACTCACTGAGAGCCGAGGACACCGCCGTCTACTACTGCG (SEQ ID CTAGAAACGCCTACGGTCACGACGGCGGCTTCGCTATGGAC NO: 444) TACTGGGGTCAGGGCACCCTGGTCACCGTGAGTTCA
GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCT
CCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCCTCCGAGTC
CGTGTCCTCCAACGTGGCCTGGTATCAGCAGAGACCTGGTC
AGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGGGCC
vL DNA ACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCAC sequence CGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACT (SEQ ID TCGCCGTGTACTACTGCGGCCAGTCCTACTCATACCCCTTCA NO: 445) CCTTCGGCCAGGGCACCAAGCTGGAAATCAAG
EVQLVESGGGLVQSGGSLRLSCAASGFSLSSYGVDWVRQAPG
KGLEWVGVIWGGGGTYYASSLMGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARNAYGHDGGF AMD YWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEWCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
HC full YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequence AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID ESNGQPENNYKTTPP VLD SD GSFFLYSKLTVDKSRWQQGNVF S NO: 446) CSVMHEALHNHYTQKSLSLSPGK
EIVMTQSPATLSVSPGERATLSCRASESVSSNVAWYQQRPGQA
LC full PRLLIYGASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYC sequence GQSYSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW (SEQ ID CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL NO: 447) S STLTL SKAD YEKHKVYACE VTHQGLS SP VTKSFNRGEC GAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTGCAGTC
AGGCGGTAGCCTGAGACTGAGCTGCGCCGCCTCCGGCTTTA
GCCTGTCTAGCTACGGCGTGGACTGGGTCCGACAGGCCCCT
GGCAAAGGCCTGGAGTGGGTCGGAGTGATCTGGGGCGGAG
GCGGAACCTACTACGCCTCTAGCCTGATGGGCCGGTTCACT
ATCTCTAGGGACAACTCTAAGAACACCCTGTACCTGCAGAT
GAACTCACTGAGAGCCGAGGACACCGCCGTCTACTACTGCG
CTAGAAACGCCTACGGTCACGACGGCGGCTTCGCTATGGAC
TACTGGGGTCAGGGCACCCTGGTCACCGTGAGTTCAGCTAG
CACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCA
AGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGA
AGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCT
GGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTG
CAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGT
GCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGT
GAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTG
GAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTG
CCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTT
CCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCC
CCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGAC
CCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAAC
AGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCA
GGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCA
ACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAA
GGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGC
CCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG
ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGT
GGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG
ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCT
HC DNA GTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAG
sequence GGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCA (SEQ ID CAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCA NO: 448) AG
GAGATCGTGATGACCCAGTCCCCCGCCACCCTGTCTGTGTCT
CCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCCTCCGAGTC
CGTGTCCTCCAACGTGGCCTGGTATCAGCAGAGACCTGGTC
AGGCCCCTCGGCTGCTGATCTACGGCGCCTCTAACCGGGCC
ACCGGCATCCCTGCCAGATTCTCCGGCTCCGGCAGCGGCAC
CGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACT
TCGCCGTGTACTACTGCGGCCAGTCCTACTCATACCCCTTCA
CCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTG
GCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCA
GCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACA
ACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGAC
AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCG
LC DNA AGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACC
sequence CTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTA (SEQ ID CGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGA NO: 449) CCAAGAGCTTCAACAGGGGCGAGTGC Example 3: In vivo efficacy of anti-CDH6 ADCs as a single agent in a xenograft mouse model of ovarian cancer
[00341] The anti-tumor activity of a selection of anti-CDH6 ADCs were evaluated in the
OVCAR-3 ovarian xenograft model. Female NOD-scid-gamma mice were implanted subcutaneously on the right flank with lOxlO6 OVCAR-3 cells containing 50% Matrigel™ (BD Biosciences, San Jose, CA) in PBS. The total injection volume containing cells in suspension was 200 μΐ. Mice were enrolled in the study 21 days post implantation with average tumor volume of 174 mm3. After being randomly assigned to one of nine groups (n = 5/group), mice were administered a single i.v. dose of PBS, (10 ml/kg), a non-target isotype control hIgGl-SPDB-DM4 (5 mg/kg), a non-target isotype control hlgGl-SMCC-DMl (5 mg/kg), NOV0712-SMCC-DM1 (5 mg/kg), or one of seven anti- CDH6-SPDB-DM4 (5 mg/kg). Tumor volumes and body weights were measured 1-2 times weekly. None of the mice showed a decrease in body weight, indicating that the ADC was well tolerated at this dosage (data not shown). Of all the anti-CDH6-SPDB-DM4 agents, NOV0712-SPDB-DM4 elicited the greatest anti-tumor effect with regression 70 days post dose. NOV0710-SPDB-DM4 was the second most active anti-CDH6-SPDB-DM4 in this study (Figure 1).
Example 4: GITR antibodies have anti-tumor activity as a single agent
[00342] hGITR knock-in mice were generated by replacing the entire coding sequence (exons and introns) of mouse GITR with the human GITR cDNA sequence. Untranslated sequences upstream of the start codon and downstream of the stop codon are from mouse genome. Gene targeting was done by standard techniques in B ALB/c ES cells with targeting vectors bearing B ALB/c derived homology arms. Several ES cell clones were identified by PCR and confirmed by Southern blotting to contain the exact human cDNA knock in. Following standard mouse embryology techniques, positive ES cell clones were injected into blastocysts, which were transferred into pseudopregnant recipient foster mothers to derive chimeric offspring. Male chimeric mice were crossed with BALB/c females expressing Cre recombinase in their germline to excise the loxP flanked neomycin resistance cassette. One clone resulted in white offspring indicating germline transmission of the targeted ES cells.
Excision of the loxP-flanked cassette was confirmed by PCR genotyping. A subsequent breeding step with BALB/c wt mice removed the Cre recombinase.
[00343] hGITRL knock-in mice were generated by replacing mouse the coding portion of exon 1 with the human GITRL cDNA sequence followed by a bovine growth hormone poly -A signal. All ES cell work and mouse embryology were done similar to the procedures described
above. hGITR-hGITRL double knock-in mice were generated by intercrossing the two founder lines for 2 generations to produce homozygous double knock-in mice. [00344] The Colon26 murine colon carcinoma cell line was obtained from the Division of
Cancer Treatment and Diagnosis at the National Cancer Institute (vial: 0507238). Murine Colon26 carcinoma cells were cultured in RPMI 1640 medium (HyClone SH30027.02) supplemented with 10% FBS (Gibco 10099-141), lOmM HEPES (Gibco 15630-080) and lmM sodium pyruvate (Gibco 11360-070). 8-10 week old female hGITR-hGITRL knock-in mice were injected subcutaneously with 0.5xl06 Colon26 cells in lOOuL of RPMI in the flank. Tumors were measured using digital calipers and tumor volume calculated using the equation (L x W2)/2. When tumors reached an average size of 180mm3, mice were randomized and dosed with a single intraperitoneal injection of vehicle (PBS) or therapeutic antibody (15mg/kg) in 200μΙ. PBS. Mice were sacrificed and tumors collected for analysis by flow cytometry 7 days after dosing with therapeutic antibodies. All animal experiments were performed in an AAALAC accredited facility using IACUC approved protocols. Statistical analysis was performed in Prism software using student's t-test with two-tailed 95% confidence interval or One-way ANOVA with Tukey correction.
[00345] hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or GITR.MAB7 (n=10/timepoint) antibody. Figure 2 depicts results of tumor measurements twice per week and tumor volume calculated using the equation (LxW2)/2. Data shown is from the fifteen (15)-day time point group. This demonstrates the anti-GITR antibodies have anti-tumor activity as a single agent.
Example 5: Generating the RENCAmCDH6 Cell line
[00346] Murine RENCA kidney adenocarcinoma cells (CRL-2947) were purchased from
ATCC and transduced with a lentiviral construct delivering murine CDH6 (obtained from Transomics and cloned into pLenti6.3). Cells expressing CDH6 were selected by two rounds of fluorescence activated cell sorting using a PE-conjugated CDH6 antibody (R&D Systems #FAB2715) and isolated into distinct cell lines. The FACs plots and an indication of tumor growth in mice are shown in Figure 3. After treatment with the CDH6 ADC for ten days, the number of Tregs associated with the tumor increase, and this is shown in Figure 4.
Example 6: RENCAmCDH6 Clone5F5 Efficacy study
[00347] The anti-tumor activity of the combination of CDH6-sulfo-SPDB-DM4 and the mouse surrogate agonistic anti-GITR IgG2a antibody niDTA-1 (Bulliard et al., J.Exp.Med. 2013;
210(9): 1685-1693) was evaluated in the RENCAmCDH6 Clone 5F5 xenograft model. This cell line model had been engineered from the syngeneic RENCA cell line to express murine CDH6, and had been validated as tumorigenic and sensitive to CDH6-sulfo-SPDB-DM4 treatment in a previous experiment. Female BALB/cJ mice were implanted subcutaneously on the right flank with 5xl05 RENCAmCDH6 clone5F5 cells Hanks Balanced salt solution (HBSS). The total injection volume containing cells in suspension was 100 μΐ. At 15 days post implantation mice were randomized into to one of eight groups of equal mean tumor volume of 176 mm3 (n = 10/group), mice were administered a single i.v. dose of either CDH6-sulfo-SPDB-DM4 5 mg/kg or a non-target isotype control hlgGl- sulfo-SPDB-DM4 5 mg/kg, in combination with either mDTA-1 5mg/kg or a non-target isotype control mIgG2a. The administration regimen was 5 mg/kg for the initial timepoint, then 3 days later or 6 days later. Tumor volumes and body weights were measured at least once weekly. In addition to tumor growth kinetics up to day 39, a time to endpoint (TTE) data are plotted in Figure 5. The endpoint employed was 1000mm3, once tumors surpassed this volume they were removed from study. Across the CDH6-sulfo-SPDB-DM4 and mDTA-1 treated groups, 5 mice were tumor free up to 112 days post tumor implant (Figure 6). This data indicated a significant survival benefit of the combination of CDH6-sulfo-SPDB-DM4 and GITR agonist antibody when compared to the combination of CDH6-sulfo-SPDB-DM4 with the mIgG2a control, ( p = 0.0061 Mantel-Cox test).
Example 7: RENCAmCDH6Clone5F5 re-challenge study
[00348] On day 112 a second implant of 5xl05 RENCAmCDH6 clone5F5 cells was administered as a re-challenge alongside a cohort of ten naive BALB/cJ control mice. In this experiment tumors and body weights were measured at least once weekly. There was no further administration of anti-CDH6 or anti-GITR agents to either the re-challenged or the control mice. All ten control mice grew tumors whereas none of the re-challenged mice grew palpable tumors up to day 51 post re-challenge. This data is shown with the mouse cohort, plotted as mean tumor volume over time in Figure 7, with individual tumor growth plotted in Figure 8. Five of these mice were euthanized alongside a new cohort of non-tumor-bearing BALB/cJ mice to provide control tissue. The same 1000mm3 endpoint was employed, TTE data are plotted illustrating the significant difference between the groups (p = 0.0005, Mantel-Cox test), and this data is shown in Figure 9.
Example 8: ELISPOT assay to confirm functional T cell response to tumor antigen
[00349] Spleens were harvested from re-challenged and control mice in preparation for the
ELISPOT assay. The spleens were smashed in a 70μπι filter placed on top of a 50ml tube using a 3ml syringe barrel. The filter was then rinsed with 2ml RPMI. The splenocytes collected in the tube were washed at 1200rpm for 5 minutes and the pellet was resuspended in lmL ACK lysis buffer to lyse the RBCs for 5 minutes and then resuspended in 9ml of RPMI to quench the reaction. The cells were washed once and resuspended in 5ml X-Vivo 15 media. The cells were then counted and kept on ice. The frozen vials of irradiated Renca 5F5 cells were thawed, suspended in the X-Vivo 15 media and counted.
[00350] These splenocytes (control mice and re-challenged mice) and irradiated tumor cells were used in the co-culture Mouse IFN-γ Single-Color ELISPOT assay (Cellular Technology Limited, Cat no: mIFNg-lM/2). An Effector: Tumor (E: T) ratio of 10: 1 was used in the assay and absolute cell numbers were as follows:
Table 7:
Figure imgf000171_0001
[00351] The positive control used in this assay was 500K splenocytes (pooled) + Dynabeads®
Mouse T-Activator CD3/CD28 (ThermoFisher Cat no: 11456D) in 1 : 1 ratio. The cells were then seeded in the IFN-gamma capture antibody coated plates as described in Table 7. This plate was placed in the incubator (37°C, 5% C02) and left undisturbed for 24 hrs. The processing of the plate was carried out as per the protocol provided with the ELISPOT kit and the developed plate was read using the ImmunoSpot® S6 CORE (Cellular Technology Limited). Results showed a significant IFN- gamma signal in the presence of splenocytes from re-challenged mice incubated with target cells compared to control splenocytes ( p<0.0001, 2 way ANOVA). Incubation in the absence of tumor antigen also elicited an IFN-gamma signal but to a significantly lower extent (p<0.001, Mann-Whitney Rank Sum Test). The difference between these two responses depicts the magnitude of the tumor specific T cell mediated response and this is shown graphically in Figure 10.
Example 9: Generating Colon26mCDH6 Cell line
[00352] Murine Colon epithelial carcinoma cells (Colon26) were purchased from ATCC
(CRL-2638) and transduced with a lentiviral construct delivering murine CDH6 (obtained from
Transomics and cloned into pLenti6.3). A population expressing CDH6 was selected by two rounds of fluorescence activated cell sorting using a PE-conjugated CDH6 antibody (R&D Systems #FAB2715). Example 10: GITR expression increases on Tress following CDH6-sulfo-SPDB-DM4 treatment in Colon26mCDH6 model
[00353] The anti-tumor activity of CDH6-sulfo-SPDB-DM4 was evaluated in the
Colon26mCDH6 model. Female B ALB/cJ mice were implanted subcutaneously on the right flank with 5xl05 Colon26mCDH6 cells Hanks Balanced salt solution (HBSS). The total injection volume containing cells in suspension was 100 μΐ. At 14 days post implantation mice were randomized into to ht groups (n=5) of equal mean tumor volume of 160 mm3, one group was dosed with CDH6-sulfo- SPDB-DM4 and the other acted as untreated control. Figure 11 demonstrates that CDH6-sulfo-SPDB- DM4 was efficacious, with a tumoristatic or tumorcidal response out to day 24. Tumors were harvested 10 days post dose (day 24) and were dissociated using a mixture of DNase 1 (Roche), Collagenase P (Roche) and Dispase (Gibco, Life Tech) in RPMI (Gibco, Life Tech). The tumor went through five total cycles of digestion, each lasting 20min and was agitated ever five minutes. Collected cells were then pelleted (1500 rpm, 5 min) and strained through a 70μπι filter. The resulting single cell suspension was then stained for flow cytometric analysis of the following markers: a-CD45-B V510, (Clone 30F-11, Biolegend), a-CD4-BUV395 (Clone GK1.5, BD Biosciences), a-CD8-BV650 (Clone 53-6.7, Biolegend), a-FoxP3-AF488 (Clone FJK-16s, eBioscience), a-GITR-BV421 (Clone DTA-1, eBiosciences), a-CDl lb-BUV737 (Clone Ml/70, BD Biosciences) a-CD 19-BUV737 (Clone 1D3, BD Biosciences). A fixable live/dead (eFluor-780,eBioscience) was used to exclude dead cells. A fixation/permeabilization buffer kit (eBioscience) was used for FoxP3 staining. As shown in Figure 12, analysis of T cell subsets demonstrated that treatment with CDH6-sulfo-SPDB-DM4 resulted in a significant increase in GITR expression on Foxp3+CD4+ T cells in comparison to untreated controls (p<0.001, unpaired t-test). Based on the level of GITR expression, Tregs could be sub gated into GITR+ and GITR1" cells. The significant increase in GITR expression observed on the Tregs correlated with a significant increase in the frequency of GITR1" cells within the Treg subset (pO.001, unpaired t-test).
[00354] It is understood that the examples and aspects described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMS What is claimed is:
1. A combination comprising: a) an anti-CDH6 antibody drag conjugate of the formula
Ab-(L-(D)m)n
or a pharmaceutically acceptable salt thereof; wherein
Ab is an antibody or antigen binding fragment thereof that specifically binds to an epitope of human
CDH6;
L is a linker;
D is a drag moiety, wherein the drag moiety is N(2')-deacetyl-N2-(4- mercapto-4-methyl- 1 - oxopentyl)-maytansine (DM4) or N(2')- deacetyl-N(2')-(3-mercapto-l-oxopropyl)-maytansine (DM1) ; m is an integer from 1 to 8; and
n is an integer from 1 to 10; and b) an anti-GITR agonist antibody or antigen binding fragment thereof.
2. The combination of claim 1, wherein in the antibody drag conjugate said n is 3 or 4.
3. The combination of claim 1 or 2, wherein in said antibody drag conjugate said antibody or antigen binding fragment thereof comprises:
(i) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:224, (b) a LCDR2 of SEQ ID NO:225, (c) a LCDR3 of SEQ ID NO:226; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 227, (e) a HCDR2 of SEQ ID NO: 228, and (f) a HCDR3 of SEQ ID NO:229;
(ii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:210, (b) a LCDR2 of SEQ ID NO:211, (c) a LCDR3 of SEQ ID NO:212; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:213, (e) a HCDR2 of SEQ ID NO: 214, and (f) a HCDR3 of SEQ ID NO:215;
(iii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:266, (b) a LCDR2 of SEQ ID NO:267, (c) a LCDR3 of SEQ ID NO:268; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 269, (e) a HCDR2 of SEQ ID NO:270, and (f) a HCDR3 of SEQ ID NO: 271; (iv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:308, (b) a LCDR2 of SEQ ID NO:309, (c) a LCDR3 of SEQ ID NO:310; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:311, (e) a HCDR2 of SEQ ID NO:312, and (f) a HCDR3 of SEQ ID NO:313;
(v) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 14, (b) a LCDR2 of SEQ ID NO: 15, (c) a LCDR3 of SEQ ID NO: 16; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 17, (e) a HCDR2 of SEQ ID NO: 18, and (f) a HCDR3 of SEQ ID NO: 19;
(vi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:28, (b) a LCDR2 of SEQ ID NO:29, (c) a LCDR3 of SEQ ID NO:30; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:31, (e) a HCDR2 of SEQ ID NO:32, and (f) a HCDR3 of SEQ ID NO:33;
(vii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:42, (b) a LCDR2 of SEQ ID NO:43, (c) a LCDR3 of SEQ ID NO:44; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:45, (e) a HCDR2 of SEQ ID NO:46, and (f) a HCDR3 of SEQ ID NO:47;
(viii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:56, (b) a LCDR2 of SEQ ID NO:57, (c) a LCDR3 of SEQ ID NO:58; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:59, (e) a HCDR2 of SEQ ID NO:60, and (f) a HCDR3 of SEQ ID NO:61;
(ix) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:70, (b) a LCDR2 of SEQ ID NO:71, (c) a LCDR3 of SEQ ID NO:72; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:73, (e) a HCDR2 of SEQ ID NO:74, and (f) a HCDR3 of SEQ ID NO:75;
(x) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:84, (b) a LCDR2 of SEQ ID NO:85, (c) a LCDR3 of SEQ ID NO:86; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO:87, (e) a HCDR2 of SEQ ID NO: 88, and (f) a HCDR3 of SEQ ID NO: 89;
(xi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:98, (b) a LCDR2 of SEQ ID NO:99, (c) a LCDR3 of SEQ ID NO: 100; and a heavy chain variable region that comprises: (d) a HCDRI of SEQ ID NO: 101, (e) a HCDR2 of SEQ ID NO: 102, and (f) a HCDR3 of SEQ ID NO: 103; (xii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 112, (b) a LCDR2 of SEQ ID NO: 113, (c) a LCDR3 of SEQ ID NO: 114; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 115, (e) a HCDR2 of SEQ ID NO: 116, and (f) a HCDR3 of SEQ ID NO: 117;
(xiii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 126, (b) a LCDR2 of SEQ ID NO: 127, (c) a LCDR3 of SEQ ID NO: 128; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 129, (e) a HCDR2 of SEQ ID NO: 130, and (f) a HCDR3 of SEQ ID NO: 131;
(xiv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 140, (b) a LCDR2 of SEQ ID NO: 141, (c) a LCDR3 of SEQ ID NO: 142; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 143, (e) a HCDR2 of SEQ ID NO: 144, and (f) a HCDR3 of SEQ ID NO: 145;
(xv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 154, (b) a LCDR2 of SEQ ID NO: 155, (c) a LCDR3 of SEQ ID NO: 156; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 157, (e) a HCDR2 of SEQ ID NO: 158, and (f) a HCDR3 of SEQ ID NO: 159;
(xvi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 168, (b) a LCDR2 of SEQ ID NO: 169, (c) a LCDR3 of SEQ ID NO: 170; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 171, (e) a HCDR2 of SEQ ID NO: 172, and (f) a HCDR3 of SEQ ID NO: 173;
(xvii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 182, (b) a LCDR2 of SEQ ID NO: 183, (c) a LCDR3 of SEQ ID NO: 184; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 185, (e) a HCDR2 of SEQ ID NO: 186, and (f) a HCDR3 of SEQ ID NO: 187;
(xviii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO: 196, (b) a LCDR2 of SEQ ID NO: 197, (c) a LCDR3 of SEQ ID NO: 198; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 199, (e) a HCDR2 of SEQ ID NO:200, and (f) a HCDR3 of SEQ ID NO:201;
(xix) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:238, (b) a LCDR2 of SEQ ID NO:239, (c) a LCDR3 of SEQ ID NO:240; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:241, (e) a HCDR2 of SEQ ID NO:242, and (f) a HCDR3 of SEQ ID NO:243; (xx) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:252, (b) a LCDR2 of SEQ ID NO:253, (c) a LCDR3 of SEQ ID NO:254; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:255, (e) a HCDR2 of SEQ ID NO:256, and (f) a HCDR3 of SEQ ID NO:257;
(xxi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:280, (b) a LCDR2 of SEQ ID NO:281, (c) a LCDR3 of SEQ ID NO:282; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:283, (e) a HCDR2 of SEQ ID NO:284, and (f) a HCDR3 of SEQ ID NO:285;
(xxii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:294, (b) a LCDR2 of SEQ ID NO:295, (c) a LCDR3 of SEQ ID NO:296; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:297, (e) a HCDR2 of SEQ ID NO:298, and (f) a HCDR3 of SEQ ID NO:299; or
(xxiii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:322, (b) a LCDR2 of SEQ ID NO:323, (c) a LCDR3 of SEQ ID NO:324; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO:325, (e) a HCDR2 of SEQ ID NO:326, and (f) a HCDR3 of SEQ ID NO:327.
4. The combination of any one of claims 1 to 3, wherein in said antibody drug conjugate said antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region (vH) that comprises SEQ ID NO: 230, and a light chain variable region (vL) that comprises SEQ ID NO:231;
(ii) a heavy chain variable region (vH) that comprises SEQ ID NO: 216, and a light chain variable region (vL) that comprises SEQ ID NO:217;
(iii) a heavy chain variable region (vH) that comprises SEQ ID NO: 272, and a light chain variable region (vL) that comprises SEQ ID NO:273;
(iv) a heavy chain variable region (vH) that comprises SEQ ID NO:314, and a light chain variable region (vL) that comprises SEQ ID NO:315;
(v) a heavy chain variable region (vH) that comprises SEQ ID NO: 20, and a light chain variable region (vL) that comprises SEQ ID NO:21;
(vi) a heavy chain variable region (vH) that comprises SEQ ID NO: 34, and a light chain variable region (vL) that comprises SEQ ID NO:35; (vii) a heavy chain variable region (vH) that comprises SEQ ID NO:48. and a light chain variable region (vL) that comprises SEQ ID NO:49;
(viii) a heavy chain variable region (vH) that comprises SEQ ID NO:62, and a light chain variable region (vL) that comprises SEQ ID NO:63;
(ix) a heavy chain variable region (vH) that comprises SEQ ID NO:76, and a light chain variable region (vL) that comprises SEQ ID NO:77;
(x) a heavy chain variable region (vH) that comprises SEQ ID NO:90, and a light chain variable region (vL) that comprises SEQ ID NO:91;
(xi) a heavy chain variable region (vH) that comprises SEQ ID NO: 104. and a light chain variable region (vL) that comprises SEQ ID NO: 105;
(xii) a heavy chain variable region (vH) that comprises SEQ ID NO: 118, and a light chain variable region (vL) that comprises SEQ ID NO: 119;
(xiii) a heavy chain variable region (vH) that comprises SEQ ID NO: 132, and a light chain variable region (vL) that comprises SEQ ID NO: 133;
(xiv) a heavy chain variable region (vH) that comprises SEQ ID NO: 146, and a light chain variable region (vL) that comprises SEQ ID NO: 147;
(xv) a heavy chain variable region (vH) that comprises SEQ ID NO: 160, and a light chain variable region (vL) that comprises SEQ ID NO: 161;
(xvi) a heavy chain variable region (vH) that comprises SEQ ID NO: 174, and a light chain variable region (vL) that comprises SEQ ID NO: 175;
(xvii) a heavy chain variable region (vH) that comprises SEQ ID NO: 188, and a light chain variable region (vL) that comprises SEQ ID NO: 189;
(xviii) a heavy chain variable region (vH) that comprises SEQ ID NO: 202, and a light chain variable region (vL) that comprises SEQ ID NO:203;
(xix) a heavy chain variable region (vH) that comprises SEQ ID NO: 244, and a light chain variable region (vL) that comprises SEQ ID NO:245;
(xx) a heavy chain variable region (vH) that comprises SEQ ID N0.258, and a light chain variable region (vL) that comprises SEQ ID NO:259; (xxi) a heavy chain variable region (vH) that comprises SEQ ID NO:286, and a light chain variable region (vL) that comprises SEQ ID NO:287;
(xxii) a heavy chain variable region (vH) that comprises SEQ ID NO:300, and a light chain variable region (vL) that comprises SEQ ID NO:301; or
(xxiii) a heavy chain variable region (vH) that comprises SEQ ID NO:328, and a light chain variable region (vL) that comprises SEQ ID NO:329.
5. The combination of any one of claims 1 to 4, wherein in said antibody drug conjugate the linker (L) is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker and a dicarboxylic acid based linker.
6. The combination of any one of claims 1 to 5, wherein in said antibody drug conjugatethe linker is derived from a cross-linking reagent selected from the group consisting of: N-succinimidyl-4-(2- pyridyldithio)2-sulfo-butanoate (sulfo-SPDB), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N- sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfo-SMCC) or 2,5- dioxopyrrolidin-l-yl 17-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-5,8,l l,14-tetraoxo-4,7,10,13- tetraazaheptadecan-l-oate (CXl-1).
7. The combination of any one of claims 1 to 6, wherein in said antibody drug conjugate the linker is derived from N-succinirnidyl-4-(2-pyridyldithio)2-sulfo-butanoate (sulfo-SPDB).
8. The combination of any one of claims 1 to 7, wherein in said antibody drug conjugateD and L consist of:
Figure imgf000179_0001
Ab-suifo-SPDB-DM4 or a pharmaceutically acceptable salt thereof.
9. The combination of any one of claims 1 to 8, wherein the anti-GITR agonist antibody or antigen binding fragment thereof , specifically binds to SEQ ID NO:336.
10. The combination of any one of claims 1 to 9, wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
(i) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427;
(ii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:352, (b) a LCDR2 of SEQ ID NO:353, (c) a LCDR3 of SEQ ID NO:354; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 355, (e) a HCDR2 of SEQ ID NO: 356, and (f) a HCDR3 of SEQ ID NO: 357;
(iii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:366, (b) a LCDR2 of SEQ ID NO:367, (c) a LCDR3 of SEQ ID NO:368; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 369, (e) a HCDR2 of SEQ ID NO:370, and (f) a HCDR3 of SEQ ID NO:371; (iv) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:380, (b) a LCDR2 of SEQ ID NO:381, (c) a LCDR3 of SEQ ID NO:382; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 383, (e) a HCDR2 of SEQ ID NO: 384, and (f) a HCDR3 of SEQ ID NO: 385;
(vi) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:394, (b) a LCDR2 of SEQ ID NO:395, (c) a LCDR3 of SEQ ID NO:396; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 397, (e) a HCDR2 of SEQ ID NO: 398, and (f) a HCDR3 of SEQ ID NO: 399;
(vii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:408, (b) a LCDR2 of SEQ ID NO:409, (c) a LCDR3 of SEQ ID NO:410; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 411, (e) a HCDR2 of SEQ ID NO: 412, and (f) a HCDR3 of SEQ ID NO: 413; or
(viii) a light chain variable region that comprises (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:436, (b) a LCDR2 of SEQ ID NO:437, (c) a LCDR3 of SEQ ID NO:438; and a heavy chain variable region that comprises: (d) a HCDRl of SEQ ID NO: 439, (e) a HCDR2 of SEQ ID NO: 440, and (f) a HCDR3 of SEQ ID NO: 441.
11. The combination of any one of claims 1 to 10, wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region (vH) that comprises SEQ ID NO: 428, and a light chain variable region (vL) that comprises SEQ ID NO:429;
(ii) a heavy chain variable region (vH) that comprises SEQ ID NO: 358, and a light chain variable region (vL) that comprises SEQ ID NO: 359;
(iii) a heavy chain variable region (vH) that comprises SEQ ID NO: 372, and a light chain variable region (vL) that comprises SEQ ID NO:373;
(iv) a heavy chain variable region (vH) that comprises SEQ ID NO: 386, and a light chain variable region (vL) that comprises SEQ ID NO:387;
(v) a heavy chain variable region (vH) that comprises SEQ ID NO: 400, and a light chain variable region (vL) that comprises SEQ ID NO:401;
(vi) a heavy chain variable region (vH) that comprises SEQ ID NO: 414, and a light chain variable region (vL) that comprises SEQ ID NO:415; or (vii) a heavy chain variable region (vH) that comprises SEQ ID NO: 442, and a light chain variable region (vL) that comprises SEQ ID NO:443.
12. The combination of any one of claims 1 to 11, wherein the anti-GITR agonist antibody or antigen binding fragment thereof comprises:
(i) a heavy chain that comprises SEQ ID NO: 432, and a light chain that comprises SEQ ID NO:433;
(ii) a heavy chain that comprises SEQ ID NO: 362, and a light chain that comprises SEQ ID NO:363;
(iii) a heavy chain that comprises SEQ ID NO: 376, and a light chain that comprises SEQ ID NO:377;
(iv) a heavy chain that comprises SEQ ID NO: 390, and a light chain that comprises SEQ ID NO:391;
(v) a heavy chain that comprises SEQ ID NO: 404, and a light chain that comprises SEQ ID NO:405;
(vi) a heavy chain that comprises SEQ ID NO: 418, and a light chain that comprises SEQ ID NO:419; or
(vii) a heavy chain that comprises SEQ ID NO: 446, and a light chain that comprises SEQ ID NO:447.
13. The combination of any one of claims 1 to 12, wherein the anti-GITR agonist antibody or antigen binding fragment thereof consists of: (i) a light chain variable region that consists of (a) a LCDR1 (CDR-Complementarity Determining Region) of SEQ ID NO:422, (b) a LCDR2 of SEQ ID NO:423, (c) a LCDR3 of SEQ ID NO:424; and a heavy chain variable region that consists of: (d) a HCDR1 of SEQ ID NO: 425, (e) a HCDR2 of SEQ ID NO: 426, and (f) a HCDR3 of SEQ ID NO: 427.
14. The combination of any one of claims 1 to 13, wherein the anti-GITR agonist antibody or antigen binding fragment thereof is selected from the group consisting of: BMS-986156, INCAGN01876, AMG 228, TRX518, MEDI1873, MK-4166, MK-1248 and FPA-154.
15. The combination of any one of claims 1 to 14, wherein the antibody drug conjugate and anti-GITR antibody or antigen binding fragment thereof are in separate forms.
16. The combination of any one of claims 1 to 15, wherein the antibody drug conjugate and the anti- GITR antibody or antigen binding fragment thereof are in a fixed combination.
17. A method of treating a CDH6 positive cancer in a patient in need thereof, comprising administering to said patient the combination of any one of claims 1 to 16.
18. The method of claim 17, wherein said cancer is selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and
cholangiocarcinoma.
19. The method of claim 17 or 18, wherein the antibody drug conjugate and the anti-GITR antibody or antigen binding fragment thereof are administered simultaneously or separately.
20. The method of claim 17 or 18, wherein the antibody drug conjugate is administered prior to the administration of the anti-GITR antibody or antigen binding fragment thereof.
21. A method of decreasing Treg cells in a tumor, the method comprising; c) administration of an antibody drug conjugate, wherein the administration of the antibody drug conjugate induces GITR expression on Treg cells;
d) administration of an anti-GITR agonist antibody or antigen binding fragment thereof, which decreases the number of Treg cells associated with the tumor.
22. The method of claim 21, which decreases reoccurrence of the tumor in a patient.
23. The method of claim 21 or 22, which increases progression-free survival in a patient.
24. Combination for use in treating cancer, wherein the combination is as in any one of claims 1 to 16.
25. Combination for use in treating cancer according to claim 24, wherein said cancer is selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and cholangiocarcinoma.
26. Use of the combination according to any one of claims 1 to 16 in the manufacture of a medicament for the treatment of cancer.
27. Use according to claim 26, wherein said cancer is selected from the group consisting of: ovarian cancer, renal cancer, hepatic cancer, soft tissue cancer, CNS cancers, thyroid cancer and
cholangiocarcinoma.
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