DK3013861T3 - Anti-fcrh5-antistoffer - Google Patents

Anti-fcrh5-antistoffer Download PDF

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DK3013861T3
DK3013861T3 DK14742047.5T DK14742047T DK3013861T3 DK 3013861 T3 DK3013861 T3 DK 3013861T3 DK 14742047 T DK14742047 T DK 14742047T DK 3013861 T3 DK3013861 T3 DK 3013861T3
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amino acid
acid sequence
antibody
sequence
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DK14742047.5T
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Allen J Ebens Jr
Meredith C Hazen
Jo-Anne Hongo
Jennifer W Johnston
Teemu T Junttila
Ji Li
Andrew G Polson
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Genentech Inc
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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    • 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
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    • 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
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    • 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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6879—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 being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Description

Description
FIELD OF THE INVENTION C0001J Provided herein are anti-FcRH5 antibodies (e g., bispecific antibodies) and immunoconjugates and methods of using the same,
BACKGROUND
[0002] The Fc receptor-like 5 (FcRLS, also known as FeRHS and IRTA2) belongs to a family of 6 recently identified genes of the immunoglobulin superfamily (IgSF), This family of genes is closely related to the Fc receptors with the conserved genomic structure, extracellular jg domain composition and the ΠΊΜ- and ITAM-like signaling motifs (Davis RS et aL, Eur J Immunol (2005) 35:674-80), Members of this family have also been calied iFGPs (from Ig super-family, FcR. gp42) and SPAPs (SH2 domain-containing phosphatases anchor proteins), Six members of the FcRH/IRTA receptor family have been described: FeRH17!RTA5, FcRH2/IRTA4, FcRH3/IRTA3,FcRH4/RTA1, FcRFISi1RTA2 and FcRH6 (Poison AG et ai„ int. Immunol, (2008) 18(9):1363-1373). Ai! FeRH/IRTAs contain some combination of canonical immunoreceptor tyrosine-based inhibitory motifs and 'immunoreceptor tyresine-based activation motifs-like' signaling motifs. The FcRH eDNAs encode type ! transmembrane glycoproteins with multiple ig-like extracellular domains and cytoplasmic domains containing consensus immunoreceptor tyrosine-based activating and/or inhi bitory signaling motifs. The FcRH genes are structurally related, and their protein products share 28-60% extracellular identity with each other. They also share 15-31 % identity with their ciosest FcR relatives. There is a high degree of homology between the different FcRHs.
[0003] The ligand(s) for FeRHS are unknown, but FeRHS has been implicated in enhanced proliferation and downstream isotype expression during the development of antigen-primed B-celis (Demerit-Brown J, et ai. J Leukoc Biol (2012) 91:59-67). The FeRHS locus has three major mRNA isoforms (FcRHSa, FcRHSb, and FcRHSc). The major FcRH5 protein isoforms encoded by these transcripts share a common amino acid sequence unti! residue 560, featuring a common signal peptide and six extracellular ig-iike domains. FcRHSa represents a 759 amino acid secreted glycoprotein with eight ig-iike domains followed by 13 unique, predominantly polar amino acids at its C-termihus, FcRHSb diverges from FcRH5a at amino acid residue 560 and extends for a short stretch of 32 additional residues, whose hydrophobicity is compatibie with Sts docking to the plasma membrane via a GP! anchor. FcRHSc is the longest isoform whose sequence deviates from FcRHSa at amino add 746, FcRHSc encodes a 977 aa type f transmembrane glycoprotein with nine extracellular lg-type domains, harboring eight potential N-linked gfycosyfation sites, a 23 amino add transmembrane, and a 104 amino add cytoplasmic domain with three consensus SH2 binding motifs with the ΓΠΜ consensus, [0004] The FcRH genes are clustered together in the midst of the classical FcR genes. FcyRi, FcyRH, FcyRiii, and FceRI, in the fq21-23 region of chromosome 1. This region contains 1 of the most frequent secondary chromosomal abnormalities associated with malignant phenotype in hematopoietic tumors, especially in multiple myeloma {Hafzivas-siiiou G. et at, immunity (2001) 14:277-89). FcRH5 is expressed oniy in the B-ceii lineage, starting as early as pre-8-ceffs, but does not attain full expression until the mature B-cett stage. Unlike most knawnoiher B-ceii-speciftc surface proteins (e.g., CD20, CD19, and CD22), FcRH5 continues to be expressed in pfasma ceiis whereas other B-celi-speciflc markers are downreguiated {Poison AG et ai., int Immunoi (2006) 18:1363-73). In addition, FCRH5 mRNA is overexpressed in multiple myeloma ceii fines with 1q21 abnormalities as detected by oligonucleotide arrays (inoue J,. Am J Paihoi (2004) 165:71-81}. The expression pattern indicates that FeRHS could be a target for antibody-based therapies for the treatment of multiple myeloma. Multiple myeloma is a malignancy of plasma ceiis characterized by skeletal lesions, renal failure, anemia, and hypercalcemia, it is essentially incurable by current therapies. Current drug treatments for multiple myeloma include combinations of the proteosome inhibitor borfezoroib (Velcade), the immunomoduiator fena-fidomide (Reviimid), and the steroid dexamethasone.
[0005] WO 2006/039238 discloses antibodies that specifically bind to the extracellular domain of FcRH5 which are not capable of specificaily binding to an isoform c-specific region of the extraceffular domain of FeRHS.
[0006] FcRH5c specific antibody-based therapies and detection methods may be particularly efficacious as they specifically recognize target cell, membrane-associated FeRHS rather than antibodies which recognize both soluble and membrane isoforms of FcRH5. However, oniy the last Ig-iike domain of FeRHS (Ig-iike domain 9) is unique extraceiiufar region that differentiates between the three major isoforms of FeRHS, and there is significant homology between the fg-fike domains within FeRHS. Further, the fast Ig-like domain is highly conserved between FcRH1, FcRH2, FcRH3, and FeRHS, Any antibody-based therapy that specifically targeted FeRHS would have to have minimal cross-reactivity with other FcRH3 to avoid adverse off-target effects (e.g., FcRH3 is expressed on norma! NK cells), There is a need in the art for agents that aid in the diagnosis and treatment of cancer, such as FcRHS-associated cancer.
SUMMARY
[0007) Provided herein are anti-EcRHS antibodies inciurfing bispectfic antibodies, immunoconjogates, and the same for medico! use, as defined in the claims. Provided herein are isolated anti-FcRHS antibodies that binds an isoform c-speciftc region of the extracellular domain of FcRHSc. The isoform c-specific region comprises ig-tike domain 9. The isoform e-specific region comprises amino acids 743-850 ofSEQ iD NO:1.
[0008) In some embodiments, the antibody comprises: a} a heavy chain comprising a HVR-B1 comprising the amino acid sequence of SEQ iD NO;38, HVR-H2 comprising the amino acid sequence of SEQ iD NO :62, and HVR-H3 comprising the amino acid sequence of SEQ iD NO.86: and h) a tight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ iD NO:2. HVR-L2 comprising the amino acid sequence of SEQ (D NO: 14, and HVR-L3 comprising the amino add sequence of SEQ !D NO:26.
[0009) In some embodiments, the antibody comprises; a) a heavy chain comprising a HVR-H1 composing the amino acid sequence ofSEQ iD NO :39, HVR-H2 comprising the amino acid sequence of SEQ IDNO:63.and HVR-H 3 comprising the amino acid sequence of SEQ !D NO:87; and b) a tight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NQ;3, HVR-L2 comprising the amino acid sequence ofSEQ iD NO: 15, and HVR-L3 comprising the amino acid sequence of SEQ iD NO;27.
[0010) In some embodiments, the antibody comprises: a) a heavy chain comprising a H-VR-H1 comprising the amino acid sequence ofSEQ iD NO:40, HVR-H2 comprising the amino acid sequence ofSEQ !DNO:64, and HVR-H3 comprising the aminoadd sequence ofSEQ iD NQ:88, and b) a tight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ iD NO:4. HVR-L2 comprising the amino acid sequence ofSEQ !D NO: 16, and HVR-L3 comprising the amino acid sequence of SEQ iD NO:28.
[0011) In some embodiments, the antibody comprises: a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ ID NO:41, HVR-H2 comprising the amino add sequence ofSEQ ID NO:65, and HVR-H3 comprising the amino acid sequence ofSEQ iD NO:89; and b) a fight chain comprising a HVR-L1 comprising the aminoadd sequence of SEQ !D NO:5, HVR-L2 comprising the amino add sequence of SEQ iD NO:17, and HVR-L3 comprising the amino acid sequence of SEQ iD NO:29.
[0012) In some embodiments, the antibody comprises: a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ iD NO:42, HVR-H2 comprising the amino acid sequence of SEQ iD NO:68, and HVR-H3 comprising the amino acid sequence of (SEQ !D NO:90; and b) a tight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO:6, HVR-L2 comprising the amino acid sequence of SEQ (D NO; 18, and HVR-13 comprising the amino acid sequence of SEQ iD NO:30.
[0013) In some embodiments, the antibody comprises; a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence ofSEQ !D NO :43, HVR- H2 comprising the amino acid sequence of SEQ ID NO:87, and HVR-H3 comprising the amino acid sequence of SEQ iD NO:91; and b) a light chain comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO::7, HVR-L2 comprising the amino acid sequence of SEQ !D NO: 19, and HVR-L3 comprising the amino acid sequence ofSEQ iD NO:31, [0014) in some embodiments, the antibody comprises; a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of (SEQ ID NO:44, HVR-H2 comprising the amino acid sequence of SEQ iD NO:S8, and HVR-H3 comprising the amino acid sequence ofSEQ: ID NQ:92; and b) a tight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO:8, HVR-L2 comprising the amino acid sequence of SEQ ID NQ:2G, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:32, [0015) In some embodiments, the antibody comprises: a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ !D NO:45, H VR-H2 comprising the amino acid sequence of SEQ I D NO: 69, and HVR-H3 comprising the amino acid sequence of SEQ SD NG:93; and b) a light chain comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO:9, HVR-L2 comprising the amino acid sequence of SEQ ID NO:21, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:33.
[0016) in some embodiments, the antibody comprises; a) a heavy chain comprising a HVR-H1 comprising the amino acid sequenceof SEQ ID NO:48, HVR-H2 comprising the amino acid sequence ofSEQ !D NO:70, and HVR-H3 comprising the amino acid sequence of SEQ iD NO:94; and b) a Sight chain comprising a HVR-L1 comprising the amino add sequence of SEQ ID NO: 10. HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and HVR-13 comprising the amino acid sequence of SEQ ID NO;34.
[0017) In some embodiments, the antibody comprises: a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ iD NO;47, HVR-H2 comprising the amino acid sequence of SEQ iD NO:? 1, and HVR-H3 comprising the amino acid sequence of SEQ iD NO:95; and b) a light chain comprising a HVR-11 comprising the amino acid sequence of (SEQ ID NO:T1. HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, and HVR-L3 comprising the amino acid sequence of SEQ iD NO:35.
[0018) In some embodiments, the antibody comprises: a} a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEG ID NO:48,HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 72, and HVR-H3comprising the amino add sequence of SEQ !D NO:96; and b) a light chain comprising a HVR-L1 comprising the amino acid sequence of SEQ iO NO: 12, BVR-12 comprising the amino acid sequence of (SEQ !D NO.24, and HVR-L3 comprising the amino acid sequence of SEQ iO NO:36. (0019} In some embodiments, the antibody comprises: a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:49« HVR-H2 comprising the amino acid sequence of SEQ i D NO: 73, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:97; and b) a light chain comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13, HVR-L2 comprising the amino acid sequence of SEQ ID NO:25, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:37. (0020} in some embodiments of any of the antibodies, the antibody comprises: a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:t1l and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 110; b) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:113 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 112; c) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:115 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 114; d) a VH sequence having at least 95% sequence identity to the amino acid sequence of (SEQ ID NO:117 and a VL sequence having at least 95% sequence Identity to the amino acid sequence of SEQ ID NG:118; e)a VH sequence having at least 95% sequence Identity io the amino acid sequence of SEQ ID NO:119 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:118; f) a VH sequence having st least 95% sequence identity to the amino acid sequence of SEQ !D NO: 121 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 120; g) a VH sequence having at least 95% sequence identity to the amino add sequence of SEQ ID NO;123 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:122; h) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:125 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:124; i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:127 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 126: J) a VH sequence having at least 95% sequence identity to She amino acid sequence of SEQ ID NO; 129 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:12S; k) a VH sequence having at least 95% sequence Identity to the amino acid sequence of SEQ ID NO:131 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO;13G;1) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 133 and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NC:132; or a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ! D NO: 135 and a VL sequence having at leas t 95% sequence identity to the amino acid sequence of SEQ iD NO;134, (0021} in some embodiments of any of the antibodies, the antibody comprises: a) a VH sequence of SEQ ID NO:t 11 and a VL sequence of SEQ ID NO: 110; b) a VH sequence of SEQ ID NO: 113 and a VL sequence of SEQ iD NO:112; c) a VH sequence of SEQ ID NO:115 and a VL sequence of SEQ ID NO:114; d) a VH sequence of (SEQ ID NO:f 17 and a VL sequence of SEQ ID NO:116; e) a VH sequence of SEQ ED NO:119 and a VL sequence of SEQ !D NO:118; f) a VH sequence of SEQ iD NO: 121 and a VL sequence of SEQ !D NO:120; g)a VH sequence of SEQ ID NO:123and a VL sequence of SEQ ID NO: 122; h) a VH sequence of SEQ ID NO: 125 and a VI sequence of SEQ ID NO:124; i) a VH sequence of SEQ ID NO:127 and a VL sequence of SEQ ID NO: 126; ]} a VH sequence of SEQ iD NO:129 and a VL sequence of SEQ ID NO;128; k) a VH sequence of SEQ: iD NO:f 31 and a VL sequence of SEQ ID NO:130;1) a VH sequence of SEQ ID NO: 133 and a VL sequence of SEQ ID NO:132, or m) a VH sequence of SEQ ID NO: 139 and a VL sequence of SEQ ID NO: 134. (0022} In some embodiments of any of the antibodies, the antibody is a monoclonal antibody. In some embodiments of any of the antibodies, the antibody Is a human, humanized, or chimeric antibody , In some embodiments of any of the antibodies, the antibody is an antibody fragment Shat binds FcRHS. Io some embodiments Of any of the antibodies, the antibody is an fgGI. lgG2a or lgG2b antibody. (0023} in some embodiments of any of the antibodies, the antibody has one or more of the following characteristics; a) cross reactive with foil length human end cyno FcRHS, b) does not cross react with FcRH3, FcRH2. FcRHS, and/or FcRH4, c) binds to endogenous FcRHS, d) does not cross react with FcRHSa, and e) does not cross react with another ig-iike domain of FcRHS. (0024} In some embodiments of any of the antibodies, the antibody is a btspecific antibody. In some embodiments, the bispecific antibody binds FcRHS and CD3. (0025} In some embodiments, an isolated nucleic acid that encodes an antibody described herein is provided, in some embodiments, a host ceii comprising the nucleic acid is provided. In some embodiments, a method of producing an antibody described herein is provided In some embodiments, the method comprises culturing the host cel comprising the nucleic acid that encodes an antibody. (0026} In some embodiments, immunoconjugates are provided as defined in the claims. An immunoconjugate comprises an anti-FcRH5 antibody and a cytotoxic agent. The anti-FcRHS antibody binds an isoform c-specific region of the
extracellular domain of FcRHSc, in some embodiments, the anti-FcRH5 antibodies binds Ig-ltke domain 9 of FcRHSc. in some embodiments, an immunoeonjugate has the formula Ab-(l-D) p, wherein: (a) Ab is an antibody described herein; fb) l is a »nker; (c) D is a drog selected from a maytansinoid, an auristatin, a calicheamicin, a pyrroiobenzodiazepine, and a nemorubicin derivative; and {d} p ranges from 1-8. in some embodiments, D is an auristatin. in some such embodiments, 0 has formula DE
wherein R2 and R6 are each methyl, Rs and R4 are each isopropyl, Rs is H, R7 is sec-butyl, each R® is independently selected from CHS, 0-CH3, OH, and H; R9 is H; and Rts is -C{R8)£-C(R8)2*8ryi, in some embodiments, D is MM.AE having the structure:
[0027] In some embodiments, D is a pyrroiobenzodiazepine of Formula A;
wherein the dotted fines indicate the optional presence of a double bond between C1 and C2 or C2 and C3; R2 is independently selected from H, OH, =0, =CHa. CN, R, OR, =CH-RB, ~C(R%.0-S02-R, C02R and COR, and optionally further selected from halo or dihalo, wherein RD is independently selected from R, C02R, COR. CHO, C02H. and halo; R® and R8 are independently selected from H, R, OH. OR, SH, SR, NHg, NHR, NRR, N02, Me3Sn and halo; R7 is independently selected from H,R,OH, OR, SH, SR, NH2. NHR, NRR‘, N02, Me3Sn and halo; Q is independenfly selected from O, S end NH; R11 is either H, or R or, where Q is 0, S03M, where M is a metal cation; R and R’ are each independently selected from optionally substituted C?,s alkyl, C,_12 alkyl. C3.B heterocyciyi, C3.2<;! heterocycfyl, and Cs_20 aryl groups, and optionally in relation to the group NRR’, R and R: together with the nitrogen atom tojwhich they are attached form an optionaly substituted 4-, 5-, 6- or 7-membered heterocyclic ring; R12, R1b> R19and R1, are as defined for RA, R6, R# and R7 respectively; R" is a C3. j2 alkylene group, which chain may be interrupted by one or more heteroatoms and/or aromatic rings that are optionally substituted; and X and X' are independently selected from O, S and N(H). In some such embodiments, D is
·> wherein n is 0 or 1, {0028] in some embodiments, D is a nemombicln derivative. In some embodiments, D has a structure selected from and
[0029] in some embodiments, an immunoconjugate comprises a linker that is eleavable by a protease, in some etr bodiments, the linker comprises a vai-cit dipeptide or a Phe-homoLys dipeptide, in some embodiments, an immunocor jugate comprises a linker that is acid-iabiie. In some such embodiments, the linker comprises hyrfrazone. {0Q30J In some embodiments, an immunoconjugate has a formula selected from:
wherein S is a sulfur atom;
an
[00311 In some embodiments, p ranges from 2-5.
[00321 in some embodiments, pharmaceutical formulations are provided as defined in the claims, in some such embodiments, a pharmaceutical formulation comprises an immunoconjugate comprising an antibody that binds FcRHS, e.g., as described herein. The anti-FcRHS antibody binds an isoform e-specific region ef the extracellular domain of FcRHSc. in some embodiments, the anti-FcRHS antibodies binds ig-like domain 9 of FcRHSc, in some embodiments, a pharmaceutical formulation further comprises an additions! therapeutic agent.
[00331 In some embodiments, anli-FcRhS antibodies and/or immunoconjugates of the invention for use in methods of treating individuals having FcRH5 (e g., FcRH5c)-positive cancers are provided as defined in the claims. In some such embodiments, a method comprises administering a pharmaceutical formulation comprising an immnunoconjugate comprising an antibody that binds FcRHS and/or an FcRHS bispecific antibody, eg., as described herein. In some embodiments, the FcRHS bispecific antibody comprises an FcRHS binding arm and a CD3 binding arm. The anti-FcR5 antibody binds an isoform c-specific region of the extracellular domain of FcRHSc. in some embodiments, the anti-FcRHS antibodies binds lg-like domain 9 of FcRHSc. In some embodiments, the FcRHS-positive cancer is a B-celi proliferative disorder, in some embodiments, the FcRHS-positive cancer is plasma ceil neoplasm, In some embodiments, the plasma cell neoplasm is multiple myeloma, in some embodiments, a method comprises administering an additions! therapeutic agent to the individual [0034] In some embodiments, anti-FcRHS antibodies and/or immunoconjugates of the invention for use in methods of inhibiting proliferation of an FcRHS (e g., FcRH5c)-posifive ceii are provided as defined in the ciaims. fn some embodiments, the method comprising exposing the cell to an immunoconjugate comprising an antibody that binds FcRHS and/or an FcRHS bispecific antibody under conditions permissive for binding of the antibody to FcRHS on the surface of the ceii, fn some embodiments, the FcRHS bispecific antibody comprises an FcRHS binding arm and a CDS binding arm. The anft-FcRHS antibody binds an isoform c-specific region of the extracellular domain of FcRHSc. in some embodiments, the anti-PcRH5 antibodies binds ig~iike domain 9 of FcRHSc, in some embodiments, the antibody that binds FcRHS is an antibody described herein, in some embodiments, the FcRHS-positive cancer is a 8-ceii proliferative disorder, fn some embodiments, the Fr-RHS-posfflve cancer is plasma ceii neoplasm, in some embodiments, the plasma ceii neoplasm is multiple myeloma, in some embodiments, a method comprises administering an additional therapeutic agent to the individual.
[0035) An antibody that binds FcRHS may be conjugated to a label. In some embodiments, the anti-FcRHS antibody binds an isoform c-speciiic region of the extracellular domain of FcRHSc. in some embodiments, the anti-FcRHS antibodies binds ig-like domain 9 of FcRHSc, in some embodiments, the antibody that binds FcRHS is an antibody described herein, in some embodiments, the label is a positron emitter, in some embodiments, the positron emitter is 89Zr, [0036} in some embodiments, a method of detecting human FcRHS in a biological sample is provided as defined in tbe ciaims, in some embodiments, a method comprises contacting the biological sampfe with an anti-FcRHS antibody under conditions permissive for binding of the anti-FcRHS antibody to a naturally occurring human FcRH5, and detecting whether a complex is formed between the anti-FcRHS antibody and a naturally occurring human FcRHS in the biological sampfe. The anti-FcRHS antibody binds an isoform c-specific region of the extraceiiuiar domain of FcRHSc. in some embodiments, the anti-FcRHS antibodies binds fg-like domain 9 of FcRHSc, in some embodiments, the anti-FcRHS antibody is an antibody described herein.
[0037} In some embodiments, a method for detecting an FcRHS-positive cancer is provided as defined in the ciaims. in some such embodiments, a method comprises (i) administering a labeled anti-FcRHS antibody to a subject having or suspected of having en FcRHS-positive cancer, and (ii) defecting the labeied anti-FcRHS antibody in the subject, wherein detection of the labeled anii-FcRHS antibody indicates an FcRHS-positive cancer in the subject. The anti-FcRH5 antibody binds an isoform c-specific region of the extraceiiuiar domain of FcRHSc, in some embodiments, the anti-FcRHS antibodies binds ig-iike domain 9 of FcRH5c. in some embodiments, an anti-FcRHS antibody is an antibody described herein, BRIEF DESCRIPTION OF THE FIGURES [00381 FIG. 1{A) depicts the three major isoforms of FcRHS, FcRHSa (IRTA2a; UniRrot identifier Q96RD9-3), FcRH5b (!RTA2b: UniProt Identifier G96RD9-4), and FcRHSc {(R7A2c; UniProt identifier Q96RD9-1). The ig-iike domains are numbered and correspond to the amino acid sequence of UniProt identifier Q69RD9-1 (SEQ ID NO:1): ig-iike domain 1 (aa ("(amino acid") 23-100), ig-iike domain 2 (aa 105-185), ig-iike domain 3 (aa 188-271), ig-iike domain 4 (287-373), ig-iike domain 5 (aa 380-466), Ig-iike domain 6 (aa 490-555), ig-iike domain 7 (aa 568-652), ig-iike domain 8 (aa 658-731), and ig-iike domain 9 (aa 754-835). f !G. 1(B) depicts part of FcRHS (SEQ ID NQ:136) and the structure and homology of FcRHS amino acids 735 to 977 of FcRHSc (SEQ ID NO:2). FIG, 2 shows binding of FcRHS antibodies to SVT2 ceils transfected with (A) human FcRHS and (S) cyno FcRHS, in different concentrations. FIG. 3 shows binding of FcRHS antibodies io (A) EJM ceils or (8) OPM2 ceils transfected with human FcRHS, and binding of subclone supernatants (C) 5A10.1, (D) 5F1.1, (E) 3G7.1, and (F) 6D2.2 to MOLP2 ceils which express FcRHS endogenously. FIG. 4 shows (A) binding of FcRH5 subcfone supernatants to 293 ceils transfected with WT or (8} mutant FcRHS with deletion of 4 membrane proximal extraceiiuiar domains. FIG. 5 shows binding of (A) the FcRHS antibodies to FcRHSa by ELISA, and (B) binding of subcione supernatants to human 8 ceils. FIG, 6 shows binding of FcRHS subcione supernatants to SVT2 ceiis transfected with (A) FcRH1, (B) FcRH2, (C) FcRHS, or (D) FcRH4, FIG. 7 shows the binding of FcRHS antibody subcione supernatants to NK ceils. FIG. 8 shows (A) killing activity of FcRHS bisFabs, FcRH5-TDB (clone 10A8) and (B) HER2-TDB and (C) killing activity of FcRHS-bisFahs and (D) FcRHS-TPBs on FcRHS transfected 293 celts. FIG. 9 shows (A) killing activity and (8) T-cefi activation of FcRHS bisFabs and FcRHS-TDBs on MOLP-2 ceiis.
DETAILED DESCRIPTION I. DEFINITIONS £0039j The term "FcRHS," as used herein, refers to any native, mature FcRHS which results from processing of an FcRHS precursor protein in a cell. The term includes FcRHS from: any vertebrate source, including mammals such as primates {e.g. humans and cynomolgus monkeys) and rodents {e.g., mice and rats), unless otherwise indicated. The term also indudes naturally occurring variants of FcRHS, e.g., splice variants or allelic variants. In some embodiments, the amino acid sequences human FcRHS proteins is FcRHSa {iRTA2a; UniProt identifier Q96RD9-3; 759 aa), FcRH5b f!RTA2b; UniProt identifier Q96RD9-4; 592 aa), FeRHSc (l:RTA2c; UniProt identifier Q96RD9-1; 977 aa (SEQ ID NO:1>, UniProt identifier Q96RD9-2 {124 aa}, and/or FcRH5d <IRTA2d; UniProt identifier Q96RD9-5; 152 aa). £0040J The term "glycosylated forms of FcRHS" refers to naturally occurring forms of FcRHS that are post-transiationafiy modified by the addition of carbohydrate residues, [00413 "Percent {%} amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity . Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly availabie computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNA8-TAR) software Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maxima! alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program AUGN-2, The AUGN-2 sequence comparison computer program was authored by Genentech, inc., and the source code has been fried with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The AUGN-2 program is publicly available from Genentech, lnc„ South San Francisco, California, or may be compiled from the source code. The AUGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D, All sequence comparison parameters are set by the AUGN-2 program and do not vary, £0042| In situations where AUGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALiGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in 8. it will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence 8, the % amino acid sequence identity of A to 8 will not equal the % amino acid sequence identify of B to A. Unless specifically stated otherwise, ail % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
[0043J The terms "anti-FcR H5 antibody" and "an antibody that binds to FcRHS" refer to an antibody that is capable of binding FcRHS with sufficien t affinity such that the a ntibody is useful as a diagnostic and/or therapeutic agent in targeting FcRH5. In one embodiment, the extent of binding of an anti-FcRH5 antibody to an unrelated, non~FcRH5 protein is fess than about 10% of the binding of the antibody to FcRHS as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to FcRHS has a dissociation constant (Kd) of < 1μ.Μ, < 100 nM, < 10 nM,, < 5 Nm,, it 4 nM„ &amp; 3 nM,, < 2 nM, < 1 nM, < 0,1 mM, £ 0,01 nM, or £ 0.001 nM (e.g.. 10 s M or less, e.g. from 10¾ to 10-13 iyjs e,g,, from 10'® M to 10*13 M}. in certain embodiments, an anti-FeRH5 antibody binds to an epitope of FcRHS that is conserved among FcRHS from different species, in some embodiments, the anit~FcRH5 antibody binds an isofcrm c-specific region of the extracellular domain of FcRH5c, in some embodiments, the antl-FcRHS antibodies binds ig-iike domain 9 of FcRHSc. £0044) The term '‘antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclone! antibodies, polyclonal antibodies, muittspectftc antibodies {e.g., bispectficantibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity, [0045J An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab-SH, F{ab’}2:; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and muitispecific antibodies formed from antibody fragments. £0046) The term "epitope" refers to the particular site on an antigen molecule to which an antibody binds.
[00471 An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.
[0048| The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, /.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts, in contrast to poiycionai antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monocionaf antibody of a monocionai antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of fee antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing ail or part of the human immunogiobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
[0049) The terms ”fuii length antibody," "intact antibody,” and "whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
[0058| A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiely (e.g.. a cytotoxic moiety) or radiofabel. The naked antibody may be present in a pharmaceutical formulation.
[00511 "Native antibodies" refer to naturaiiy occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000daltons, composed of two identical light chains and two identical heavy chains thatare disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy drain variable domain, followed by three constant domains (CH1, CH2, and GH3). Similarly, from N- to C-termlnus, each light chain has a variable region (VL), also called a variable light domain or a fight chain variable domain, foiiowed by a constant fight (CL) domain. The fight chain of an antibody may be assigned to one of two types, catted kappa (k) and lambda (λ), based on the amino acid sequence of its constant domain, [0052) The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, white the remainder of the heavy and/or light chain is derived from a different source or species, f0053J A "human antibody" is one which possesses an amino add sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specificaiiy excludes a humanized antibody comprising non-human antigen-binding residues.
[0054) A "humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino add residues from human FRs, In certain embodiments, a humanized antibody will comprise substantially allot at least one, and typically two, variable domains, in which all or substantially all of the HVRs (eg,, GPRs) correspond to those of a non-human antibody, and aft or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at ieast a portion of an antibody constant region derived from a human antibody. A "humanized form* of an antibody, e,g., a non-human antibody, refers to an antibody that has undergone humanization.
[0055) The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are fi ve major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., igG<, !gG2, tgG3, IgG* IgA}, and lgA2, The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, κ, y, and μ, respectively.
[8055) The term ”Fc region" herein is used to define a C-terminai region of an immunogiobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy drain. However, fee C-terminai lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also caiied the EU index, as described in Rabat ef ai., Sequences of Proteins of immunologien! interest. 5th Ed. Public Health Service, National Institutes ol Health. Bethesda, MO, 1991.
[0057) The term "variable region" or “variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light drain (VH and VI.., respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs), (See, e.g.. Kindt ef ai, Kuby Immunology, 6th ed., W.H. Freeman and Co„ page 91 (2007),) A single VH or VL domain may be sufficient to confer antigen-binding specificity,
Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively, See, e.g., Portolano et al-, J. Immunol, 150:880-88? (1993); Clarkson ef el., Mature 352:824-628 ¢1991).
[8058) "Framework" or TR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FRi-H1(L1}-FR2-H2(L2)-FR3~H3(L3)-FR4. [9059) A "human consensus framework” is a Framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences Is a subgroup as in Kahafetal., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91 -3242, Bsthesda MD (1991), vois. 1 -3. In one for the VL, the subgroup is subgroup kappa ! as in (Kabat ef at, supra, in one embodiment, for the VH, the subgroup is subgroup Hi as in Rabat ef a/„ supra, [0060] The term "hypervariabie region" or "HVR,” as used: herein, refers to each of the regions of an antibody variable domain which are hypervariabie in sequence and/or form structurally defined loops ("hypervariabie loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariabie loops and/or from: the "complementarity determining regions" {CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariabie loops occur at amino acid residues 28-32 (LI), 50-52 (12), 91-98 (L3), 26-32 (HI), 53-56 (H2), and 96-101 (H3). (Chothia and: Lesk, J. Moi, Biol, 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-12, CDR-L3, CDR-H1, CDR-H2, and COR-H3) occur at amino acid residues 24-34 of L1, 50-58 of L2, 89-97 ofL3, 31-35B of H1,50-65 of H2, and 95-102 of H3. (Rabat et ai., Sequences of Proteins of immunological interest, 5fh Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991),) With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariabie loops. CDRs also comprise ’’specificity determining residues," or “SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviaied-CDRs, or »-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31 -34 of L1,50-55 of L2,89-96 of L3,31-358 of H1,50-58 of H2. and 95-102of H3. (See Almagroand Fransson, Front. Biosci. 13:1819-1633(2008).} Unless otherwise indicated, HVR residues and other residues in the variabie domain (e.g., FR residues) are numbered herein according to Kabat ef aL supm, [0861) An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes, in some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, in some embodiments, the VL acceptor human framework is identicai in sequence to the VL human immunogiobufin framework sequence or human consensus framework sequence.
[0062) "Affinity" refers to the strength of the sum tote! of noncovaient interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair {e.g,, antibody and antigen). The affinity of a molecule Xforits partner Yean generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
[8063) An "affinity matured" antibody refers to an antibody with one or more afterations in one or more hypervariabie regions (HVRs), compared to a parent antibody which does not possess such afterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
[0064) An "immunoconjugate" is an antibody conjugated to one or more heteroiogous moiecufe(s), including but not limited to a cytotoxic agent, [0065) The term "cytotoxic agents" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes ceil death or destruction. Cytotoxic agents indude, but are not limited to, radioactive isotopes (eg. . At211, p al pas ysøt ReiS6 ReiSS, Sm1s'\ Si212, P32,Pb212and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinbiastine, etoposide), doxorubicin, meiphaian. mitomycin C, chlorambucil, daunorubiciri or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nudeoiytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animat origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.
[0066) "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions indude: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of celt surface receptors {eg. 8-cel! receptor); and B-ceii activation.
[0067] An "isolated antibody)'" is one which has been separated from a component of its natural environment, to some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (o.g., SDS-FAGE, isoelectric focusing (IEF). capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g„ Fiatman et a/,. J. Chromatagr, 8 848:79-87 ¢2007).
[0088) An “isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its naturai environment. An isolated nucleic acid includes a nucfeic acid molecule contained in ceiis that ordinarity contain the nucleic add molecule, but the nucleic add molecule is present extrachromosomaily or at a chromosomal location that Is different from its natural chromosomal location, [0089) "Isolated nucleic acid encod ing an anti-FcRH5 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecuie{s) in a single vector or separate vectors, and such nucleic acid moiecuie(s) present at one or more locations in a host cell.
[0070) The term "vectors," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a seif-replicating nucleic acid structure as well as the vector incorporated into the genome of a host ceil into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors," [0071) The terms "host cell," "host ceil line,” and "host cell culture" are used interchangeably and refer to ceiis info which exogenous nucleic acid has been introduced, i ncluding the progeny of such ceiis. Host ceiis include ’’transformants" and “transformed ceils,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations, Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed ceil are included herein.
[0072J As used herein, "treatment" (and grammatics! variations thereof such as "threat" or "treating") refers to clinical intervention in an attempt to alter the natura! course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis, in some embodiments, antibodies provided herein are user! to delay development of a disease or to slow the progression of a disease.
[0073) The terms "cancer" and "cancerous” refer to or describe the physioiogieai condition in mammais ihat is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are net limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin’s lymphoma), blastema, sarcoma, and ieukemia, More particular examples of such cancers include squamous cell cancer, small-cel! lung cancer, non-smaii cel! lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocefluiar cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, biadder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, small intestine cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vuivai cancer, thyroid cancer, hepatic carcinoma, ieukemia and other iymphopro-liferative disorders, and various types of head and neck cancer.
[0074) A "8-cel! malignancy" herein includes non-Hodgkin’s lymphoma (NHL), including low grade/follicuiar NHL, small lymphocytic (St) NHL, intermediate grade/follicuiar NHL. intermediate grade diffuse NHL, high grade immunobiastic NHL. high grade lymphoblastic NHL. high grade smaii non-cieaved ceil NHL, bulky disease NHL, mantle ceii lymphoma, AiDS-related lymphoma, and Waldenstrom's Macroglobuiinemla, non-Hodgkin’s lymphoma (NHL), lymphocyte predominant Hodgkin's disease (LPHD), smaii iymphocytic lymphoma (SLL), chronic lymphocytic ieukemia (CLL), indolent NHL, including relapsed indolent NHL, and rituximab-refraclory indolent NHL; leukemia, including acute lymphoblastic leukemia (ALL), chronic iymphocytic leukemia (CLL), Hairy ceii ieukemia, chronic myeiobiastic ieukemia; mantle cell iymphoma; and other hematologic malignancies, Such malignancies may be treated with antibodies directed against B-ceil surface markers, sucb as FcRH5 (e.g,. FcRHSc). Such diseases are contemplated herein to be treated by the administration of an antibody directed against a B-ceii surface marker, such as FcRHS {e.g.. FcRHSc), and includes the administration of an unconjugated ("naked"} antibody or an antibody conjugated to a cytotoxic agent as disclosed herein. Such diseases are also contemplated herein to be treated lay combination therapy including an anti-FcRHS antibody (including FcRHS bispecific antibody) or anti-FcRH5 antibody drug conjugate in combination with another antibody or antibody daig conjugate, another cytotoxic agent, radiation or other treatment administered simultaneously or in series.
[0075) The term "non-Hodgkin's lymphoma" or “NHL", as used herein, refers to a cancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin's lymphomas can generally be distinguished from non-Hodgkin’s lymphomas by the presence of Reed-Sfemberg ceiis in Hodgkin’s lymphomas and the absence of said ceiis in non-Hodgkin’s lymphomas. Examples of non~Hodgkin‘s lymphomas encompassed by the term as used herein include any that woufd be identified as such by one skilled in the art (e,g, an oncologist or pathologist) in accordance with classification schemas known in the art< such as the Revised European-Ameriean Lymphoma (REAL) scheme as described in Color Alias of Clinical Hematology (3rd edition), A. Victor Hoifbrand and John E. Pettit (eds.) (Harcourt Pu blishers Ltd. , 2000). See, in particular, the iists in FIGS. 11.57, 11.58 and 11.59. More specific examples include, but are not limited to, relapsed or refractory NHL, front fine low grade NHL, Stage tll/fV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/or fymphoma, smail lymphocytic lymphoma, 8-cell chronic lymphocytic leukemia and/or proiymphocytic leukemia and/or smaif lymphocytic lymphoma, B-ceii proiymphocytic lymphoma, immunocytoma and/or Symphopiasroaeytic lymphoma. fymphoplasmacytic lymphoma, marginal zone B-ceii lymphoma, splenic marginal 2one lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy ceii leukemia, plasmacytoma and/or plasma ceff myeloma, tow grade/fofficufar iymphoma, intermediate grade/foificufar NHL, mantle ceil lymphoma, foilicfe center fymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-ceii lymphoma, aggressive NHL, (inciuding aggressive front-fine NHL and aggressive reiapsed NHL), NHL, relapsing after or refractory to autoiogous stem cell transplantation, primary mediastinai iarge B-cefi iymphoma, primary effusion iymphoma, high grade immunobiastic NHL. high grade lymphoblastic NHL, high grade smail non-cieaved ceii NHL, bulky disease NHL, Burkitfs iymphoma, precursor (peripheral) large granular iymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin (cutaneous) lymphomas, anapiastic large ceii lymphoma, angiooentric iymphoma, (0076) Plasma celfs disorders result from the uncontroiied division or multiplication of a plasma cell clone. Plasma cells arise from activated B lymphocytes (/e, B-cells), Each B-eell produces a unique receptor, known as the B-ceii receptor, arrayed on its ceii surface that is specific for a foreign substance, i.e,, antigen. When a B-call receptor binds its cognate antigen, the ceii expressing the receptor is activated to re-enter the ceii cycle, producing many cionaf copies of itself. The clones mature info piasma ceits that reside principally in the bone marrow and that are specialized to produce copies of the B-cefl receptor that are reieased into the blood stream as antibodies. In a piasma ceii. disorder, the piasma ceii or the parent B-ceii suffers genetic damage resuiting in suppression of or insensitivity to the normal restraints on ceii division and/or activity. Daughter piasma cells derived from such coifs are malignant in that they may divide unchecked and/or generate excess amount of the same immunoglobulin (antibody). Often the immunogtobutin produced is incomplete or has an incorrect conformation that can result In accumufation of the protein (aiso known as monoclonal protein, M protein, paraprotein oramytoid protein, dependent on the specific disorder) In the serum, tissues or organs (especially the kid neys), leading to organ dysfunction and/or failure. Plasma cell disorders include monoclonal gammopathies of undetermined significance (MGUS). multiple myeloma (MM), macrogfobulinemia, heavy chain diseases, and systemic fight-chain amyloidosis (AL), which are differentiated based on the proliferative nature of the eione, the extent of marrow involvement, and the type of U protein expressed. Additional plasma cell disorders are solitary plasmacytoma, extramedullary plasmacytoma, multiple solitary plasmacytomas, piasma ceff leukemia, Waldenstrom's mac-rogiobuiinaemia, δ-cel non-Hodgkin lymphoma, B-ceil chronic lymphocytic leukemia. (0077) The term ”FcR HS-positive cancer" refers to a cancer comprising cells that express FcRHS on their surface. For the purposes of determining whether a cell expresses FcRHS on the surface, FcRHS mRNA expression is considered to correlate to FcRHS expression on the cell surface. In some embodiments, expression of FcRHS mRNA is determined by a method selected from In situ hybridization and RT-PCR (including quantitative RT-PCR), Alternatively, expression of FcRHS on the cell surface can be determined, for example, using antibodies to FcRHS in a method such as immu-nohisiochemistry, FACS, etc. in soma embodiments, FcRHS is one or more of FcRHSa, FcRHSb, FcRHSc, UniProt Identifier QS6RDS-2, and/or FcRHSd. in some embodiments, the FcRHS is FcRHSc, (0078) The term "FcRHS-positive cell” refers to a ceii that expresses FcRHS on its surface, in some embodiments, FcRHS is one or more of FcRHSa, FcRRSb, FcRHSc, UniProt identifier Q96RD9-2, and/or FcRHSd. in some embodiments, the FcRHS is FcRHSc, (0079) An "effective amount” of an agent, eg., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. (0080) An "individua!” or ‘’subject" is a mamma!. Mammals include: but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e,g., humans and non-human primates such as monkeys), rabbits, and rodents (e,g„ mice and rats). In certain embodiments, the individual or subject is a human. (0081) The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combi nation therapy, contraindications and/or warnings concerning the use of such therapeutic products. (0082) The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained there in to be effecti ve, and which contains no additional com ponents which are unacceptably toxic to a subject to which the formulation would be administered. (0083) A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceuiicaiiy acceptøble carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative, (0084) "Alkyl" is CrCi8 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyf (Ms, -CH3), ethyl (Et, -CH2CH3), 1-propyi (n-Pr, n-propyf, -CH2CH2CH3), 2-propyl (S-Pr, i-propyl, -CH{CH3}2}; 1-butyl {n-Bu, n-butyl, -CH2CH2CH2CH3}, 2-methyi-T-propy! (i-Bu, i-buiyf, -CHjCHCCH^), 2-butyl {s-Bu, s-butyt, -CH(CH3)CH2CH3), 2-melhyi-2-prapyf {t-8u, t-butyi, -0(0¾}^. 1-peoiyi (n-pentyl, -CHjtCHgCHjjCHjjCH^, 2-pentyl {-CH{CH3)CH2CH2CH3}, 3-pentyl (-CH(CH2CH3)2), 2-methyi-2-butyi (-C(CH3}2CHaCH3}, 3-methy!-2-butyf {-CH{CH3)CH(CH3)2}, 3-metbyl-1 -butyl (-CHgCHgCHiCH^), 2~methy!-1 -butyl {-CH2CH{CH3)CH2CH3). 1-hexyl {-CH2CH2CH2CH2CH2CH3), 2-hexyI¢-CH(CH3)CHaCH2CH2CH3), 3-hexyl (-CHiCH2CH3HCH2CH2:CH3}}, 2-metbyl-2-peniyi{-C(CH5)2CH2CH2CH3}! 3-raeihyl-2-pentyi {-CH{CH3)CH(eH3)CH2CB3}, 4-meihyl-2-penfyt (-CH(CH3}CH2CH(CH3)s), 3-mefhyS~3~peniy! [-C{eH3}{CH2CH3}2), 2-methyl-3-pe:ntyl{-CH{CH2CH3}CH(C H3)2). 2,3-dimethyi-2-butyi <-C{CH3)2CH(CH3)2>, 3,3-dimethyl-2-butyl (-CH[CH3)C(CH3)3. The term ”CrCg alkyl," as used herein refers to a straight diairt or branched, saturated or unsaturated hydrocarbon having from i to 8 carbon atoms. Representative *CrC8 aikyf groups include, but are not limited to, ’’methyl, -ethyl, -n-propyi, “n-butyl, -n-pentyl, -n-hexyi, -n-heptyl, -n-octyf, -n-nonyl and -n-decyt; while branched Ct-Cg alkyls include: but are not limited to,-isopropyl, -sec-buty!, -isobutyl, -teff-butyi, -isopentyi, 2-mathyibutyl, unsaturated C-j-Cg alkyls indude, but are not limited to, -vinyl, -alyl, -1-butenyl, -2-buteny!, -isobutylenyi, -t-pentenyi, -2-pentenyl,-3-methyl-1~butenyi, -2-methyi-2-butenyl, -2,3-dimetbyi-£-butenyi, 1-hexyl. 2-hexyi, 3-hexyl,-acetylenyi, -propynyi. -1-butynyl, -2-butynyl. -1-pentynyl, -2-peniynyi, -3-methyi-l butynyi. A C^-C8 alky! group can be unsubstituted or substituted with one or more groups including, but not limited fo,-CrC8 alkyl, -0-(CrCs alkyl}, -aryl, -C{0)R’, -0C(0)R*, -C(0)0R\ -C(0)NH2, -C(O}NHR’-C(0)N(R'i2 -NHC(0)R’ -SOjR’, -S{0)2R\ -S{0)R', -OH, -halogen, -N3>-NH2, -NH{R’}, -N(R')2 and -CN; where each R’ is independently selected from H, -C-i-Cy alkyl and aryl, [0085} The term "C;-C.i2 alkyl," as used herein refers to 3 straight chain or branched, saturated or unsaturated hydrocarbon having from 1 to 12 carbon atoms. A C j-C12 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, ~tVCs alkyl, -O-iC^Cg alkyl),-aryl, -C(0)R\ -0C(0}R’, -C(0}0R\ ~C{0)NHz> -C[0)NHR\ -C{0}N(R')2 -NHC{0}R’, -S03RVS{0)2R\ -S{Q}R\ -OH, -halogen, -N3, -NH2, -NH(R’), -N[R')2 and -CN; where each R' is independently selected from H, -CrC8 alkyl and aryi.
[0986J The term "CrC3 alkyi," as used herein refers to a straight chain or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms, Representative "Cs-C3 alkyi" groups Include, but are not limited to, -methyl, -ethyl, -n-propyi, -n-butyl, -n-pentyl, -and n-hexyl; while branched C-j-C6 alkyls Include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyi,-isopentyi, and 2-metbyibutyl; unsaturated CrCg alkyl include, but are not limited to, -vinyl, -aliyl, -1 -butenyl, -2-butenyl, and -isobutylenyi, -1 -pentenyl, -2-pentenyi, -3-methy!-1-butenyf, -2-meihyl-2-butenyi, ~2,3-dimethyl~2-buienyl. 1-hexyl, 2-hexyi, and 3-hexyl, A C5-Ce group can be unsubstituted or substituted with one or more groups, as described above for CrC3 alkyl group.
[0087} The term "CrC4 alkyl" as used herein refers to a straight chain or branched, saturated or unsaturated hydrocarbon having from 1 to 4 carbon atoms. Representative "C--C4 alkyi" groups include, but are not limited to, -methyi, •ethyl, -n-propyi, -n-butyi* while branched C;-C,, alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyi, -fert-butyl: unsaturated CrC4 alkyis include, but are not limited to, -vinyl, -ally!, -t-butenyi, -2-buienyi. and -isobutylenyi, A C4 -C4 alky! group can be unsubstituted or substituted with one or more groups, as described above for C rC3 aikyl group. [0088} "Aikoxy" is an alkyi group singly bonded to an oxygen. Exemplary aikoxy groups include, but are not limited to, methoxy (-OCH3) and ethoxy (~QGH2CH3). A X t-Cs aikoxy" Is an aikoxy group with 1 to 5 carbon atoms, Aikoxy groups may can be unsubstituted or substituted with one or more groups, as described above for aikyl groups.
[0089} "Alkenyl" is C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at (east one site of unsaturation, i.e. a carbon-carbon, sp2 double bond. Examples include, but are not iimited to: ethylene or vinyi {-CH=GH2}, ally! (-CH2CH=XH2). cycfopentenyl (-CSH?), and 5-hexeny! fCH2 CH2CH2CH2CH==CH2). A “CrCa alkenyi" is a hydrocarbon containing 2 to 8 normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp2 double bond.
[0990} "Atkyoyr is C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a cafbon-cafbon, sp tripie bond, Exampies indude, but are not limited to; acetylenic (-feCH) and propargyt {-CH2C»CH}. A "C2-C8 aikynyi" is a hydrocarbon containing 2 to 8 normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp triple bond.
[9091} "Alkyiene" refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent aikane. Typical alkyiene radicals indude, but are not iimited to; methylene (-CH.,-) 1,2-ethy!{*CH2CH2-), 1,3-propyl t-CH2CH2CH2-. 1,4-butyi {-CH2CH2CH2CH2-), and the like.
[0992} A "CrC10 alkyiene” is a straight chain, saturated hydrocarbon group of the formula -(CH2)M0-. Examples of aC4-Cieaiky!eneinciude methylene, ethylene, propylene, butylene, pentyiene, hexylene, heptylene, ocytylene, nonylene and decaiene, [9093) "Aikenyiene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent aikene. Typical aikenyiene radicals include, but are not limited to: 1.2-ethylene (-CH=CH-), [0994] ” Alky ny! ene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typica! alkynylene radicals include, but are not limited to: acetylene {-C*C->, propargyl (-€Η20«0*}( and 4-pantynyi {-CHjCHjCHjCsC-}.
[00951 "Aryl* refers to a carbocyolic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and aothracenyi. A carbocyoiic aromatic group or a heterocyclic aromatic group can be unsubstituted or substituted with one or more groups including, but not limited to, -CrC8. alkyl, -©-{CrCgaikyt,), -aryl, -C(0)R’< -OC(0}R’, -C(0)0R\ -C(0)NH2> -CfOJNHR’, -CplNfR’^-NHCfOiR', -S{0)2R‘, -S(0)R', -OH, -halogen, -% ,-NH2,-NH{R'), -N(R’)2 and -CN; wherein each R’ is independently selected from H, -C:-Cg aikyi: and aryl. {90961 A "Cfl'C2ø aryT is an aryl group with 5 to 20 carbon atoms in the carbocyoiic aromatic rings. Examples of C5-C20 aryl groups include, but are not limited to, phenyl, naphthyl and anthraeenyl. A Cs-C20 aryl group can be substituted or unsubstituted as described above for ary! groups. A HC5-C14 aryl” is an aryl group with 5 to 14 carbon atoms In the carbocyclic aromatic rings. Examples of Cg-C«. ary! groups include, but are not limited to, phenyl, naphthyl and anthra-cenyf. A C5-C14 aryl group can be substituted or unsubstituted as described above for aryl groups. {0097] An "aryiene" is an aryl group which has two covalent bonds and can be in the ortho, meta, or para configurations as shown in the following structures:
in which the phenyl group can be unsubstituted or substituted with up to four groups including, but not limited to, -C^Cj alkyls. -0-{CrC6 alkyl), -aryl, -C{0)R\ -0C(O)R*, -C{0}OR\ -C(0}NH2, - CfOJNHR', -C(0}N<R‘}2-NHC{0)R’, -S(0)2R’, -S(0)R!, -OH, -halogen, -N3, -NH2, -NH{R), -N{R’)2 and -CN; wherein each R' is independently selected from H, -C aikyi and aryl. {0098] "Aryialkyi” refers to an acyclic afky! radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenyfetharvt -yf, 2-pheny!ethen~1-yi, naphthytmethyi, 2-naphthyiethan-1-yl, 2-naphihyletben-1-y!, naphthobenzyi, 2-naphthophenyiethan-1-yi and the like. The arylalkyl group comprises 6 to 20 carbon atoms, e.g, the alkyl moiety, including alkanyi, alkenyl or aikynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and ihe aryi moiety is 5 to 14 carbon atoms.
[0099] "Heteroaryiaikyf refers to an acyclic alky! radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal orsp3 carbon atom, is replaced with a heteroaryl radical. Typical heteroaryiatkyl groups include, but are not limited to, 2~benzimidazo!y!methyl, 2-furyietbyI. and the like. The heteroarylalky! group comprises 0 to 20 carbon atoms, &amp;,g. the aikyi moiety, Including alkanyi, alkenyl or alkynyl groups, of the heteroaryiatkyl group is 1 to 8 carbon atoms and the heteroary! moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, Ο, P, and S. The heteroaryi moiety of the heteroarylalky! group may be a monocycle having 3 to 7 ring members ¢2 to 6 carbon atoms or a bicycle having 7 to 10 ring members {4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: a blcyclo {4,51, {5,6], {5,6] or [6,8] system, [0100] ‘’substituted aikyi,” “substituted aryi,” and "substituted arylalkyl1' mean alkyl, aryl, and arylalkyl respectively, in which one or more hydrogen atoms are each independent^ replaced with a substituent. Typica! substituents include, but are not Imited to, -X, -R, -G\ -OR, -SR, -5a -NR2, -NR3, ~NR, -CX3, -CN, -OCR, -SCN, -N-C-O, -NCS, -NO, -N02< =N2, -N3, NC(*0)R, -C{*G)R,-C{=Q}NR2, ~SQ3a -SGaH, -S{*0}2R;, -OS{*0}aGR, ~S{=0)2NR, ~S(»0)R. -OR(=0)(OR}2, -P(=0)(OR)2, -P03, -PO3H2, -C{-0}R, -C{=0)X, -C{~S)R, -C02R, -C02, -C{=S)OR, -C{=0)SR;! -C(*S)SR, -€<*0)NR2, -C(*S)NR2, C(*NR)NR2, where each X is independently a halogen: F, Cl, Br. or i: and each R is independently -H, C2-C18 alkyl, Cft-C2ft aryi, C3-CM heterocycie, protecting group orprodrug moiety. Alkyiene, aikenylene, and afkynylene groups as described above may also be similarly substituted. {0101] "heteroaryr and ”heterocyo!e“ refer to a ring system in which one or more ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. The heterocycie radical comprises 3 to 20 carbon atoms and 1 to 3 heteroaloms selected from N, O, P, and S. A heterocycie may be a monocycie having 3 to 7 ring members (2 to 8 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members {4 to 9 carbon atoms and 1 to 3 heteroaloms selected from N, O, P, and S), for example: a blcyclo [4,5], [5,5], [5,6], or [8,6] system.
[0102] Exemplary heteroeycles are described, e.g,, in Paquette, Leo A., "Principles of Modem HeterocyciicGhernistry" (W.A, Benjamin, New York, 1968), particularly Chapters 1,3,4,6,7, and 9; "Trie Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley &amp; Sons, New York, 1950 to present), in particular Volumes 13,14,16,19, and 28; and J, Am, Cheat, See. (1960) 82:5566.
[0103J Examples of heterocycies indude by way of example and not limitation pyridyl,dfhydroypyridyi,tetrahydropyrtdyi {piperidyl}, thiazolyl, teirahydrothiophenyf, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrroiyi, pyrazolyi, imidazoiyi, tetrazotyl, benzofuranyl, ihianaphthalenyt. indolyi, indolenyi, quinolinyl, isoquinolinyl, benztmtda-zoiyl, piperidinyt, 4-pipeddonyl, pyrrolidmyl, 2-pyrroiitlonyi, pyrroiinyt, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahy-dfopytanyi. bis-tetrahydrapyranyt, tetrahydroquinolinyl, teirahydroisoquinolinyl, decahydroqutnoiinyi, octahydrotsoquin-oiinyi, azocinyi, triazinyi, 6N-1,2,5-ihiadiassnyi. 2H,6H~1,5,2~dithiazinyt, tbienyi, ihtanihrenyl, pyranyl, isobenzofuranyt, chromenyi, xanfhenyi, phenoxathinyl, 2H-pyrrolyi, isothiazoiyi, tsoxazofyi, pyrazmyl, pyridazinyS, indoiizinyi, Ssoindotyi, 3B~indaiyl, 1 H-indazolyf, purinyl, 4H-quinolfzinyl, phthaiazinyl, naphthyridinyf, quinoxaftnyl, quinazolinyi, einnolinyl, pte-ridinyl, 4aH-carbazolyl, carbazolyl, p-carbolinyl, phenanthridinyl, acridfrtyl, pyrimidinyl, phenanthrolinyl, phenazinyi, phe-notbSazinyl, furazanyl, phanoxazinyl, tsochromanyi, chromanyi, imidazoiidinyl, imidazolinyl, pyrazoiidinyl, pyrazoiinyi, piperazinyi, indolinyl, isoindoinyl, quinuclidinyl, morphoiinyi, axazolidinyi, ben20tfiazotyi, benzisoxazofyi, oxindoiyl, ben-zoxazolinyl, and isatinoyl.
[01041 By way of example and not limitation, carbon bonded heterocycies are bonded at position 2, 3,4,5, or 6 of a pyridine, position 3,4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 6« or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, fetrahydrofuran, thiofurart, thiophene, pyrrole or tetrahydropyrroie, position 2,4, or 5 of an oxazole, imidazole or thiazole, position 3,4, or 5 of an isoxazole, pyrazole, or isothiazoie, position 2 or 3 of an aziddine, position 2, 3, or 4 of an azetiidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more typically, carbon bonded heterocycies include 2-pyridyl, 3-pyridyi, 4-pyridyl, 5-pyridyi, 8-pyridyi, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyi, 4-pyrimidinyl, 5-pyrimidinyf, 6-pyrimidirsyi, 2-pyrazlnyl, 3-pyrazinyi, 5-pyrazmyf, 6-pyrazinyl, 2-thiazoiyl, 4-thiazolyl, or $-thiazo!yl, [01051 By way of example and not limitation, nitrogen bonded heterocycies are bonded at positron 1 of an aztridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidtoe, 2-imidazofine, 3-imidazoiine, pyrazole, pyrazoilne, 2-pytazoiine, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of a isoindoie, or isoindoline, position 4 of a morpholine, and position 9 of a carbazote, or p-carboline. Sfill more typically, nitrogen bonded heterocycies include 1-azitidyl, 1-azeiedyl, 1 -pyrroiyi, 1-imidazoiyi, 1-pyrazolyi, and 1-ptperidinyf.
[01061 A "Cg-Cg heterocycle” refers to an aromatic or non-aromatic C3-G3 carbocycie in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N, Representative examples of a C3-C8 heterocycle include, but are not limited to, benzofuranyl, benzofhiophene. indolyi. benzopyrazolyl, coumarinyl, isoquinolinyl, pyrroiyi, thiophenyl, furanyl, thiazolyl, imidazoiyi, pyrazoiy!, triazolyl, quinoimyl, pyrimidinyl, pyridinyi, pyridonyi, pyrazinyi, pyridazinyl, isofhiazoiyi, isoxazolyt and tetrazoiyl. A C3-Cs heterocyde can be unsubstituted or substituted with up to seven groups including, but not limited to, -0^-0¾ alkyl, -0-{CrC8 alkyl}, - aiyi, -C(0)R\-0C(0)R‘, C{0}0R',-C{O)NH2, CfOJNHR', -C(0)N(R’)2 -NHC(0)R', -S{0}£R'.-S(0)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R’ is independently selected from H, -CrCB alkyl and aryl, [01071 C^-Cg heterocycio" refers to a G3-Cfi, heterocyde group defined above wherein one of the heterocycle group’s hydrogen atoms is replaced with a bond . A C3-Cs heterocycio can be unsubstituted or substituted with up to six groups including, but not limited to, -CrCs alkyl, -0-(CrC3 alkyl), -aryl,-€fO)R\ -OC{G)R\ -C(0)OR\ -C(0)NH2, -C(0)NHR', -C(0)N(R')a -NHC{0)R', -S{0)?R\ -S(0)R\ -OH, -halogen, -N3, -NH2, -MH(R’). -N{R}2 and -CN; wherein each R’ is independently selected from H: -CrC8 alkyl and aryl.
[0108} A "Cj-C^o heterocycle” refers to an aromatic or non-aromatic C3-C3 carbocycie in which one to four of the ring carbon atoms are independently replaced with a beteroatom from the group consisting of O. S and N. A C3-C20 heterocycle can be unsubstituted or substituted with up to seven groups including, but not limited to, -0,-0¾ aikyf, -0-(0.,-0,- alky!}, -aryl, -C(0)R’, -OC(0)R\ -C[0}OR’, - C(0)NH2, -C{0)NHR’, -0{O)N(R )2 -NHC(0)R\ -S{0)2R\ -S(0)R'. -OH, -halogen, -Ns, -NH2, - NH(R'), -N(R');; and -CN; wherein each R’ is independently selected from H, -C ,-Cg alky! and aryl.
[0109} "CrCss heterocycio" refers to a C3-C20 heterocycle group defined above wherein one of the heterocyde group's hydrogen atoms is replaced with a bond. "Carbocycie” means a saturated or unsaturated ting having 3 to 7 carbon atoms as a monocycie or 7 to 12 carbon atoms as a bicyde, Monocyclic carbocydes have 3 to 6 ring atoms, stiil more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as a bicycle [4,5), [5,5), [5,6) or [6,6] system, or 9 or 10 ring atoms arranged as a bicycie (5,6] or [6,6) system. Examples of monocydic carbocycie inciude cyctopropyt, cyclobuiyf, cyclopentyl, 1-cy-ciopeoM-enyi, l-cydopent-2~enyi, 1-cydopent-3-eny!f cycfohexyf, 1-cyciohex-l-enyl, 1-cyclohex-2-enyl, 1-cydobex-3-enyl, cycfoheptyl, and cyciooctyt.
[0110} A "C3-C8 carbocycie" is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyciic ring. Representative C3-C3 catbocycfes inciude, but ate not iimited to, - cydopropyi, -cyclobutyi, cyciopentyi, -eyciopen-tadienyl, -cydobexyl, -cyciohexenyl, -1,3-cyciohexadienyi, -1,4-cyciohexadtenyi, -cycioheptyf, -1,3-cyciohepfadierty!, -1,3,5-cydobeptatrienyl, - cyctooctyi, and -cyclooctadienyl. A C3~CS carbocycie group can be unsubstituted or substituted with one or more groups induding, but not limited to, -0,.0¾ alkyl, -O~(Cr08 alkyl), -aryl, -C{0)R',-0C(0)R’, -C(0)OR’, -C(0)NH2, -C(0}NHR\ -C(ø)N(R!}2 -NHC(0)R\ -S(ø)2R\ -S(0)R\ -OH, - halogen, -N3. -NH2, -NH(R'), -N(R)2and -CM; where each R' is independently selected from H, -C5-C§ alky! aed aryl.
[0111] A ”CS-CS carbocycia" refers to a C^-C^ carbocyde group defined above wherein one of the carbocycte groups' hydrogen atoms is replaced with a bond. É0112J “Linker* refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, linkers include a divalent radicaf such as an afkytdiyf. an aryldiyl, a heteroaryldiyS, moieties such as: -(CR^CKCR;,),,··. repeating units of atkyioxy (e.g. poSyethyieaoxy, PEG, pofymethyl-eneoxy) and aikytamino (e.g. poiyethyfeneamino, Jeffamine™}; and diacid ester and amides including succinate, sue-cinamide, digiycoiate, malonate, and caproamide, in various embodiments, tinkers can comprise one or more amino acid residues, such as valine, phenylalanine, iysine, and homofysine. (01131 The term "chiral" refers to molecules which have the property of non-supenmposabity of the mirror image partner, while the term "achiral" refers to molecules which are superimposabfe on their mirror image partner, [0114] The term "stereoisomers* refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space, [0115| ’"Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another, Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
[0110J "Enantiomers" refer to two stereoisomers of a compound which are non-superimpesabte mirror images of one another.
[0117J Stereochemical definitions and conventions used herein generally folfow S, P, Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Efief, E. and Wilen, 3., Stereochemistry of Organic Compounds (1994) John Wiley &amp; Sons, Inc,, New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized iighl. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration ofthe molecule about its chiral centers). The pref ixes d and 1 or {+} and (-) are employed to designate the sign of rotation of piane-poiartzed light by thecompound, with (-) or 1 meaning that the compound is ievoratatory. A compound prefixed with {+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture, A 50:50 m ixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or sfereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
[011 β| "Leaving group" refers to a functional group that can be substituted by another functional group. Certain leaving groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesuifonyl (rnesyl), p-toluenesuifonyl {tosyl}, trifluoromethylsuifonyl (inflate), and trifluorometbylsultonate.
[0119| The term; "protecting group” refers to a substituent that is commonly employed to block or protect a particular functionality while reacting other functional groups on the compound. For example, an "amino-protecting group" is a substituent attached to an amino group that blocks or protects the amino functionality in the compound. Suitable amino protecting groups include, but are not limited to, acetyl, tnfiuoroaceiyi, t-butoxycarbonyf (BOC), benzyioxycarbonyi (CSZ) and 9-fluorenyimethylenoxycarbonyS (Fmoc). For a genera! description of protecting groups and their use, see T. W, Greene, Protective Groups in Organic Synthesis, John Wiley &amp; Sons, New York, 1991, or a later edition.
[01201 As is understood by one skilled in the art, reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X", [0121J It is understood that aspect and embodiments described herein include "consisting" and/or "consisting essentially of aspects and embodiments. As used herein, the singular form "a", "an”, and "the" includes plural references unless indicated otherwise.
ft. COMPOSITIONS AND METHODS
[0122J Provided herein are antibodies that bind to FcRH5 including Bispedfic antibodies and immunoconjugates comprising such antibodies. Antibodies and immunoconjugates may be useful, e.g., for the diagnosis or treatment of FcRHS-positive cancers, in some embodiments, the anti-FcRH5 antibody binds an isoform c-specifie region of the extracellular domain of FcRHSc, in some embodiments, the anti-FcRHS antibodies bind !g~!ike domain 9 of FCRH5c, [0123J Without being bound by theory, the selection of the precise antigen for the antibodies of the present invention was driven by at least three important considerations. First, there was a need for little to no cross-reactivity with FcRHS isoforms other than FcRHSc, such as isoform a and isoform h, to avoid the resulting therapeutic from binding to nontarget molecules and thus reducing its effectiveness. As illustrated in Figure 1. domain 9 of FcRHS is an example of a unique sequence among the three isoforms. Next there was a need for tittle to no cross-reactivity with FcRH family members other than FcRHS. such as FcRHl, FeRH2, FcRHS, and FcRM4. This is difficult because of the generaffy highiy conserved nature of the last ig-iike domains in many of the FcRH famify members. Bui because of the paraiiei need for FcRH5 isoform c specificity, an antibody that binds the last fg-!ike domain was pursued. Finaiiy. for antibodies to be used in therapeutic molecules that work to bring targe structures in close proximity, such as T-ceiis and tumor celts using a bispecific antibody fonnat, K is known that tumor epitopes doser to the cefi membrane are more effective (see, e.g., Bfuemei et ai. Cancer immune! immunother. (2010) 59:1197 1209). Sometimes described as the theory of kinetic segregation, the eel! membrane proximal location of domain 9 of FcRH5 is a desirable antigen target in this context To meet these considerations and as described in detail below, certain embodiments of the antibodies of the present invention were developed. (61243 Provided herein are isolated anti-FcRH5 antibodies thai binds an isoform e-specific region of the extraceiiufar domain of FcRHSc. in some embodiments, the isoform c-specific region comprises ig-iike domain 9. in some embodiments, the lg*iike domain 9 is aiso eaiied Ig-iike C2~fype 8. in some embodiments, the isoform c-specific region comprises amino acids 754-835 of SEQ (0 NO:1. in some embodiments,, the isoform c-specific region comprises amino acids 752-834 of SEQ iO NO:1. in some embodiments, the isoform c-specific region comprises amino acids 743-850 of SEQ iø NQ:1, in some embodiments, the isoform c-specific region comprises amino acids 745-851 of SEQ ID NG:1. in some embodiments, the isoform c-specific region comprises amino, acids about any of 1, 2,3,4, 5, 6, ?, 8, 9.10,11,12,13, 14, or 15 from the N-terminaf and/or C-terminai boundary, in some embodiments, the isoform c-specific region comprises amino acids from about any of 750,751,752,753, or 754 to about any of 830, 831, 832.833, 834, 835, or 836 of SEQ iD NO:1. in some embodiments, the antibodies binds FcRHSc and/or the isoform c-specific region with an affinity of < 5 nM, or < 4 nM, or < 3 nM, or < 2 nM, or <1 nM, and optionaiiy >. 0.0001 nM, or > 0.001 nM, or > 0,01 nM. (0125J in some embodiments of any of the antibodies, the antibody has one or more of the following characteristics: a) cross reactive with foil length human and cyno FcRH 5 (/,e, binds full length human FcRHS and binds foil length cyno FcRHS), b) does not significantly cross react with FcRH 1 FcRH2, FcRHS, and/or FcRH4 {/.©>, does not significantiy bind FcRHl, FcRH2, FcRH3, and/or FcRH4), c) binds to endogenous FcRHS, d) does not cross react with FcRHSa (/_e., does not significantiy bind FcRH5a), and e) does not cross react with another ig-iike domain of FcRHS (/,e, does not significantiy bind another ig-iike domain of FcRHS}. Methods of determining the ability to bind are known in the art and described befow. (01263 Provided herein, and in some embodiments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:3S, HVR-H2 comprising the amino acid sequence of SEQ ID No:62, and HVR-H3 comprising the amino acid sequence of SEQ iD NO:86; and/or b) a fight chain comprising a HVR-L1 comprising the amino acid sequence of (SEQ !D NO;2, HVR-L2 comprising the amino acid sequence of SEQ iD NO:14, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:26. in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:50 HVR-H2 comprising the amino acid sequence of SEQ ID NO:?4, and HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:88. in some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD NO;111 and/or a VI sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD NO: 110. In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:111 and/or a VL sequence of SEQ ID NO:110, In some embodiments of any of the antibodies, the antibody comprises six HVRs of 1C8.1. In some embodiments, the antibody comprises VH domain and VL domain of 1C8.1. In some embodiments, the antibody binds an isoform c-specific region of the extracellular domain of FcRHSc (e.g., ig-iike domain 9), So some embodiments, the antibody is cross reactive with foil length human and cyno FcRHS. in some embodiments, the antibody does not significantly cross react with FcRHl, FcRH2, FcRHS, and/or FcRH4, In some embodiments, the antibody binds to endogenous FcRHS. In some embodiments, the antibody binds 8-ceiis. in some embodiments, the antibody does not significantly bind NK ceils and/or monocytes.
[0127] Provided herein, and in some embodiments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NQ:39, HVR-H2 comprising the amino acid sequence of SEQ ID NO:63, and HVR-H3 comprising the amino acid sequence of SEQ iD N0.87; and/or b) a light chain comprising a HVR-L1 comprising the amino add sequence of SEQ ID MO:3. HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and MVR-L3 comprising the amino acid sequence ofSEQ !D NO:27. In some embodiments, the heavy chain comprising a HVR-Ht comprising the amino acid sequence of SEQ ID NOtSt, HVR-H2 comprising the amino acid sequence of SEQ ID NO:75, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:99, in some embodi ments, the antibody comprises a VH sequence having at feast about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD NO: 113 and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino add sequence ofSEQ iO NO :112, In some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ !D NO-135 and/or a VL sequence having at feast about any of 90%., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% sequence identity to the amino acid sequence of SEQ fD NO:134. in some embodiments, the antibody comprises a VH sequence of SEQ iO NO: 113 and/or a VL. sequence of SEQ fD NO: 112. fn some embodiments, tbe antibody comprises a VH sequence of SEQ iD NO:135 and/or a VL sequence of SEQ ID NO: 134, in some embodiments of any of the antibodies, the antibody comprises six HVRs of 1 G7.2. !n some embodiments, the antibody comprises VH domain and VL domain of 1G7.2, in some embodiments of any of tbe antibodies, the antibody comprises six HVRs of 1G7.2'. in some embodiments, the antibody comprises VH domain and VL domain of 1G7.2’. In some embodiments, the antibody binds an isoform c-specific region of the extraceiiuiar domain of FcRH5c (e,g,; ig-like domain 9). In some embodiments, the antibody is cross reactive with fuii (ength human and cyno FcRHS. in some embodiments, the antibody does not significanfiy cross react with FcRHI, FcR2, FcRH3, and/or FcRH4. in some embodiments, the antibody binds to endogenous FcRHS. in some embodiments, the antibody binds 8-ceiis. in some embodiments, the antibody does not significantly bind NK ceiis and/or monocytes, in some embodiments, the antibody does not significantly cross react with FcRHSa, [0128] Provided herein, and in some embodiments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of (SEQ !D NO-40, HVR-H2 comprising the amino acid sequence of SEQ ID NO:64, and HVR-H3 comprising the amino acid sequence of SEQ fn NO:88; and/or b) a fight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ iD NO:4, HVR-L2 comprising the amino acid sequence of SEQ iD NO:16, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:28, in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:52. HVR-H2 comprising the amino acid sequence of SEQ fD NO:76, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 in some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of (SEQ fD NO:115 and/or a VL sequence having af ieast about any of 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:114. In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:115 and/or a VL sequence of SEQ ID NO:114, fn some embodiments of any of the antibodies, the antibody comprises six HVRs of 2H7.3, in some embodiments, the antibody comprises VH domain and VL domain of 2H7.3, fn some embodiments, the antibody binds an tsoform c-specific region of the extraceiiuiar domain of FcRHSc (e.g., ig-iike domain 9). in some embodiments, the antibody is cross reactive with full length human and cyno FcRHS. in soma embodiments, the antibody does not significantly cross react with FcRHI, FcRHS, and/or FcRH4. in some embodiments, the antibody binds to endogenous FcRHS, In some embodiments, the antibody binds 8~ceils. in some embodiments, the antibody does not significantly bind NK cells and/or monocytes.
[81291 Provided herein, and in some embodiments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NQ:41, HVR-H2 comprising the amino acid sequence of SEQ ID NO:65, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:89; and/or b) a tight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ fD NO:5, HVR-L2 comprising the amino acid sequence of SEQ ID NO:17, and HVR-L3 comprising the amino add sequence of SEQ iD NO:29. in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID :NQ:53, HVR-H2 comprising the amino acid sequence of SEQ ID NO:77. and HVR-B3 comprising the amino acid sequence of SEQ ID NO:101. in some embodiments, the antibody comprises a VH sequence having at feast about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ SD NO:117 and/or a VL sequence having at ieast about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ !D NO:116. In some embodiments, the antibody comprises a VH sequence of SEQ iD NO: 117 and/or a VI sequence of SEQ ID NO: 118. in some embodiments of any of the antibodies, the antibody comprises six HVRs of 3A4.2. in some embodiments, the antibody comprises VH domain and VL domain of 3A4.2. in some embodiments, the antibody binds an isoform c-specific region of the extraceiiuiar domain of FcRHSc (e.g.t ig-itke domain 9). in same embodiments, the antibody is cross reactive with fuii length human and cyno FcRHS. fn some embodiments, the antibody does not significantly cross react with FeRH1, FcR2, FcR3, and/or FcRB4. In some embodiments, the antibody binds to endogenous FcRHS. in some embodiments, the antibody binds 8-ceiis. in some embodiments, the antibody does not significantly bind NK cells and/or monocytes.
[0139] Provided herein, and in some embodiments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid; sequence of (SEQ ID NO.42, HVR-H2 comprising the amino acid sequence of SEQ ID NO;66, and HVR-H3 comprising the amino acid sequence of SEQ ID NQ:90, and/or b) a Sight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ 10 NO:6, HVR-L2 comprising the amino acid sequence of (SEQ ID NO: 18, and HVR-L3 comprising the amino acid sequence of SEQ iD NG:3G. in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:54, HVR--H2 comprising the amino acid sequence of SEQ ID NO:78, and HVR-H3 comprising the amino acid sequenceof SEQ: iD NO: 102. in some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ !D NO:119 and/or a VI sequence having at ieast about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identify ίο the amino acid sequence of SEQ ID NG:118. In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:119 and/or a VI. sequence of SEQ ID NO: 118, in some embodiments of any of the antibodies, the antibody comprises six HVRs of 3812.1.1, In some embodiments, the antibody comprises VH domain and VL domain of 3812.1.1, In some embodiments, the antibody binds an isoform c-specific region of the extracellular domain of FcRH5c {e.g.. Ig-like domain 9), in some embodiments, the antibody is cross reactive with full iength human and cyno FcRHS. In some embodiments, the antibody does not significantly cross react with FeRHI, FcRH2, FcRHS, and/or FcRH4. tn some embodiments, ihe antibody binds to endogenous FcR5, In some embodiments, the antibody binds B-ceiis. In some embodiments, the antibody does not significantly bind NKceiis and/or monocytes. In some embodiments, the antibody does not significantly cross react with FeRH5a. (0131} Provided herein, and in some emboidments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of (SEQ ID NO:43, HVR-H2 comprising the amino acid sequence of SEQ ID NO:67, and HVR-H3 comprising the amino acid sequence of SEQ iD NO:91; and/or b) a light chain comprising a BVR-L1 comprising the amino acid sequence of SEQ ID NO:7, HVR-L2 comprising the amino acid sequence of SEQ iD NO: 19, and HVR-L3 comprising the amino acid sequence of (SEQ ID NO;31. In some embodiment, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ iD NO:55, HVR-112 comprising the amino acid sequence of SEQ ID NO: 79, and HVR-H3 comprising ihe amino acid sequence of SEQ ID NO:1Q3. in some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 121 and/or a VL sequence having at ieast about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:120, In some embodiments, the antibody comprises a VH sequence of SEQ !D NO:121 and/or a VL sequence of SEQ ID NO: 120. In some embodiments of any of the antibodies, the antibody comprises six HVRs of 3C10. In some embodiments, the antibody comprises VH domain and VL domain of 3C10. In some embodiments, the antibody binds an isofotrn c-specific region of the extracellular domain of FcRHSc (e.g., ig-like domain 9). in some em bodimen ts , the antibody is cross reacti ve with full iength human and cyno FcRHS. In some embodiments, the antibody does not significantly cross react with FcRH1, FeRH2, FcRH3, and/or FcRM4, in some embodiments, the antibody binds to endogenous FcRHS. In some embodiments, the antibody binds B-ceiis. in some embodiments, the antibody does not significantly bind NK ceils and/or monocytes, [0132} Provided herein, and in some emboidments, are antibodies comprising a} a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO;44, HVR-H2 comprising the amino acid sequence of SEQ ID NO:68, and HVR-H3 comprising the amino acid sequence of SEQ iD NQ;92; and/or b) a fight chain comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO:8. HVR-L2 comprising the amino acid sequence of SEQ iD NO:20, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:32. In some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:58, HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and HVR-H3 comprising the amino acid sequertceof SEQ ID NO: 104, in some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD Neo;123 and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD NO: 122. In some embodiments, the antibody comprises a VH sequence of SEQ 10 Neo:123 and/or a VL sequence of SEQ ID NO:122, In some embodiments of arty of the antibodies, the antibody comprises six HVRs of 3E10, in some embodiments, the antibody comprises VH domain and VL domain of 3F10. in some embodiments, the antibody binds an isoform c-specific region of the extracellular domain of FcRHSc (e.g., Ig-like domain 9), So soma embodiments, the antibody is cross reacti ve with fall length human and cyno FcRHS. in some embodiments, the antibody does not significantiy cross react with FcRR1, FcRE2, FcRHS, and/or FcR4. In some embodiments, the antibody binds to endogenous FcRHS. In some embodiments, the antibody binds B-cells. In some embodiments, the antibody does not significantiy bind NK cells and/or monocytes, in some embodiments, the antibody does not significantiy cross react with FcRHoa.
[0133} Provided herein, and irt some emboidments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ: ID NO:45, HVR-H2 comprising the amino acid sequence of SEQ ID NO:69, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and/or b) a Sight chain comprising a HVR-L1 comprising the amino add sequence of SEQ ID NO:9, MVR-L2 comprising the amino acid sequence of SEQ ID Neo:21, and HVR-L3 comprising the amino add sequence of SEQ ID NO:33. In some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ: ID NO:57, HVR-H2 comprising the amino acid sequence of SEQ ID NO:81, and HVR-H3 comprising the amino acid sequence of SEQ ID N0:105. in some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:125 and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 36%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:124. In some embodiments, the antibody comprises a VH sequence of SEQ ID NO: 125 and/or a VI sequence of SEQ iO NO:124. in some embodiments of any of the antibodies, the antibody comprises six HVRS of 3G3. in some embodiments, the antibody comprises VH domain and VL domain of 3G3. in some embodiments, the antibody binds an isoform c-spedfc region of the extracellular domain of FcRHSc {e.g., Ig-iike domain 9). In some embodiments, the antibody is cross reactive with full length human and cyno FcRHS in some embodiments, the antibody does not significantly cross react with FcRH1, FcRH2, FcRHS, and/or FcRH4, In some embodiments, the antibody bi nds to endogenous FcRHS. In some embodiments, the antibody binds 8-cells, in some embodiments, the antibody does not significantly bind INK ceils and/or monocytes. In some embodiments, the antibody does not significantly cross read with FcRH5a, {0134] Provided herein, and in some emboidments, are antibodies comprising a) 3 heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NQ:46. HVR-H2 comprising the amino acid sequence of SEQ IO NO:70, and HVR-H3 comprising the amino add sequence of SEQ ID NQ:94; and/or b) a light chain comprising a HVR-L1 comprising the amino add sequence of SEQ ID N0;1O, HVR-L2 comprising the amino acid sequence of SEQ in NO:22, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:34. in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:58, MVR-H2 comprising the amino acid sequence of SEQ !DN:Q:82, and HVR-H3 comprising the amino acid sequence of SEQ iD NO: 108. Insome embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ in NO;127 and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD NO: 126, In some embodiments, the antibody comprises a VH sequence of SEQ iD NG;127 and/or a VL sequence of SEQ ID NO:126, in some embodiments of any of the antibodies, the antibody comprises she HVRs of 307.1.5, in some embodiments, the antibody comprises VH domain and VL domain of 3G7.1.5. in some embodiments, the antibody binds an isoform c-specific region of the extracellular domain of FcRHSc (e.g., ig-iike domain 9), in some embodiments, the antibody is cross reactive with full length human and cyno FcRHS. in some embodiments, the antibody does not significantly cross react with FcR.H.1, FeRH2, FcR3, and/or FcRH4. in some embodiments, the antibody binds to endogenous FcRHS. in some embodiments, the antibody binds 8-cells, in some embodiments, the antibody does not significantly bind NK celis and/or monocytes.
[0135] Provided herein, and in some emboidments, are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ !D NO:47, HVR-H2 comprising the amino acid sequence of SEQ iD 140:71, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:95; and/or b) a light chain comprising a HVR-L1 comprising the amino acid sequence of SEQ !D NO:11, HVR-L2 comprising the amino acid sequence of SEQ iD NO;23, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:35. in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:59, HVR-H2 comprising the amino acid sequence ofSEQ !D NO:83, and HVR-H3 comprising the amino acid sequence of SEQ !D NO;107. in some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ !D NO: 129 and/or a VL sequence having at feast about any of 90%, 91%, 92%, 93%, 94%, 95%, 36%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 128. in some em bodiments, the antibody comprises a VH sequence of SEQ (D NG;129 and/or a VI sequence ofSEQ ID WO:128. in some embodiments of any of the antibodies, the antibody comprises six HVRs of 5A10.1.3. in some embodiments, the antibody comprises VH domain and VL domain of 5A10,1,3. in some embodiments, the antibody binds an isoform c-specific region of the extraceiiuiar domain of FcRHSc (e.g., ig-iike domain 9), in some embodiments, the antibody is cross reactive with full length human and cyno FcRRS, In some embodiments, the antibody does not significantly cross react with F'cRH 1, FcRH2, FcRH3, and/or FcRH4. In some embodiments, the antibody binds to endogenous FeRH5, in some embodiments, the antibody binds B-ceBs. in some embodiments, the antibody does not significantly bind NK ceiis and/or monocytes. (01363 Provided herein, and in some emboidments, are antibodies comprising a) a heavy chain comprising a ITVR-H1 comprising the amino acid sequence ofSEQ ID NG;48, HVR-H2 comprising the amino acid sequence ofSEQ ID NO:72, and HVR-H3 comprising the amino acid sequence of (SEQ ID NO:96; and/or b) a light chain comprising a BVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:24, and HVR-L3 comprising the amino acid sequence of SEQ ID NQ:36, in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO:60, HVR-H2 comprising the amino acid sequence ofSEQ !D NO:84, and HVR-H3 comprising the amino acid sequence of SEQ in NG:108. In some embodiments, the antibody comprises a VH sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD NO:131 and/or a VL sequence having at feast about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ iD NO::13Q, In some embodiments, the antibody comprises a VH sequence ofSEQ ID NO:131 and/or a VL sequence of SEQ iD NO: 130. In some embodiments of any of the antibodies, the antibody comprises six HVRs of 5F1.1.5. in some embodiments, the antibody comprises VH domain and VL domain of 5F 1.1.5. Insome embodiments, the antibody binds an isoform e-spedf/c region of the extracellular domain of FcRHSc (e.g,, Ig-iike domain 9). In some embodiments, the antibody is cross reactive with foil tength human and cyno FcRHS. in soma embodiments, the antibody does not significantly cross react with FcRHI, FcRH2, FcRHS, and/or FcRH4. In some embodiments, the antibody binds to endogenous FcRHS. In some embodiments,, the antibody binds 8-ceiis. in some embodiments,, the antibody does not significantly bind NK ceils and/or monocytes, in some embodiments, the antibody does not significantly cross react with FcRHSa, [0137) Provided herein, and in some emboidments. are antibodies comprising a) a heavy chain comprising a HVR-H1 comprising the amino acid sequence of SEQ fD NO:49, HVR-H2 comprising the amino acid sequence of SEQ iD NO:73, and HVR-H3 comprising the amino acid sequence of SEQ ID NO;97; and/or b) a fight chain comprising a HVR-L1 comprising the amino acid sequence of (SEQ iD NO: 13, MVR-L2 comprising the amino acid sequence of SEQ ID NO:25. and HVR-L3 comprising the amino acid sequence of SEQ iD NQ:37. in some embodiments, the heavy chain comprising a HVR-H1 comprising the amino add sequence of SEQ iD NO:61HVR-H2 comprising the amino acid sequence of SEQ ID NQ:85, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:109. in some embodiments, the antibody comprises a VH sequence having atieast about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, or 100% sequence identity to the amino acid sequence of (SEQ iD NO:133 and/or a VL sequence having at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:132. In some embodiments, the antibody comprises a VH sequence of SEQ iD NO;133 and/or a VL sequence of SEQ ID NG:132. in some embodiments of any of the antibodies, the antibody comprises six HVRs of 6D2, in some embodiments, the antibody comprises VH domain and VL domain of 6D2. In some embodiments, the antibody binds an isoform c-specific region of the extraeeiiuiar domain of FcRH5e (e.g., ig-iike domain 9). in some embodiments, the antibody is cross reactive with fall length human and cyno FcRHS. in some embodiments, the antibody does not significantfy cross react with FcRHI, FcRH2, FcR3, and/or FcRH4. in some embodiments, the antibody binds to endogenous FcRHS, In some embodiments, the antibody binds S-ceffs. in some embodiments, the antibody does not significantly bind NK ceils and/or monocytes.
[0138) In a further aspect provided herein, an anti-FcRHS antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody, in one embodiment, an anti-FcRHS antibody is an antibody fragment, e,g„ a Fv, Fab, Fab’, scFv, diabody, or F(afo')2 fragments, in another embodiment, the antibody is a substantially full length antibody, e.g„ an tg61 antibody or other antibody class or isotype as defined herein. [01391 in a further aspect provided herein, an antibody binds to the same epitope as an anti-FcRHS antibody provided herein, in certain embodiments, an antibody is provided that binds an isoform c-specific region of the extraeeiiuiar domain of FcRHSc from, within, or overlapping amino acids 754-835 of SEQ ID NO:1, [0140] In some embodiments of arty of the anti-FcRHS antibodies, the FcRHS antibody, particuiariy an FcRHS bispecific {e,g,, anti-CD3/anti-FcRH5 bispecific), may have features, singly or in combination, based upon NEK ceii fine assays {HEK ceils reconstituted with necessary signaling com ponents for the TCR triggering as described i n James and Vaiie, Nature 487:64-69 (2012). in some embodiments, the features, singly or in combination, may include tumor cel! inter-phase/immunoiogicai synapse, Lek-mecfiated TCR phosphorylation, ZAP7Q activity including phosphorylation state and localization, CD58 activity including ioeaiizatton and binding, føAr activity including localization and binding, CAAX activity including iocaiization and binding CD45 activity including localization:, pMHC activity including Iocaiization, and/or TCR activity and triggering features, [0141] In a further aspect, an anti-FeRH5 antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in (a)~(e) and/or Sections 1-7 below. (a) binds an hokum c-specific mgkms of the ex treæliular domain of FcRH5c [0142] ktethodsof determining whetheran anti-FcRHS antibody bindstoenisoformc-speciflc region of the extraeeiiuiar domain of FcRHSc are known in the art, in some embodiments, binding of an anti-FcRHS antibody to an isoform o-specific region of the extraeeiiuiar domain of FcRHSc may be determined by expressing FcRHS polypeptides with bland C-terminai deletions in 293 cells and/or SVT2 cells and testing by FACS as described in the Examples binding of the anti body to the truncated polypeptides, in some embodiments, a substantia! reduction (> 70% reduction) or elimination of binding of the antibody to a truncated polypeptides relative to binding to full-length FcRR5 expressed in 293 celts indicates that the antibody doss not bind to that truncated polypeptides.
[0143] In some embodiments, the isoform c-specific region comprises !g-like domain 9. in some embodiments, the Ig-iike domain 9 is also called ig-iike C2-type 8. In some embodiments, the isoform ©-specific region comprises amino acids 754-835 ofSEQ !D NQ:1. in soma embodiments, the isoform c-specific region comprises amino acids 752-834 of SEQ ID NQ:1. in some embodiments, the isoform c-speciftc region comprises amino acids 743-850 ofSEQ iD NG:1. In some embodiments, the isofarm c-specific region comprises amino acids 745-851 of SEQ iD NO:1 In some embodiments, the isoform c-specific region comprises amino adds about any of 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13, 14, or 15 from the N-termsna! and/or G-terminal boundary. In some embodiments, the isoform c-specific region comprises amino acids from about any of 750, 751, 752, 753, or 754 to about any of 830, 831, 832, 833, 834, 835, or 836 ofSEQ !0 NO:1, In some embodiments, FcH5 is human FcRHS. In some embodiments, FcRHS is human FcRH5 or cynomolgus monkey FcRHS.
(b) cross reads with (binds) human and cyno FcRHS with an affinity of s'5 nM, or < 4 nM, ar £ 3 nM, or-&amp; 2 nM, or < 1 nM, and optionally > 0.0001 nM, or >0.001 nM, or.>0,01 nM
[01441 Methods of determining binding affinity are known in the art. In some embodiments, the binding affinity may be determined according to a BIAcore® assay, ELiSA, Facs, and iHC. for example, as described in the Examples, [014¾ In some embodiments, the anti-FcRHS antibody binds human and/or FcRH 35 with an affinity of about any of < S nM, or < 4 nM, or < 3 nM, or < 2 nM, or < 1 nM, fn some embodiments, the anti-FcRH5 antibody binds human and/or cyno FcRHS with an affinity of about < 5. In some embodiments, the anti-FcRHS antibody binds human and/or cyno FcRHS with an affinity of about < 4 nM, In some embodiments, the anb-FcRHS antibody binds human and/or cyno FcRHS with an affinity of about < 3 nM, in some embodiments, the anb-FcRHS antibody binds human and/or cyno FcRHS with an affinity of about <2 nM, In some embodiments, FcRHS is human FcRHS, in some embodiments, FcRHS is cynomolgus monkey FcRHS. (c) does not cross react with (does not bind} FcRH1t FcRH2. FcRHS, andior FcRH4 [0146J Methods of determining binding are known in the art. in some embodiments, the binding affinity may be determined according to a BIAcore® assay, Facs, ELISA, and iHC. for exampie, as described in the Examples.
[0147J In some embodiments, the anfi-FcRHS antibody binds FcRHS with an affinity of more than about any of 2, S, 10, 20, 50,100, S00, or 1000-fofd greater than FcRH1, FcRH2, FcRH3, and/or FcRH4. in some embodiments, FcRR is human FcRH. (d) does not cross read with (does not bind) FcRHSa [0148} Methods of determining binding are known in the art. In some embodiments, the binding affinity may be determined according to a BIAcore® assay, Facs, ELISA, and iHC, for exampie, as described in the Examples, [0149} in some embodiments, the anti-FcRH5 antibody binds FcRHSc with an affinity of more than about any of 2, 5. 10, 20, 50,100, 500, or 1000-fold greater than FcRESa. In some embodiments, FcRH is human FcRH,
(e) does nd cross read with another Ig-iike domain (does not bind) of FcRHS
[0150} Methods of determining binding are known in the art. in some embodiments, the binding affinity may be determined according to a BIAcore® assay, Facs, ELiSA,, and IHC, for exampie, as described in the Examples.
[01513 In some embodiments, the anti-FcRHS antibody binds Ig-lke domain 9 of FcRHS with an affinity of more than about any of 2, 5,10,2D, 50,100, 500, or 1000-fofd greater than ig-iike domain 1,2, 3, 4, 5,6, 7, and/or 8 of FcRHS, in some embodiments, FcRH is human FcRH. in some embodiments, the ig-iike domain is ig-iike domain 1 {aa 23-100 of SEQ ID 140:1), Ig-lke domain 2 {aa 105-185 ofSEQ ID NO:1), ig-iike domain 3 {aa 188-271 ofSEQ ID NO:1), Ig-iike domain 4 {287-373 of SEQ ID NO:t), Ig-iike domain 5 {aa 380-466 of SEQ: ID NO:1), ig-iike domain 6 {aa 480-555 of SEQ ID NO:1), Ig-iike domain 7 {aa 568-652 of SEQ ID NO:1), fg-iike domain 8 {aa 658-731 of SEQ ID NQ:1),
Binding Assays and Other Assays [01523 In one aspect, an anti-FcRH5 antibody is tested for its antigen binding activity. For exampie, in certain embodiments, an anti-FcRHS antibody is tested for its ability to bind to FcRHS expressed on the surface of a cell. A FACS assay may be used for such testing.
[01S3J in an exemplary competition assay, immobilized FcRHS is incubated in a solution comprising a first labeled antibody that binds to FcRHS and a second uniabeled antibody that is being tested for its ability to compete with the first antibody for binding to FcRHS. The second antibody may be present in a hybridoma supernatant. As a control. immobilized FcRHS is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to FcRHS, excess unbound antibody is removed, and the amount of label associated with immobilized FcRHS is measured. If the amount of label associated with immobilized FcRHS is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to FcRHS. In certain embodiments, immobilized FcRHS is present on the surface of a ceil or in a membrane preparation obtained from a ceil expressing FcRH 5 on its surface, [0154} In one aspect, purified anti-FcRHS antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chroma- tøgraphy (HPLC), mass spectrometry, ion exchange chromatography and papain digestion, in one embodiment, contemplated are an altered antibody that possesses some hot not aii effector functions, which make it a desirable candidate for many applications in which the half life of fee antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In certain embodiments, the Fc activities of the antibody are measured to ensure that only the desired properties are maintained, in vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/deptetion ofCDC and/or ADCC activities. For example, Fc receptor {FcR} binding assays can be conducted So ensure that She antibody lacks Fc/R binding (hence iikely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RliS only, whereas monocytes express Fc(Ri, Fc(RII and Fc(RIII, FcR expression on hematopoietic ceils is summarized in Table 3 on page 484 of Ravetch and Kinet, Annu, Rev, Smmunol, 9:457-82 (1991). An example of an in vitro assay to assess ADCC activity of a mofecule of interest is described in U.S. Pat, No, 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear ceils (P8MC)snd Natural Killer (NK) ceils. Alternatively, or additionally, ADCC activity of the moiecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clyneset a!, PNAS (USA) 95:652-656 (1898). Ciq binding assays may also be carried out to confirm feat the antibody is unable to bind Ctq and hence lacks CDC activity, To assess complement activation, a CDC assay, e.g. as described In Gazzano-Santoro et ai„ J. Immunol. Methods 202:163 (1996), may be performed, FcRn binding and in vivo clearance/haif life determinations can aiso be performed using methods known in the art. f< Antibody Affinity [0155) In certain embodiments, an antibody provided herein has a dissociation constant {Kd} of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, &amp; 0,01 niM, or < 0.001 nM, and optionally is > 1Q'13 M. (e.g, 10*8 M or less, e.g, from 1Q'SM to 10'13 M< e.g., from 1Q"S Mi to 10'13 Mi), [0156) In some embodiments, Kd may be measured by a radiolabeled antigen binding assay (RiA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity ofFebs for antigen may be measured by equilibrating Fab wife a minimal concentration of (1:2S!}-iabeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated pfate (see, e.g., Chen et a!, J, Mo Biol. 283:865-881(1998)). To establish conditions for the assay, MICROTfTER® multi-well plates (Thermo Scientific) may be coated overnight with 5 pg/mi of a capturing anti-Fab antibody (Cappei Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hoorsaf room temperature (approximately 23*0), in arson-adsorbent plate (Nunc #269620), 100 pMor26 pM [mj-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody. Fab-12, in Presta et al., Cancer Res, 67:4593-4598 (1997)). The Fab of interest is then incubated overnight: however, the incubation may continue for a ionger period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures may be transferred to the capture plate for incubation at room temperature (e,g„ tor one hour). The solution may be then removed and the plate washed eight times with 0.1% poiysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 pL/weii of scintiiiant (MiCROSCINT-20™; Packard) may be added, and the plates may be counted on a TGPCOUNT™ gamma counter (Packard) tor ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding may be chosen for use in competitive binding assays.
[0157) According to another embodiment, Kd is measured using surface plasmon resonance assays using a Bl-ACORE® -2000 or a B!ACORE€MOGO (BiAcore. inc., Piscataway, NJ) at 25”C with immobilized antigen CMS chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (GM5, BIACORE, Inc.) may be activated with N-etbyW~{3-dimefeytominopropy!)-carbodiimide hydrochloride(EDC) and W-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen may be diluted with 10 mM sodium acetate, pH 4.S, to 5 ftg/mi (-0.2 p.M) before injection at a flow rate of 5 pL/minute to achieve approximately 10 response units (RU) of coupled protein, Foifowing the injection of antigen, 1 M ethanoiamine may be injected to biock unreacted groups. For kinetics measurements, twofold serial difutions of Fab (0,78 nM to 500 nM) may be injected in PBS with 0.05% poiysorbate 20 (TWEEN-2G™) surfactant (PBST) at 25*C at a flow rate of approximately 25 pL/min, Association rates (k,ifj and dissociation rates (k0#) may be calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) may be calculated as the ratio koff/ko!i, See, e.g,, Chen et al., J. Mol. Biot. 293:865-881 (1999). if the on-rate exceeds 10 M s by the surface piasmoo resonance assay above, then the on-rate may be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation * 295 nm; emission * 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS. pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMiNCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. 2. Antibody Fragments [0158] !n certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments inciude, but are not limited to, Fab, Fab', Fab’-SH, F(ab‘}?, Fv, and seFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Hal Med. 9:129-134 (2003). For a review ofscFv fragments, see, e.g., Piuckibun, in The Pharmacology of Monoclonal Antibodies, vot. 113. Rosenburg and Moore eds., (Springer-Veriag, Hew York), pp, 289-315 (1994); see also WO 93/16185; and U.S Patent Nos. 5,571,894 and 5,587,458, For discussion of Fab and F(ab‘}2 fragments comprising salvage receptor binding epitope residues and having increased in vivo baif-iife, see U.S. Patent No. 5,869,046.
[0159] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et a!., Hat. Med. 9:129-134 (2003); and Hoiiinger et al, Proc. Natl Acad. Sci. USA 90: 6444-6448 (1993). Tdabodies and tetrabodies are atso described in Hudson et al, Nat. Med. 9:129-134 (2003).
[0160] Single-domain antibodies are antibody fragments comprising ai! or a portion of the heavy chain variabie domain or ait or a portion of the fight chain variable domain of an antibody, in certain embodiments, a singie-domain antibody is a human singie-domain antibody (Oomantis, inc„ Waitham, MA; see, e.g., U.S, Patent No, 6,248,516 81).
[0161] Antibody fragments can be made by various techniques, inciuding but not iimited to proieoiyiic digestion of an intact antibody as weii as production by recombinant host ceils (e.g, B. coil or phage), as described herein. 3. Chimeric and Humanized Antibodies [0162] In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et at, Proc. Nat!. Acad, Sci. USA, 81:6851-6855 (1984)). in one example, a chimeric antibody comprises a non-human variable region (e g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region, in a further example, a chimeric antibody is a "class switched* antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies inciude antigen-binding fragments thereof.
[01631 In certain embodiments, a chimeric antibody is a humanized antibody. Typicaiiy, a non-human antibody is humanized to reduce irmmmogenicity to humans, white retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variabie domains in which HVRs, e.g., CORs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionaliy wifi aSso comprise at least a portion of a human constant region, in some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a nonhuman antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
[01643 Humanized antibodies and methods of making them are reviewed, e.g,, in Aimagro and Fransson, Front. Biosd. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al. Nature 332:323-329 (1988); Queen et al, Proc. Natl Acad. Sci. USA 88:10029-10033 (1989); US Patent' Nos, 5, 821,337,7,527,791,6,982,321, and 7,087,409; Kashmiri et al, Methods 36:25-34 ¢2005) (describing SDR (a-CDR) grafting); Radian, Mot. Immunol. 28:489-498 (1991) (describing '’resurfacing"}: Dall'Acqua et at., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al, Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection’’ approach to FR shuffling).
[0165] Human framework regions that may be used for humanization inciude but are not limited to: framework regions selected using the "best-fif method {see, e.g., Sims et ai. J. Immunol 151:2298 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of tight or heavy chain variable regions (see, e.g., Carter et al Proc. Natl Acad. Sci. USA, 89:4265 (1992); and Presta et ai. J. Immunol 151:2623 (1993)}; human mature (somatically m utated) framework regions or human germiine framework regions {see, e.g., Aimagro and Fransson, Front. Biosci, 13:1619-1633 (2008)}; and framework regions derived from screening FR libraries (see. e.g., Baca etal, J, Biot. Chem. 272:10678-10684 (1997) and Rosok et a!., J. Biol Cbem, 271:22611-22618 (1996)), 4. Human Antibodies [0166] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Di|k and van de Winkei, Curr, Opin, Pharmacof 5: 368-74 (2091) and Lonberg, Curr. Opin. Immunol, 20:450-459 (2008).
[0167J Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typicaiiy contain all or a portion of the human immunoglobuiin ioci, which replace the endogenous immunogiobuiin loci, or which are present extrachromosomaiiy or integrated randomly into the animat's chromosomes, in such transgenic mice, the endogenous immunoglobulin ioci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005), See also, e.g., U.S. Patent Nos, 6,075,181 and 6,150,584 describing XENOMOUSE™ technofogy; U.S. Patent No. 5.770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0081900, describing VELOCi MOUSE® technoiogy). Human variable regions from inlact antibodies generated by such animais may be further modified, e.g., by combining with a different human constant region. mm Human antibodies can also be made by hybridoma-based methods. Human myeioma and mouse-human heteromyeloma cell tines tor the production of human monoclonal antibodies have been described. (See, e g., KozborJ. Immunol,, 133: 3001 {1984): Brodeur et a!.. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 {Marcel Dekker. too.. New York, 1987); and Boerner et a!„ J. Immunol., 147:86 {1991},) Human antibodies generated via human B-ceii hybridoma technoiogy are also described in Li et at. Proc. Nati, Acad, Sci. USA, 103:3557-3562 (2008), Additionaf methods include those described, for example, in U.S, Patent No. 7,189,826 {describing production of monoclonal human IgM antibodies from hybridoma cel! lines) and NL Xiandai Mianyixue, 26(4):265-268 {2006) {describing human-human bybridomas), Human hybridoma technology {Trioma technology) is also described in Voiimers and Bran-diein, Histology and Histopathoiogy, 20(3):927-937 (2005) and Voiimers and Brandleln, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005), (0169) Human antibodies may aiso be generated by isolating Fv clone variable domain sequences selected from human-derived phage dispiay libraries. Such variabie domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below, 5. L/brary-Derived Andbodies (0170J Antibodies provided herein may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage dispiay libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et a!, in Methods in Molecular Biology 178:1-37 (O'Brien et at., ed,, Human Press, Totowa, NJ, 2001) and further described, e.g„ in the McCafferty et at., Nature 348;552-554; Ciackson eta!.. Nature 352: 824-628 (1991): Marks et si., J, Mol, Biol 222: 581 -597 {1992): Marks and Bradbury, in Methods in Mofecuiar Biology 248:181-175 (to, ed.. Human Press, Totowa, NJ, 2003); Sidhu et ai., J. Mol. Biol. 338(2): 299-310 (2004); Lee et af„ J, Mol Biol. 340(5): 1073-1093 (2004): Felfouse, Proc, Natl. Acad. Sci, USA 101(34): 12467-12472 (2004); and Lee eta!., J, Immunol. Methods 284(1 -2): 119-132(2004), [0171) In certain phage display methods, repertoires of VH and VL genes are separately cioned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which cm then be screened: for antigen-binding phage as described in Winter et al., Ann Rev. Immunol., 12: 433-455 (1994), Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-seif and aiso self antigens without any immunization as described by Griffiths et at, EMBO J. 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PGR primers containing random sequence to encode the highly variable C0R3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J Mol, BioL, 227: 381-388 (1992), Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574,2005/0119455, 2005/02:86000, 2007/0117126, 2007/0180598, 2007/0237764, 2007/0292936, and 2009/0002360.
[0172J Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. δ. Multispecific Antibodies (0173) lh certain embodiments, an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, in certain embodiments, one of the binding specificities is for FcRHS and the other is for any other antigen, in certain embodi ments, one of the binding specificities is for FcRHS and the other is for CD3. See, e.g., U.S. Patent No. 5,821,337. in certain embodiments, bispecific antibodies may bind to two different epitopes of FcRHS, Bispecific antibodies may also be used to localize cytotoxic agents to cells which express FcRHS. Bispecific antibodies can be prepared as full length antibodies or antibody fragments, [0174) In some embodiments, the FcRHS antibodies are FcRHS bispecific antibodies. Bispecific antibodies are anti- bodies that have binding specificities for at feast two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of an FcRHS protein as described herein. Other such antibodies may combine an FcRHS binding site with a binding site for another protein. Alternatively, an anfi-Fc.RH5 arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-ceii receptor moiecuie (e.g. CD3), or Fc receptors for igG (FcyR), such as Fcj-Ri {C064). FcyRii (C032) and FcyRiii (CD16), so as to focus and localize ceiiuiar defense mechanisms to the FeRHS-expressIng ceil. Bispedfic antibodies may also be used to iocaiize cytotoxic agents to ceils which express FcRHS. These antibodies possess an FcRHS-binding arm and an arm which binds toe cytotoxic ageni {e.g., saporio, anti-interferon a vinca alkaloid, rtdn A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can he prepared as fuif length antibodies or antibody fragments (e.g., F{ab'}2 bispecific antibodies), in some embodiments, the anti-FcRHS antibody binds an Isoform c-specific region of the extracellular domain of FcRHSc. in some embodiments, the anti-FcRHS antibodies binds ig-fike domain 9 of FcRHSc. (91753 In some embodiments, the FcRHS bispedfic antibody comprises a first arm, wherein the first arm binds FcRHS and a second arm, wherein the second arm binds a Fc. The second arm of toe FcRHS bispecific antibody may be any anti-Fc antibody known in toe art. For example, WO 96/16673 describes a bispedfic anti~ErbB2/anti-FcvRili antibody and U.S. Pat. No, 5,837,234 diseioses a bispecific anti-ErbS2/anti-FcyRi antibody. A bispedfic anti-ErbB2/Fca antibody is shown in WO98/02463, in some embodiments, the anti-FcRHS antibody binds an isoform e-spedfse region of the extracellular domain of FcRHSc. in some embodiments, the anii-FoRHS antibodies binds Ig-iike domain 9 of FcRHSc. {61763 in some embodiments, toe FcRHS bispedfic antibody comprises a first arm, wherein the first arm binds FcRHS and a second arm, wherein the second arm binds CD3, The second arm of the FcRHS bispecific antibody may be any anti-CD3 antibody known in the art. U.S. Pat. Nos, 5,821,337 and 6,407,213 teach bispedfic anti-ErbB2/anti-CD3 antibodies. Additional bispedfic antibodies that bind an epitope on the C03 antigen and a second epitope have been described. See, for example, U.S, Pat, No. 5,078,998 {anti-CDS/tumorceii antigen); U.S. Pat. No. 5,601,819{anfi-CD3/iL-2R; anti-CD3/CD28; anti-C03/CD45); U.S. Pat No. 6.129.914 (anti-C03/maiignant B-cell antigen); U.S. Pat No. 7,112,324 {anti-CD3/CD19); U.S. Pat. No. 6,723,538 {anti-CD3/CCR5}; U.S. Pat No. 7,235,641 {antoC03/EpCAM); U.S. Pat. No. 7,262,276 (anti-CD3/ovarian tumor antigen); and U.S. Pat. No. 5,731,168 (anti-CD3/CD4lgG). in some embodiments, the anti-CD3 antibody of the second arm is an antibody described in any one of WO 2005/118635, W02007/042261, W02008/119567, US5929212, US6750325, US6491916, US7994289, US7993641, US6?06265, US5585097, US5968509. US5932448, US6129914, US7381803, US5834597, and US7862613. in some embodiments, the anfi-FcRH5 antibody binds an isoform c-specific region of the extraceiiuiar domain of FcRHSc. in some embodiments, the anti-FcRHS antibodies binds ig-iike domain 9 of FcRHSc. (0177] Techniques for making muifispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-f ight chain pairs having different specificities (see Milstein and Cueiio, Nature 305:537 (1983)), WO 93/08829, and Traunecker et a!„ EMBO J. 10:3855 (1991)), and “knob-in-hole" engineering (see, e.g,, U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heierodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et at.. Science, 229:81 (1985)}: using leudne zippers to produce bi-speeifie antibodies (see, e.g., Kosteiny et ai., J, Immunol,, 148(5); 1547-1553 (1992)); using ’’diabody" technology for making bispecific antibody fragments (see, e.g., Rollinger etai., Proc. Nati. Acad. Set. USA, 90:6444-6448 (1993)};. and using single-chain Fv (sFv) dimers (see, e.g. Gruber et ai., J. immunol, 152:5368 {1994}); and preparing trispecific antibodies as described, e.g., in Tutt et ai, J. immunol. 147; 60 (1991).
[0178} Engineered antibodies with three or more functional antigen binding sties, including “Octopus antibodies," are also included herein (see, e g. US2008/0025576A1). {0179] The antibody or fragment herein also includes a "Dual Acting FAb“ or “DAP comprising an antigen binding site that binds to FcRHS as well as another, different antigen (see, US 2008/0069820, for example). {01803 According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, toe fusion is with an ig heavy chain constant domain, comprising at least part of the hinge. CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for fight chain bonding, present in at ieast one of the fosions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin iigbt chain, are inserted Into separate expression vectors, and are co-1ransfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three poiypeplide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired trispecific antibody, it is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equai ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination. (0181] In some embodiments, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-fight chain pair (providing a second binding specificity) in toe other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecifie molecule provides for a facile way of separation. This approach is disclosed in WO 34/04690. For further details of generating bispecifie antibodies see. for example, Suresh et ai., Methods in Enzymology 121:210(1986). £01823 According to another approach described in U.S. Pat, No, 5,731,168, the interface between a pair of antibody molecules can be engineered ίο maximize the percentage ofheterodimers which are recovered from recombinant cell Culture. The preferred interface comprises at least a part of the Cw domain, in this method, one or more small amino acid side chains from the interface of the first antibody moiecuie are replaced with larger side chains (e.g.. tyrosine or tryptophan). Compensatory "cavities'' of Identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.. alanine or threonine). This provides a mechanism for increasing the yield of the heferodimer over other unwanted end-products such as homodimers. Bispecifie antibodies produced in accordance with this approach are referred to herein as "protuberance-into-cav:ty" antibodies.
[01833 Bispecific antibodies include cross-linked or “heteroconjugate" antibodies. For example, one of the antibodies in the heteroeonjugate can be coupled to svidtn, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells {U.S, Pat, No, 4,676,980), end for treatment of HIV infection (WO 91 /00360, WO 92/200373, and EP 03089). Heteroeonjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are weii known in the art, and are disclosed in U.S. Pat, No. 4,676,980, along with a number of cross-linking techniques, [01841 Techniques for generating bispecifie antibodies from antibody fragments have also been described in the literature. For example, bispecifie antibodies can be prepared using chemical linkage, Brennan et a).. Science 229:81 (1985} describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab’}2 fragments. These fragments are reduced in {he presence of the dithiol com plexing agent, sodium arsenste. to stabilize vicinal dithiots and prevent intermotecuiar disu lfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB derivatives is then reconverted to the Fab'-thiof by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNS derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
[01851 Fab -SH fragments from £, call can be directly recovered and chemically coupled to form bispecific antibodies. Shalaby et at, J, Exp. Med, 175: 217-225 {1992} describe the production of a fuiiy humanized bispecifie antibody F{ab')2 moiecuie. Each Fab’ fragment was separately secreted from coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecifie antibody thus formed was able to bind to ceils overexpressing the ErbB2 receptor and normaf human T eeiis, as well as trigger the iytic activity of human cytotoxic lymphocytes against human breast tumor targets, [0186| Various techniques for making and isolating bispecific anti body fragments directly from recontbi nant ceil culture have also been described. For example, bispecifie antibodies have been produced using leucine zippers, Kosteiny et at., J. Immunol. 148(5): 1547-1553 (1992), The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody" technofogy described by Hoiiinger et at,, Free. Natt. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecifie antibody fragments. The fragments comprise a VH connected to a VH by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VI domains of one fragment are forced to pair with the complementary VI and V domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecifie antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et at., J. immune!., 152:5368 (1994). [01873 Antibodies with more than two valencies are contemplated. For example, (»specific antibodies can be prepared. Tutt et a!., J. immunof, 147:60 (1991), 8. Antibody Varimts [0188J In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other bioiogical properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications includes, for example, deletions from, and/or insertions into and/or substitutions of residues within foe amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the finat construct possesses the desired characteristics, e.g., antigen-binding. a) Substitutions, Insertion, and Deletion Variants [016¾ In certain embodiments, antibody variants having one or more amino add substitutions are provided. Sites of interest for substitutions! mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions" More substantia! changes are provided in Tabie 1 under the heading of "exempfary substitutions." and as further described below in reference to amino acid side chain ciasses. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or COC, TABLE 1
[0190] Amino acids may be grouped according to common side-chain properties; (1) hydrophobic: Norieucine, Met, Ata, Vat, Leu, lie; {2} neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3} acidic: Asp, Giu; (4) basic: His, Lys, Erg; {5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. mm Non-conservative substitutions will entaii exchanging a member of one of these classes for another class, [01921 One type of substitutiona! variant involves substituting one or more hypervariabie region residues of a parent antibody {e.g. a humanized or human antibody). Generally, the resuiting variants) selected for further study will have modifications {e,g„ improvements} in certain biofogica! properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or wii! have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
[0193] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in H VR ’’hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e g., Chowdhury, Methods Mol. 8iol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reseiecting from secondary libraries has been described, e.g.. in Hoogeriboom ef ai in Methods in Molecular Bioiogy 178:1-37 (O'Brien et ai.s ed„ Human Press, Totowa, N3, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variabfe genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonudeotlde-direeted mutagenesis), A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues Involved in antigen binding may be specificafiy identified, e.g., using aianine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
[0194] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative aiterations {e g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
[0195] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as ang, asp, his, iys, and gfu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the ant i body with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
[0196] Amino acid sequence insertions inetude amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-ferminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody , b) Glycosyiation variants [0197] in certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosyiation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosyiation sites is created or removed, [0198] Where the anti body comprises an Fe region, the carbohydrate attached thereto maybe altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-ilnkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TI8TECH 15:26-32 (1997), The oligosaccharide may Include various carbohydrates, e.g., mannose, N-acetyt glucosamine (GIcNAc), galactose, and sialic add, as we!! as a fucose attached to a GlcNAc in the "stern” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody provided herein may be made in order to create antibody variants with certain improved properties, [0199] In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, toe amount of fucose in such antibody may be from 1% to 80%, from 1 % to 65%, from 5% to 65% or from. 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297. relative to the or aii glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDf-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in toe Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about .t 3 amino adds upstream or downstream of position 297, i.e., between positions 294 and 300, due fo minor sequence variations in antibodies. Such fucosyiation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. Ups 2003/0157108 {Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to"defucosytaied" or "fuoose-defidenf antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO2003/084570; WO 2005/035586; WO 2005/035778; WG2005/G53742; W020Q2/031140; Okazaki eta!. J. Mot. Biol. 336:1239-1249(2004); Yamane -Ohnuki et ai. Biotech. Bioeog. 87:614 (2004). Examples of ceil lines capable of producing deiucosylated antibodies include Leo13 CHO ceils deficient in protein fuccsylation (Ripka et a!. Arch. Biocbem. Biophys, 249:533-545 (1986); US PatAppf No US 2003/0157108 A1«Presta. L; and WO 2004/056312 A1, Adams etal., especially at Example 11), and knockout ceil lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO ceils (see, e.g., Yamene-Ohnuki et at. Biotech, Bioeng. 87:614 (2004); Kande, Y. et ah. Bioiechnoi. Bioeng., 94(4):680-688 (2006); and W02003/085107).
[0200) Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a hiantennary oligosaccharide attached to the Fc region of the antibody is bisected by GicNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e. g.. in WO 2003/011878 (Jean-Mairet et al); US Patent No. 6,602,684 (Umana et ai.); and US 2005/0123546 (Umana et ai.}. Antibody variants with at least one galactose residue in the oiigosaeeharide attached to the Fc region are aiso provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in W0 1997/30087 (Patel et ah); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S,). c) Fc region variants [0201! In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence {e.g.t a human igGI, igG2, igG3 or igG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions, [0202| In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which {he half-life erf the antibody in vivo is Important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious, in vitro and/or in vivo cvioioxicity assays can be conducted ίο confirm the reduetion/depietion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FeRn binding ability. The primary cells for mediating ADCC, NK ceils, express Fc(Rll! only, whereas monocytes express Fc(Ri, Fc(RII and Fc(Rf!i. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. immuno! 9:457-492 (1991). Non-iimfting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S, Patent No, 5,500,362 (see, e.g. Hellstrom, h et at. Proc, Natl Acad, Sci, USA 83:7059-7063 ¢1988)) and Heiistrom, i et a!., Pro. Nat'! Acad, Sci. USA 82:1499-1502 (1985); 5,821.337 (see Bruggemann, M, etai,, J. Exp, Mad. 186:1351-1381 (1987)), Alternatively, non-radioactive assays methods may be employed (see, for example, ACTS™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnofogy, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wl). Useful effector ceiis for such assays include peripheral blood mononuclear ceils (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., In an animal model such as that disclosed in dynes et al, Proc, Nat’l Acad. Sci. USA 95:852-658 (1998). C1q binding assays may also be canted out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g.., C1q and C3c binding ELISA in WO 2008/029879 and WO 20Q5/1QQ4Q2. To assess complement activation, a CDC assay may be performed (see, for example, Gazzaino-Santoroetai., J, Immunol. Methods 202:163 (1998); Cragg. M.S. etal., Blood 101:1045-1052 (2003); and Cragg, M.S. and M. Giennie, Blood 103:2738-2743 (2004)). FeRn binding and in vivo ciearance/haiMife determinations can also be performed using methods known in the art (see, e.g,, Petkova, S.8. et al., infl Immunol. 18(12):1759-1769(2066)).
[02033 Antibodies with reduced effector function include those with substitution of One or more of Fc region residues 238,265,269.270,297,327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants Include Fc mutants with substitutions at two or more of amino acid positions 285, 269, 270, 297 and 327, including the so-caiied "DANA” Fc mutant with substitution of residues 285 and 297 to alanine (US Patent No. 7,332,581).
[02043 Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g.. U.S. Patent No. 8,737,058; WO 2004/058312, and ShieSds et ai, J. Biol. Chem, 9(2): 6591-6804 (2001).) [0205) In certain embodiments, so antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298,333, and/or 334 of the Fc region (EU numbering of residues). [02063 In some embodiments, alterations are made in the Fc region that result in altered {>.©., either improved or diminished) CTq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6.194,551, WO 99/51642, and Idusogie el ai. J. Immunol. 164: 4178-4184 (2000).
[0207) Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FeRn). which is responsible for the transfer of maternal SgGs to the fetus (Guyer et al„ J. Immunol. 117:587 (1976) and Kim et al, J. Immunol 24:249 (1994)), are described in US2GQ5/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn, Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303. 305, 307, 311, 312. 317, 340, 356, 360,362, 376, 378, 380, 382,413, 424 or 434, e.g., substitution of Fc region residue 434 {US Patent No. 7,371.826). {0208J See also Duncan &amp; Winter, Nature 322:738-40 (1388); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821: and WO 94/29351 concerning other examples of Fc region variants. d) Cysteine engineered antibody variants {02093 to certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues, in particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive ihiof groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or (inker-drug moieties, to create an immunoconjugate, as described further herein, in certain embodiments, any one or more of the following residues may be substituted with cysteine; V2Q5 (Kabat numbering) of the Sight chain; A118 (EU numbering) of the heavy chain; and S400 {EU numbering} of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g,, in U.S, Patent No, 7,521,541, e) Antibody Derivatives [02101 fe certain embodiments, an antibody provided herein may be further modified to contain additional nonprotein-aceous moieties that are known in the art and readily available. The moieties suitabie for derivatisation of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include. bu( are not Imited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymeJhylceSSufose, dex-tran, polyvinyl alcohol, polyvinyl pyrroiidooe, poly-1, 3-dioxolaoe, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poiyaminoadds (either homopolymers or random copolymers), and dextran or po!y(n-vinyf pyrrolidone)po!yelhyl-ene glycol, propropytene glycol homopolymers, proiyprapylene oxide/ethylene oxide co-polymers, polyoxyethylafed polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol prop ion aldehyde may have advantages In manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one poiymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for denvatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used In a therapy under defined conditions, etc.
[0211| In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam el at., Proc. Natl, Acad. Sci. USA 102:11600-11605 (2005)), The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety toa temperature at which ceils proximal to the antibody-nonproteinaceous moiety are killed. 8. Recombinant Methods and Compositions [0212} Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,818,567. in one embodiment, isolated nucleic acid encoding an anti-FcRHS antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or art amino acid sequence comprising the VH of the antibody {e.g., the light and/or heavy chains of the antibody), in a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided, in one such embodiment, a host ceil comprises (e.g., has been transformed with): (1} a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VI of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the Vt of the antibody and a second vector comprising a nucleic acid that encodes art amino acid sequence comprising the VH of the antibody, in one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YQ, NS0, Sp20 cell). In one embodiment, a method of making an anti-FcRHS antibody is provided, wherein the method comprises culturing a host cel! comprisi ng a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody , and optionally recovering the antibody from the host cell (or host cell culture medium).
[0213} For recombinant production of an anti-FcRHS antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
[0214} Suitable host ceils tor cloning or expression of anybody-encoding vectors include prokaryotic or eukaryotic ceils described herein. For example, antibodies may be produced in bacteria, in particular when giycosyiation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria,, see, e.g., U.S, Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Chariton, Methods in Molecular Biology. Voi. 248 (B.K.C. Lg, ed„ Humana Press, Totowa, NJ« 2003), pp, 245*254, describing expression of antibody fragments in £ cofi.) After expression, the antibody may be isolated from the bacterial eel! paste in a soiubie fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as fiiamentous fungi or yeast are suitable cioning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose giycosyiation pathways have been "humanized,” resulting in the production of an antibody with a paritaliy or My human giycosyiation pattern. See Gerngnoss, Nat Biotech, 22:1409-1414 (2004), and LI elai., Nat. Biotech, 24:210-215 (2006).
[0215} Suitabfe host ceiis for the expression of giycosyiated antibody are aiso derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect ceils. Numerous bacuioviraf strains have been identified which may be used in conjunction with insect ceiis, particuiariy for transfection of Spodoptera frugiperda ceiis.
Riant eeii cultures can aiso be utilized as hosts. See, e.g., US Patent Nos. 6,959,177,6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODiES™ technology for producing antibodies in transgenic- plants).
[0216} Vertebrate ceiis may aiso be used as hosts. For example, mammafian ceii tines that are adapted to grow in suspension may be useful. Other examples of useful mammafian host ceii fines are monkey kidney CV1 fine transformed by SV40 (COS-7); human embryonic kidney iine (293 or 293 ceiis as described, e.g, in Graham et ai, J. Gen Virol. 36:59 {1977}); baby hamster kidney ceiis (BHK): mouse sertoli ceiis (TM4 ceils as described, e.g., in Mather, Bioi. Reprod, 23:243-251 (1980}); monkey kidney ceiis (CV1); African green monkey kidney ceiis (VERO-76); human cervicai carcinoma cells (HELA); canine kidney cells (MOCK; buffalo rat tiver ceiis (8RL 3A); human iung ceiis (W138); human fiver ceiis (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Set. 383:44-88 (1982); MRC 5 ceiis; and FS4 ceils. Olher useful mammalian host ceii lines include Chinese hamster ovary (CHO) cells, including DHFR CHO ceiis (Urtaub et ai., Proc. Natl. Acad, Set. USA77:4216 (1980)}; and myeloma celt Sines such as Y0, NS0 and Sp2/G, For a review of certain mammalian host ceii lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vioi. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). C. Assays [0217} Anti-FcR H5 antibodies provided herein may be identified. screened for, or characterized for their physical/chem-ical properties and/or biological activities by various assays known in the art.
[0218J In one aspect, an antibody provided herein may be tested for its antigen binding activity , e.g., by known methods such as ELiSA, BIACore®. FACS, or Western blot.
[0219} In another aspect, competition assays may be used to identify an antibody that competes with any of the antibodies described herein for binding to FcRHS, In certain embodiments, such a competing antibody binds to the same epitope (e,g,, a linear or a conformational epitope) that is bound by an antibody described herein. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols," in Methods in Molecular Biology voi, 66 (human Press, Totowa, NJ).
[0220} in an exemplary competition assay, immobilized FcRHS is incubated in a solution comprising a first labeled antibody that binds to FcR5 {e.g., any of the antibodies described herein) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to FcRHS. The second antibody may be present in a hybridoma supernatant. As a control, immobilized FcRHS is incubated in a solution comprising the first labeled antibody but not the second uniabelerf antibody. After incubation under conditions permissive for binding of the first antibody to FcRHS, excess unbound antibody is removed, and the amount of label associated with immobilized FcRHS is measured, if the amount of label associated with immobilized FcRH5 is substantially reduced in the test sample relative to the control sampie, then that indicates that the second antibody is competing with the first antibody for binding to FcRHS, See Hariow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), in some embodiments, the FcRHS Is FcR-H5c, in some embodiments, the anti-FcRH5 antibody binds an tsoform c-specltic region of the extracellular domain of FcRRSc, in some embodiments, the anfi-FeRM5 antibodies binds ig-tike domain 9 of FcRHSc. D. immunoconjugates [0221} Also provided herein are immunoconjugates comprising an anti-FcRHS antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g,, protein toxins, enzymatically active toxins of bacteria! , fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes {/,<?., a radioconjugate). In some embodiments, the anti-FcRHS antibody binds an isoform c-speeific region of the extracellular domain of FcRHSc. In some embodiments, the anti-FcRHS antibodies binds ig-iike domain 9 of FcRHSc.
[0222) immunoconjugates allow for the targeted delivery of a drug moiety to a tumor, and, in some embodiments, intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to norma! ceils (Polakis R, {2005} Current Opinion in Pharmacology 5:382-387), [0223J Antibody-drug conjugates (ADC) are targeted chemotherapeutic molecules which combine properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor ceils {Teicher, 8 A, (2009) Current Cancer Drug Targets 9:982-1004}, thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ. and Senter P.D, (2008) The Cancer Jour. 14(3):154-169: Chari, R.V. (2008) Acc. Ghem, Res. 41:98-107.
[0224) The ADC compounds provided herein include those with anticancer activity, in some embodiments, the ADC compounds include an antibody conjugated, f.e. covaientfy attached, to the drug moiety, tn some embodiments, the antibody is covalently attached to the drug moiety through a tinker. The antibody-drug conjugates (ADC) provided herein selectively deliver an effective dose of a drug to tumor tissue whereby greater selectivity, f.e. a tower efficacious dose, may be achieved white increasing the therapeutic index (“therapeutic window").
[0225) The drug moiety (D) of the antibody-drug conjugates (ADC) may include any compound, moiety or group that has a cytotoxic or cytostatic effect Drog moieties may impart their cytotoxic and cytostatic effects by mechanisms including but not limited to tubulin binding, DNA binding or intercalation, and inhibition ofRNA polymerase, protein synthesis, and/or topoisomerase. Exemplary drug moieties include, but are not limited to, a maytansinoid, dolastatin, auristatin, eaiieheamicin, pyrrolobenzodiazepine (PBD), nemoatbicin and its derivatives, PNU-159682. anthracydine, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1G65, camptothecin, elinafide, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity. Nonlimiting examples of such immunoconjugates are discussed in further detail below, 1. Exemplary Antibody-drug Conjugates [0226J An exemplary embodiment of an antibody-drug conjugate (ADC) compound comprises an antibody (Ab) which targets a tumor cett, a drug moiety (D), and a linker moiety (L) that attaches Ab to D, In some embodiments, the antibody is attached to the linker moiety (L) through one or more amino acid residues, such as lysine and/or cysteine, [0227) An exemplary ADC has Formula i:
Ab-(L-D)p ! where p is 1 to about 20. in some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine residues, in some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADC of Form ula! include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et ai (2012) Methods in Enzym, 502:123-138), in some embodiments, one or more free cysteine residues are already present in an antibody, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody to a drug. In some embodiments, an antibody is exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues. In some embodiments, the anti-FcRH5 antibody binds an isoform o-specific region of the extracellular domain of FcRHSc. In some embodiments, the anti-FcRHS antibodies binds Ig-like domain 9 of FcRHSc, [0228) A "Linker" (L) is a bifunctiona! or muififunctiona! moiety that can be used to Sink one or more drug moieties (D) to an antibody (Ab) to form an antibody-drug conjugate (ADC) of Formula I, In some embodiments, antibody-drug conjugates (ADC) can be prepared using a Linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, in some embodiments, a cysteine thiof of an antibody (Ab) can form a bond with a reactive functional group of a linker or a drug-tinker intermediate to make an ADC.
[0229) In one aspect, a linker has a functionary that is capable of reacting with a free cystetae present on an antibody to form a covalent bond. Nonlimitlng exemplary such reactive functionalities include mafeimide, haloacetamldes, «-haloacetyl, activated esters such as succinimide esters, 4-nitropheny! esters, pentafluorophenyi esters, tetrafiuorophenyl esters, anhydrides, acid chlorides, suffonyi chlorides, isocyanates, and isothiocyanaies. See, e. g., the conjugation method at page 766 of Kiussman, et a! (2004), Bioconjugate Chemistry 15(4):765-773, and the Examples herein.
[0230) In some embodiments, a iinker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemptary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups.
In some embodiments, a heteroatom of the reactive functionality of the finker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit, Nonlimiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. £0231} A linker may comprise one or more linker components, Exemplary Sinker components include 6-maieimidoc-aproyi ("MC"), maleimidopropanoyi CMP"), valine-cfintiline f’val-cif or "vc"), alanine-phenylalanine ("sla-phe"}, p-ami-nobenzyioxyearbonyf {a "PAB”), N-Succioimidy! 4-(2-pyridylthio) pentanoafe (*SPP”), and 4-{N~maieimidomethyi} cyclohexane-1 carboxylate fMGC”). Various linker components are known in the art, some of which are described below, £0232] A linker may be a "cieavabte linker," facilitating release of a drug, Nonfimiting exempiary cieavable tinkers include acid-labile linkers £e.gf., comprising hydrozone), protease-serisitive (e.g., peptidase-sensitive) tinkers, pbotolabile linkers, or disulfide-containing finkers (Chari et ai, Cancer Research 52:127-131 (1992); US 5208020). £0233} In certain embodiments, a linker has the following Formula If: A -w -Y - it wherein A is a "stretcher unit", and a is an integer from 0 to 1; W is an "amino acid unit", and w is an integer from 0 to 12; Y is a "spacer unit”, and y is 0, 1, or 2, An ADC comprising the linker of Formula it has the Formula !{A); Ab-fA3-Ww-Yy-D)p, wherein Ab, D, and p are defined as above for Formuia I, Exempiary embodiments of such Sinkers are described in U,$. Patent No, 7,498,298, £0234| In some embodiments, a linker component comprises a "stretcher unit” (A) that links an antibody to another linker component or to a drug moiety, Noniimiting exemplary stretcher units ate shown below (wherein the wavy line indicates sites of covalent attachment to an antibody, drug, or additional tinker components):
£9235] In some embodiments, a linker component comprises an "amino acid: unit" (W). in some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the Immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina ef a!, (2003) Nat. Biotechnol 21:778*784). Exemplary amino acid units include, butare not limited to, dipeptides, tripeptldes, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, vaiine-citrulline (vc or val-cit), aianine-pheny-ialanine (af or ata-phe); phenylalanine-lysine (fk or phe-fys); phenyialanine-homolyslne (phe-homolys); and N-mothyl-valine-Citruiilne (Me-vef-cft). Exemplary tripeptldes include, but are not limited to, glyeine-vaiine-Cifrulline (gly-val-Clt) and glycine-gfycine-gfycine (gly-gfy-gly). An amino acid «nit may comprise amino add residues that occur naturally and/or minor amino adds and/or non-naturaity occurring amino acid analogs, such ascitruiiine. Amino acid units can be designed and optimized for enzymatic cieavage by a particular enzyme, for example, a tumor-associated protease, cathepsin 8, C and D, or a plasmin protease.
[02363 Typically, peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments, Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (e.g., E. Schroder and K. Lubke ( 1965) “The Peptides", volume 1, pp 76-136, Academic Press}.
[0237} In some embodiments, a linker component comprises a "spacer unit” (Y) that links the antibody to a drug moiety, either directly or through a stretcher unit and/or an amino acid unit. A spacer unit may be "seif-immoiative” or a "non-sefWmmofative “ A ”non-se!f'immof3tίve', spacer unit is one in which part or ail of the spacer unit remains bound to the drug moiety upon cleavage of the ADC. Examples of non-seif-immoiative spacer units include, but are not limited to, a glycine spacer unit and a giycine-glycine spacer unit. In some embodiments, enzymatic cieavage of an ADC containing a glycine-glycine spacer unit by a tumor-cell associated protease results in release of a giycine-gtycine-drug moiety from the remainder of the ADC, in some such embodiments, the gfycine-glycine-drug moiety is subjected to a hydrolysis step in the tumor cell, thus cleaving the glycine-giycine spacer unit from the drug moiety.
[0238] A "seif-immoiative” spacer unit allows for release of the drug moiety, in certain embodiments, a spacer unit of a linker comprises a p-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol is attached to an amino acid «nit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyf alcohol and the drug {Hamann et a! {2005} Expert Opin, Ther. Patents {2005} 15:1087-1103}, In some embodiments, the spacer unit comprises p-aminobenzy!oxyearbony! (PA8). In some embodiments, an ADC comprising a seif-immoiative linker has the structure :
wherein Q is -C-pCg alky!, -0-{C1-Cg alkyl), -halogen, -vitro, or -cyano: m is an integer ranging from 0 to 4; X may be one or more additional spacer units or may be absent; and p ranges from 1 to about 20. In some embodiments, p ranges from 1 to 10,1 to 7, 1 to 5, or 1 to 4. Nonlimiting exemplary X spacer units include:
wherein R s and ere independently selected from H and C rCf; alkyf. in some embodiments, R1 and R2 are each [0239} Other examples of seif-immoiative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAS group, such as 2-aminoimidazoi-5-rnethanoi derivatives (U.S. Patent No, 7,375,078; Hay et ai. (1999) Bioorg. Med. Chem. Lett. 9:2237} and ortho- or para-aminobenzyiacetals. in some embodiments, spacers can be used that undergo cycilzation upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobotyric acid amides {Rodrigueseta! {1995} Chemistry Biology2:223), appropriately substituted bicycio[2.2.1j and bicyde[2.2.2j ring systems (Storm et ai {1972} J. Amer. Chem, Sac, 94:5815} and 2-aminopheny!proptonic acid amides (Amsberry, et al (1990) J. Org. Chem, 55:5867). Linkage of a drug to the o.-carbqn of a glycine residue is another example of a seif-immoiative spacer that may be useful in ADC (Kingsbury et al {1984) J, Med, Chem, 27:1447).
[0240} In some embodiments, linker L may be a dendritic type linker for covalent attachment of more than one drug moiety to an antibody through a branching, multifunctional linker moiety {Sun et a! {2002} Bioorgantc &amp; Medicinal Chemistry Letters 12:2213-2215; Sun et ai (2003) Bioorganic &amp; Medictnai Chemistry 11:1781-1768), Dendritic linkers can increase the molar ratio of drug to antibody, /.e. loading, which is related to the potency of the ADC. Thus, where an antibody bears only one reactive cysteine thioi group, a multitude of drug moieties may be attached through a dendritic tinker.
[0241} Noniimiting exemplary linkers are shown below in the context of an ADC of Formula I:
wherein R1 and R2 am independently selected from H end CrC§ efkyi. In some embodiments, R, and R2 are each - CH;
Phe-homoLys-PAB-Ab; wherein n is 0 to 12, in some embodiments, n is 2 to 10. In some embodiments, n is 4 to 8. [02421 Further nonlimiting exemplary ADCs include the structures: where X is
Y is:
each R is independently H or C-s-Cg alkyl; and n is 1 to 12,
[0243J In some embodiments, a linker is substituted with groups that modulate solubility and/or reactivity. As a non-limiting example, a charged substituent such as sulfonate (-SO3) or ammonium may increase water solubility of the linker reagent and facilitate the coupling reaction of the inker reagent with the antibody and/or the drug moiety, or facilitate the coupling reaction of Ab-L {antibody-linker intermediate) with 0, or D-L (drug-inker intermediate) with Ab, depending on the synthetic route employed to prepare the AOC. in some embodiments, a portion of the Sinker is coupled to the antibody and a portion of the linker is coupled to the drug, and then the Ab-{finker portion)* is coupled to drug-(itnker portion)1-1 to form the ADC of Formula L
[0244} The compounds provided herein expressly contemplate, but are not limited to, ADC prepared with She following tinker reagents: bis-maieimida-tnoxyathyiene glycol (BMPEO), N-(j)-mai8imidøpropyløxy)-N-hydroxy succtnimide ester (BMPS), N-is-msIeimidocaproyloxy) succinimide ester (EMCS), N-fy-maleimidobu{yryloxy}succinimide ester (GMBS), 1,8-hexane-bis-vinyisuifone (H3VS). succinimidyf 4--{N-maleimiciomethyi)cyciohexane-1 -caft>oxy-{6-amidocaproate) (LC-SfvICC), m-malelmldobenzayl-N-hydroxysuecinirntde ester (MBS), acid hydrazide (MPBH), succinimidyl 3-(bromoa-cetamidojpropionate (SBAP). succinimidyl iodoacetate (SIA), succinimidyl (4~iodoacetyl)aminobenzoaie (SIAB). N-suc-cintmidy!-3-(2-pyndyidlthlo} propionate (SPDP). N-succinimidy!-4-{2-pyridyitMQ)pentanoate (SPP), succinimidyl 4-(N-mafeirnidorneihyljcyeiQhexane-t-carboxyiate (SMCC), succinimidyl 4-{p-ma!eimfdophenyf}buiyrate (SMPB), succinim-idy! 8-{(beia-maieimidopropionamido)hexanoate3 (SMPH), tmlnothiolane (IT), sulfo-EMCS, sutfo-GMBS, suifo-KMUS, sutfo-MBS, sulfo-SlAB, suifo-SMCC, and suifo-SMPB, and succintmidyK4-vinyisuifone}benzoaie(SVSB), and including bis-maieimide reagents: dithiobismaieimidoethane (DTME), 1,4-Bismaleimidobutane (BMB), 1,4 Bismafeimidyi-2,3-di“ hydroxybutane (8M0B). bismaleimidohexane (8MH), bismaieimidoethane (BMOE), BM(PEG)2 {shown below), and BM(PEG )3 (shown below): bifunctionai derivatives of imidoesters (such as dimethyl adipimidate NCI), active esters (such as disuccinlmidyi suberate}, aldehydes (such as giutaraldehyde), bis-azido compounds [such as bis {p-azsdobenzoyl) hexanadiamine), bis-diazonium derivatives (such as bis-{p-diazoniumbenzoyS)-ethyienediamfne), dlisocyanates (such as toluene 2,6-disocyanate), and bis-active fluorine compounds (such as I.S-difiuoro^^-dinitrobenzene}, In some embodiments, bis-maieimide reagents aiiow the attachment of the thiol group of a cysteine in the antibody to a thiol-containing drug moiety, linker, or linker-drug intermediate. Other functional groups that are reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyf disulfide. Isocyanate, and isothiocyanate,
8M{PE©)8 BM(PES)3 [9245} Certain useful linker reagents can be obtained from various commercial sources, such as Pierce Biotechnology, Inc. (Rockford, ill), Molecular Biosciences Inc.(Boulder, CO), or synthesized in accordance with procedures described in the art; for example, in Toki et al (2002) J, Grg. Chem. 67:1866-1872; Dubowchfk, et al. (1997) Tetrahedron tellers, 38:5257-60; Walker, M.A, (1995) J. Grg. Chem. 60:5352-5355; Frisch et al (1996) Bioconjugate Chem. 7:180-186; US 6214345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828. [0248} Carbon-14-labeied l-iSOlhiocyanatobenzyl-3-methyidietbylene toaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See. e.g.. WO94/11028, (1) Maytansine and maytansinoids [0247} In some embodiments, an immunoconjugate comprises an antibody conjugated to one or more mayiansinoid molecules. Maytansinoids are derivatives of maytansine, and are mitototie inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Mayfenus serrata (U.S. Patent No. 3898111).
Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinoi and C-3 maytansinoi esters {U.S. Patent Mo. 4,151.042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,748; 4,260,008; 4,285,814; 4,294,757; 4,307,016; 4,308,288; 4,308,269; 4,309,428; 4,313,948; 4,315,929: 4,317,821; 4,322,348; 4,331,698; 4,361,850; 4,384,886: 4,424,219; 4,450,264; 4,362,883; and 4,371,533, [0248) Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification or derivatization of fermentation products, (is) amenable to derivatization with functional groups suitable for conjugation through non-disulfide tinkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor celt fines.
[0249) Certain maytansinoids suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see, eg.. Yu et a! (2002) PNAS 99:7968-7973). Maytansinoids may also be prepared synthetically according to known methods.
[0250) Maytansinoid drug moieties include, hut are not limited to, those having a modified aromatic ring, such as: C-19-dechforo (US Pat, No. 4256748) (prepared, for example, by lithium aiuminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethy!} +/-C-19-dechforo (US Pat. Nos. 4361650 and 4307018) (prepared, for example, by demethyiation using Streptomyces or Actmomyces or dechlorination using LAH); and C-20-demethoxy. C-20-acyioxy (-OCOR), +/-dechioro (U.S, Pal No. 4,294,75?) (prepared, for example, by acylation using acyi chiohdes), and those having modifications at other positions of the aromatic ring, [0251) Maytansinoid drug moieties also include those having modifications such as; C-9-SH (US Pat. No. 4424219) (prepared, for example, by the reaction of maytansinoi with H2S or P2S5); C"14-alkoxymethyi(demethoxy/CH2OR)(US 4331598); C-14-hydroxymethyi or acyioxymethyl (CH2OH or CHjOAc) (US Pat. No. 4450254) (prepared, for example, from Nocardia); C-15-hydroxy/acyioxy (US 4364866) (prepared, for example, by the conversion of maytansinoi by Strep-fomyces): C-15-methoxy (US Pat. Nos. 4313946 and 4315929) (for example, isolated from Trewia nudffiora); C-18-N-demethyi (US Pat. Nos, 4362683 and 4322348) (prepared, for example, by the demethyiation of maytansinoi by Strep-tomyces); and 4,5-deoxy (US 4371533) (prepared, for example, by the titanium trichioride/LAH reduction of maytansinoi).
[0252) Many positions on maytansinoid compounds are useful as the linkage position. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupfing techniques in some embodiments, the reaction may occur at the C-3 position having a hydroxy! group, the C-14 position modified with hydroxymethyl, the ΟΙ 5 position modified with a hydroxy! group, and the C-20 position having a hydroxy! group. In some embodiments, the linkage is formed at the C-3 position of maytansinoi or a maytansinoi analogue.
[0253) Maytansinoid drug moieties include those having the structure:
where the wavy fine indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to a linker of an ADC, Each R may independently be H or a CrCs alkyl. The aikytene chain attaching the amide group to the sulfur atom may be methanyl, ethanyl, or propyl, Le„ m is i, 2, or 3 (US 633410; US 5208020; Chari et af (1992) Cancer Res, 52:127-131; Liu etal (1996) Proc. Nati. Acad. Sci USA 93:8618-8623).
[0254) All stereoisomers of the maytansinoid drug moiety are contemplated for the ADC provided herein, i.e any combination of R and S configurations at the chirai carbons (US 7276497; US 6913748; US 6441163; US 633410 (RE39151); US 5208020; Widdison et al (2006) d. Med. Chem. 49:4392-4408, which are incorporated by reference in their entirety), in some embodiments, the maytansinoid drug moiety has the following stereochemistry:
[0255] Exemplary embodiments of maytansinoid drug moieties include, but are not limited to, DM1; DM.3; end DM4, having the structures:
wherein the wavy tine indicates the covalent attachment of the sulfur atom of the drug to a linker (L) of an antibody-drug conjugate, [0256J Other exemplary rnaytansinoid antibody-drug conjugates have the following structures and abbreviations (wherein Ab is antibody and p is 1 to about 20. in some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4):
Ab -SPP-DM1
AB-SMCC-DM1 [0257] Exemplary antibody-drug conjugates where DM1 is linked through a BMP£0 linker to a thiol group of the antibody have the structure and abbreviation:
where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20, in some embodiments, p is 1 to 10. p is 1 to 7, p is 1 to 5, or p is 1 to 4.
[0258] Immunoconjugates containing maytansinoids, methods of making the same, and their therapeutic use are disclosed, for example, in U.S, Patent Nos. 5,208,020 and 5,418,064; US 2005/0276812 A1; and European Patent EP 0 425 235 B1. See also Liu et at. Proc. Nail. Acad. Sci. USA 93:8618-8623 (1996); and Chari et al. Cancer Research 52:127-131 (1992).
[0259] In some embodiments, antibody-maytansinoid conjugates may be prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biologtcai activity of either the antibody or the maytansi-noid molecule. See, e.g., U.S. Patent No. 5,208,020. in some embodiments, ADC with an average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody. In some instances, even one molecule of toxin/antibody is expected to enhance cytotoxicity over the use of naked antibody, [0260] Linking groups for making aotibady-maytansinoid conjugates include, for example, those described herein and those disclosed in U.S. Patent No. 5208020; EP Patent 0 425 235 B1; Chari etal. Cancer Research 52:127-131 (1992); US 2005/0276812 A1; and US 2005/016993 A1. (2) AurisMins and doiastatins {0261] Drug moieties include dolastatins, auristatins, and analogs and derivatives thereof {US 5635483; US 5780568; US 5767237; US 8124431}. Auristatins are derivatives of the marine mollusk compound dotastaiin-10. White not intending to be bound by any particular theory , doiasiatins and auristatins have been shown to interfere wit h microtubule dynamics, GTP hydrolysis, and nuclear and cellutar division (Woyke et at {2001) Antimicrob, Agents and Chemother. 45(12):3580-3584} and have antic.ancer (US 5863149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother- 42:2961-2965). The doiastatin/auristatin drug moiety may be attached to the anti body through the N (amino) terminus or the C (carboxyt) terminus of the peptidic drug moiety (WO 02/088172; Domnina et ai (2003) Nature Biotechnology 21(7):77S-7S4; Francisco etat (2003) Biood 102(4):1458-1465). {6262] Exemplary auiistaiin embodiments include the N-terminus linked monomethyiauristatin drug moieties De and Dp, disclosed in US 7498298 and US 7869241:
wherein the wavy line of De and Dp indicates the covalent attachment site to an antibody or antioody-iinker component, and independently at each location: R? is selected from H and C,-C8 aikyt; R3 is selected from H, C5-C8 alkyl, C3-Cs carbocyde, aryl, C-pCg alkyl-aryl. CrCe alkyl-(C;i-C8 carbocyde), Cs-C8 heterocyde and CrC- alkyl-(C3-C8 heterocyde); R4 Is selected from H, CrC8 alkyl, C3-C8 carbocyde, aryl, CrCfi alkyl-aryl, Ci-Ca alkyi-(C3-Ca carbocyde). CrCs heterocyde and CrCe alkyl-(C3-Cg heterocyde); R5 is selected from H and methyl; or R4 and R5 jointly form a carhocyciic ring and have the formula -(CRfiRb)„- wherein R« and R8 are independentiy selected from H, CrC8 alkyl and C3-Cs carbocycle and π is selected from 2, 3. 4, 5 and 6; R8 is selected from H and C t-C8 aikyi; R7 is selected from H, C^-Cg aikyi, C3-C8 carbocyde, aryi, CrC8 alkyl-aryl, C<-C8 alkyf-(C3-C5 carbocycle), C3-Cs heterocyde and CrCs alkyl-(C3-Cg heterocyde); each R8 is independently selected from H, OH, CrC8 aikyi, C3-Cg carbocycle and 0-(CrCs afkyl);
Rg is selected from H and C|-C8 aikyi; R10 is sefected from aryi or C3-Cs heterocyde; Z is O. S, NH. or NR12, wherein R12 is CrC8 aikyi; R11 is selected from H, C5-C20 aikyi, aryi, Cg-Cs heterocycles, -{R13G)m-R14, or -(R,30}m-CH(R15)2; m is an integer ranging from 1-1000; R13 is C2-Cs aikyi; R14 is H or C^-Cg aikyt; each occurrence of R15 is independently H, COOH, -{0Η2)η-Ν{Κ18}2, -(CH2)t>-S03H, or -(CH2),,-SOs-C rC8 aikyi; each occurrence of R16 is independently H, CrC8 alkyl, or -(CHa)n-COQH; R18 is selected from -C(R8)rC(R%-aryi, -C{Ra)2-C(Rs)2-(Ca-Ca heterocyde), and -C(R8)rC(R8)2-(C;rCg carbocyde); and n is an integer ranging from 0 to 6.
[0263} In one embodiment R3 R4 and R7 are independently isopropy! or sec-butyl and R5 is -H or methyl, in an exemplary embodiment, R3 and R4 are each isopropyl, R5 is -H, and R7 is sec-butyi [0264} In yet another embodiment, R2 and R® are each methyl, and R8 is -H.
[0266} In still another embodiment, each occurrence of R8 is -OCH3, [0266} In some embodiments, R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is -H. R7 is sec-butyi, each occurrence of R® is -OCHs, and R® is -H.
[02671 In one embodiment, 2 is -0- or -NH-, [0266} in one embodiment, R10 is aryl, [0266} In an exemplary embodiment, R18 is -phenyl.
[0270} In an exemplary embodiment when Z is -O-, Rr5 is -H, methyl or t-butyl, [0271} In one embodiment when 2 is -NH, R" is -CH(R1S)? wherein R15 is -{CB-)(!~N{R13);>, and R13 is -C«-C?; alkyl or -(CH2)n-COOH.
[0272J In another embodiment when Z is -NH, R11 is -CH{R1S)2, wherein R15 is -{CH2),t~SQ-H.
[0273} An exemplary auristatin embodiment of formula Dg is MMAE, wherein the wavy fine indicates the covalent attachment to a linker (L) of an antibody-drug conjugate:
[0274} An exempfary auristatin embodiment of formula Df.· is MMAF, wherein the wavy fine indicates the covalent attachment to a linker (L) of an antibody-drug conjugate:
[0275J Other exemplary embodiments include monomethyivaftne compounds having phenylalanine carboxy modifications at the C-terminus of the pentapepfide auristatin drug moiety {WO 2007/008848} and monomethyivaiine compounds having phenylalanine stdecham modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008603), [0276} Noniimiting exemplary embodiments of ADC of Formula I comprising MMAE or MMAF and various finker components have the following structures and abbreviations (wherein "Ab" is an antibody; p is 1 to about 8, 'Vai-Cif is a vaiine-ctiruiline dipepfide; and "S“ is a sulfur atom:
Ab-MC-ve-PAB-MMAF
Ab-MC-vc-PAB-MMAE
Ab-MC-MMAE
Ab-MC-MMAF
[0277] Nonlimiting exemplary embodiments of ADCs of Formula 1 comprising MMAF and various Sinker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF, Immunoconjugetes comprising MMAF attached to an antibody by a linker that is not protoofytically cieavabie have been shown to possess activity comparable to immunoeonjugstes comprising MMAF attached to an antibody by a proteoiyticaliy cieavabie Sinker (Domnina at a!. (2006) Bioconjugate Chem. 17:114-12:4). in some such embodiments, drug release is believed to be effected by antibody degradation in the ceii.
[0278] Typicaily, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to a l iquid phase synthesis method (see, 9.9., E. Schroder and K, Lubke, "The Peptides", volume 1, pp 78*138,1965, Academic Press). Aurista-tin/dofastetin drug moieties may, in some embodiments, be prepared according to the methods of: US 7498298; US 5635483: US 5780688; Pettit et a! (1989) J. Am, Cham. Soc, 111:5463-5465: Pettit et a) (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G.R., et at. Synthesis, 1996, 719-725; Pettit et ai (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784, [0279] In some embodiments, auristati n/d o I a statin drug moieties of formulas De such as MMAE, and Dp, such as MMAF. and drug-finker intermediates and derivatives thereof, such as MC-MMAF, MC-MMAE, MC-vc-PA8-M MAP, and MC-vc-PAB-MMAE, may be prepared using methods described in US 7498298; Doronina et al. (2006) Bioconjugate Chem, 17:114-124; and Doronina et ai. (2003) Nat. Biotech. 21:778-784 and then conjugated to an antibody of interest. (3) Gaiicheamitin [0286] In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more caiicheamicin moiecuies. The calicheamicin family of antibiotics, and analogues thereof, are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al.. (1993) Cancer Research 53:3336-3342; Lode et af, (1998) Cancer Research 58:2925-2928). Calicheamicin has intracellular sites of action but, in certain instances, does not readily cross the piasma membrane. Therefore, ceiiutar uptake of these agents through antibody-mediated internalization may, in some embodiments, greaiiy enhances their cytotoxic effects. Nonlimiting exemplary methods of preparing antibody-drug conjugates with a caiicheamicin drug moiety are described , for example, in US 5712374; US 5714586; US 5739116; and US 5767285. (4} Pyrroiobenzocfiazepines [0281] in some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, PDS dimers recognize and bind to specific DNA sequences. The natural product anthramycin, a PBD, was first reported in 1965 (Leimgruber. et at,, (1985) d. Am. Chem. Soc,, 87:5793-5795: Leimgruber, et al., ¢1965) J. Am. Chem, Soc., 67:5791-5793). Since then, a number of PBDs, both naturally-occurring and analogues, have been reported (Thurston, et ai., ¢1994) Chem:, Rev, 1994, 433-465 including dimers of the tricyclic PBD scaffold (US 6884799; US 7049311; US 7067511; US 7265105; US 7511032; US 7528126: US 7557099), Without intending to be bound by any particular theory, it is believed that the dimer structure imparts the appropriate three-dimensionai shape for isoheiicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, in Antibiotics Ilf, Springer-Verlag. New York. pp. 3-11 (1975): Hurley and Needham-VanOevanter, {1988} Acc, Chem, Res., 19:230-237}, Dimeric PSD compounds bearing 02 ary! substituents have been shown to be useful as cytotoxic agents {Hartley et al (2010) Cancer Res. 70(17):884S-6858; Aotonow (2010) J. Med. Chem, 53(7):2927-2941; Howard et at ¢2009} Bioorganic and Med. Chem. Letters 19(22):6463-6468). ¢0282} PSD dimers have been conjugated to antibodies and the resuiting ADC shown to have anti-cancer properties, Noniimiting exemplary linkage sites on the PBD dimer include the five-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group {WO 2009/018516; US 2009/304710; US 2010/047257; US 2009/036431: US 2011/0256157; WO 2011/13(593). ¢0283} Noniimiting exempiary PBD dimer components of ADCs are of Formula A:
and salts and solvates thereof, wherein: the wavy line Indicates the covalent attachment site to the linker, the dotted fines indicate the optional presence of a double bond between C1 and C2 or C2 and C3; R2 is independently selected from H. OH, =0. =CH2. CN. R, OR. «CH-R&amp; C(RD)2.0-S0rR, CO£R and COR, and optionally further selected from halo or dihaio, wherein RD is independently seiected from R, C02R, COR, CHO, C02H, and halo; R® and R- are independently seiected from H, R, OH, OR, SH, SR, NHj, NHR, NRR', H02. MesSn and halo; R7 is independently selected from H, R. OH, OR, SH, SR, NH;., NHR, NRR’, N02. Me3Sn and haio; 0 is independently seiected from O, S and VH; R11 is either H, or R or, where Q is O, S03M, where M is a metal cation: R and R' are each independently seiected from optionally substituted C1.g alkyl, C1.,? aikyi. C3.fi heterocyciyi, heterocycle, and Cs.20 ary! groups, and optionally in relation to the group NRR", R arxj R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-. 8*or7-membered beterocydie ring:; R12, H16, R19 and R17 are as defined for R2, R6, R9 and R7 respectively; R" is a C3^2 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, N{H), NMe and/or aromatic rings, e g, benzene or pyridine, which rings are optionally substituted; end X and X’ are independently selected from O, S and N(H) ¢0284} In some embodiments, R and R' are each independently selected from optionally substituted C^ aikyi, C3,2ø heterocycle, and ary! groups, and optionally in rela tion to the group NRR’, R and R’ together with the nitroge n atom to which they are attached form an optionally substituted 4-,5-,6- or 7-membered heterocyclic ring. ¢0285} In some embodiments, Rs and R17 are H. ¢0286} in some embodiments, R® and R1S are H.
[0287} In some embodiments, R7 are R17 are both OR7A, where R7A is optionally substituted aikyi, in some embodiments, RrA is Me, in some embodiments, R?A is is Ch2Ph, where Ph is a phenyl group. ¢0288} In some embodiments, X is O. ¢0289} In some embodiments, R11 is H. ¢0290} In some embodiments, there is a double bond between C2 and C3 in each monomer unit ¢0291} In some embodiments, R2 and R12 are independently seiected from H and R, in some embodiments, R2 and R'2 are independently R. In some embodiments, R2 and R12 are independently optionally substituted Ο5„20 aryl or C5„7 aryl or C&amp;>10 aryi. In some embodiments, R2 and R12 are independentiy optionally substituted phenyi, thienyl, napthyi, pyridyi, quinoiinyi, or isoquinofinyl. in some embodiments, R2 and R12 are independently seiecteci from =0, =CH2< =CH-RD, and *C{RD)S, In some embodiments, R2 and R12 each ~CH2, in some embodiments, R2 and R12 are each H. in some embodiments, R2and R12 are each =0, In some embodiments, R2 and R12 are each =CF2. in some embodiments, R2 and/or R12 are independently =C(RD>2·!n some embodiments, R2 and/or R,a are independent!'/ =CH-R°, [0292] in some embodiments, when R2 and/or R12 is ~CH-RD, each group may independently have either configuration shown below:
m [0293] In some embodiments., a =CH-RD is in configuration {I}.
[0294] In some embodiments, R” is a C3 alkytens group or a C5 aikyiene group.
[0295] in some embodiments, an exemplary PSD dimer component of an ADC has the structure of Formula A{!}:
A{I); wherein n is 0 or 1, [0296] In some embodiments, an Exemplary PSD dimer component of an ADC has the structure of Formula A{il):
A{H); wherein n is 0 or 1, [0297] In some embodiments, an exemplary PBD dimer component of an ADC has the structure of Formula A(!!l):
wherein Re and RE* are each independently selected from H or RD, wherein RD is defined as above; and wherein n is 0 or 1.
[0298] in some embodiments, n is 0, In some embodiments, n is 1. In some embodiments, Re and/or Re* is H. In some embodiments, RE and are H, in some embodiments, Re and/or RE" is R°, wherein R° is optionally substituted C^12 alkyl, in some embodiments, Rs and/or RE" is R°, wherein RD is methyl.
[0299] In some embodiments, an exemplary PBD dimer component of an ADC has the structure of Formula A{fV):
AflY); wherein Ar1 and Ar2 are each independently optionally substituted 0¾.¾ sryt; wherein Ar1 and As·1 may be the same c different; and wherein n is 0 or 1.
[0300] In some embodiments, an exemplary PBD dimer component of an ADC has the structure of Formula A(V);
A(V); wherein Ar1 and Ar2 are each independently optionally substituted Cs.2a aftf! wherein Ar1 end Ar2 may be the same t different; and wherein n is 0 or 1. C0301J In some embodiments, Ar1 and Ar2 are each independently selected from optionally substituted phenyl, furany thiophenyi and pyrtdyi. in some embodiments, Ar* and Ar2 are each independently optionally substituted phenyl, i n som embodiments, Ar1 and Ar2 are each independently optionally substituted thian-2-yl or thien-3-yl. In some embodiments Ar1 and Ar2 are each independently optionally substituted quinoiinyl or isoquinoinyi. The quinoiinyl or isoquinoiinyi grou may be bound to the PBD core through any available ring position. For example, the quinoiinyl may be quinoiin-2-y quinoiin- 3-yi, quinolin-4yi, quinolin-5-yi, quinolin-e-yl, quinoiin-7-yi and quinoiirt-8-yl, in some embodiments, the quinoiim is selected from quinoiin-3-y! and quino!fo-6-yi. The isoquinoitnyl may be isoqulnotin-t-yi, isoquinofin-3-yi, isoquinofir 4yi, isoquinolin'5-yl, isoquiaoiB>6-yl, isoqutnolfn-7-yt and isoquinoiin-8-yl. in some embodiments, the isoquinoiinyi I selected from lsoquinoiin-3-yl and isoquinotto-8-yi.
[0302] Further noniimiting exempiary PBD dimer components of ADCs are of Formula B:
and salts and solvates thereof, wherein: the wavy line indicates the covalent attachment site to the linker; the wavy line connected to the OH indicates the S or R configuration; RV1 and R'v'2 are independently selected: from H, methyl, ethyl and phenyl (which phenyi may be optionally substitute withffuoro, particularly in the 4 position) and C5^ heterocyclyi; wherein RV1 and Rv2 may be the same or different; an n is 0 or 1.
[0303] In some embodiments. RV1 and RV2 are independently selected from H, phenyi, and 4~fiuorophenyi.
[0304] In some embodiments, a linker may be attached at one of various sites of the PBD dimer drug moiety, inciudin the N1Q imine of the B ring, the C-2 endo/exo position of the C ring, or the tether unit linking the A rings (see structure C(l) and C(!i) befow).
[0305J Nonlimiting exemplary PBD dimer components of ADCs include Formulas Oft) and C(fl):
Formulas C(i) and C{ii) are shown in their N10-C11 irnine form. Exemplary PBD drug moieties also include the ca; btnolamine and protected carbinolamtne forms as well, as shown in the table below:
wherein: X is CH2 {n = i to 5), N, or O; Z and Z1 are independently selected from OR and NR;>, where R is a primary, secondary or tertiary alkyl chai containing 1 to 5 carbon atoms; R,. R',, R2 and R’2 are each independently selected from H, CrC8 alkyl, C2-Cs alkenyl, C2-C8 alkyoyl, Cg..20 an (including substituted aryls), heteroatyl groups, -NH2, -NHlvte, -OH, and -SH, where, in some embodiment; alkyl, alkenyl and afkynyl chains comprise up to 5 carbon atoms; R3 and R’g are independently selected from H, OR, NMR, and NR2, where R is a primary, secondary or tertiary aik; chain containing 1 to 5 carbon atoms; R* and R*4 are independently selected from H, Me, and One;
Rg is selected from CrC8 alkyl, C2-C8 alkenyl, C^-Cg aikynyi, Cs..2 aryl (including aryls substituted by halo, nit« cyano, alkoxy, alkyl, heterocydyl) and C5,2o heteroaryl groups, where, in some embodiments, alkyl, alkenyl an alkynyl chains comprise up to 5 carbon atoms; R^ its H, C-rCg alkyl, or a protecting group (such as acetyl, thfluoroacetyi, t-butoxycarbonyl (SOC), benzyioxycai bony! (C5Z), 9-fiuorenyimethylenoxycarbonyl (Fmoc), or a moiety comprising a self-immolating unit such as valint citrulline-PAB}; R.a is is H, C-j-Cg alkyl, or a protecting group; wherein 3 hydrogen of one of R,. R'v R2, R 2, or R12 or a hydrogen of the -OCHjCH^iX^CHjC^O-spacer betwee the A rings is replaced with a bond connected to the linker of the ADC. £03063 Exemplary PD8 d imer portions of ADC include, but are not lim ited to (the wavy tine ind icates the site of covaier attachment to the linker).
PBØ dimer;
Noniimiting exemplary embodiments of ADCs comprising PBD dimers have the following structures:
PBD dimer-val-cii-PAB-Ab:
PBD d i mer- m aie i mide- acetal - Ab;
PBD dimer-Phe-homoLys-PAB-Ab, wherein: n is 0 to 12. In some embodiments, rs is 2 to 10, fn some embodiments, n is 4 ίο 8. in some embodiments, n is selected from 4, 5,6, 7, end 8.
[03Q7J The Sinkers of PBD dimer-vai-cii-PAB-Ab end the PBD dimer-Phe-homoLys-PAB-Ab are protease cieavabie, while the Sinker of PSD dimer-maleimide-acetal is acid-labile.
[0308] PBD dimers and ADC comprising PBD dimers may be prepared according to methods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598, (S) Anthracyciines [0303] In some embodiments, an ADC comprising anthracyciine. Anthracyciines are antibiotic compounds that exhibit cytotoxic activity, Whiie not intending to be bound by any particular theory, studies have indicated that anthracyciines may operate to kil cells by a number of different mechanisms, including; 1) intercalation of the drug moiecuies into the DNA of the cei thereby inhibiting DMA-dependent nucleic acid synthesis; 2} production by the drug of free radicals which then react with cellular macromoiecutes to cause damage to the cells, and/or 3) interactions of the drug moiecuies with the cell membrane {see, e.g., C, Peterson et a!., ‘Transport And Storage Of Anthracyciine In Experimental Systems And Human Leukemia" in Anthracyciine Antibiotics In Cancer Therapy; N.R, Bachur, "Free Radical Damage" id. at pp.9?-1G2), Because of their cytotoxic potential anthracyciines have been used in the treatment of numerous cancers such as leukemia, breast carcinoma, lung carcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P.H-Wiernik, in Anthra-cycline: Current Status And Mew Developments p 11), [0313] Nonlimiting exemplary anthracyciines include doxorubicin, epirubicin, idarobicin, daunomycin, nemorubicin, and derivatives thereof, immunoconjugafes and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al (2006) Current Med. Chem, 13:477-523: Jeffrey et ai {2006} Bioorganic &amp; Med. Cham, Letters 16:358-362; Torgov et ai (2005) Bioconj. Chem. 16:717-721; Nagy et al {2000) Proc. Natl. Acad. Sci. USA 97:829-834; Dubowchik et a! (2002) Bioorg, &amp; Med. Chem. Letters 12:1529-1532; King et a! ¢2002} J, Med. Chem. 45:4338-4343; EP 0328147; US 6830579), The antibody-drug conjugate BR98-doxorubicSn reacts specifically with the tumor-associated antigen Lewis-Y and has been evaluated in phase I and SI studies (Saleh et al (2000) J, Clin. Oncology 18:2282-2292; Ajani et al (2000) Cancer Jour. 6:78-81; Toleheretai ¢1999) J. Clin. Oncology 17:478-484).
[0311] PNU-159682 is a potent metaboiite (or derivative) of nemorubicin (Quintiert, et al. ¢2005) Clinical Cancer Research 11 (4):1608-1817). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-melhoxymorpholmo group on the glycoside amino of doxorubicin and has been under clinical evaluation (Grandi et ai (1990) Cancer Treat. Rev. 17:133; Ripamonti et ai (1992) Brit, J. Cancer 66:703;), including phase it/ltt trials for hepatocellular carcinoma (Sun et al ¢2003) Proceedings of the American Society for Clinical Oncology 22, Abs1443; Guintien (2003) Proceedings of the American Association of Cancer Research, 44;1st Ed, Abs 4649; Paeoiarini et at (2008) Jour, Clin. Oncology 24:14116).
[0312] A noniimifing exemplary ADC comprising nemorubicin or nemorubicin derivatives is shown in Formula la:
wherein R1 is hydrogen atom, hydroxy or methoxy group and Ra is a CrC5 alkoxy group, or a pharmaceutically acceptable sail thereof; L1 and 2 together are a linker (l) as described herein; T is an antibody (Ab) as described herein; and m is 1 to about 20, in some embodiments, m is 1 to 10,1 to 7,1 to 5, or f to 4, [0313] In some embodiments, R< and Rs are both methoxy (~QMe).
[0314] A further nonlimiting exemplary ADC comprising nemorubidn or nemorubiein derivatives is shown in Formula I b:
wherein R1 is hydrogen atom, hydroxy or methoxy group and R2 is a C1-C5 alkoxy group, or a pharmaceutically acceptable sail thereof; L2 and Z together are a linker (L) as described herein: T is an antibody {Ab} as described herein; and m is 1 to about 20. in some embodiments, m is 1 to 10,1 to 7,1 to 5, or 1 to 4.
[0315] In some embodiments, R< and R? are both methoxy f OM©}.
[0316] In some embodiments, the nemorubiein component of a nemorubicin-containing ADC is PNU-159682. In some such embodiments, the drug portion of the ADC may have one of the following structures: or
whereon the wavy lino indicates the attachment to the linker (U, {0317} Anthracyclines, including PNU-159682, may be conjugated to antibodies through severai linkage sites and a variety of linkers (US 2011/0076287; WO20Q9/099741; US 2010/0034837; WO 2010/009124), including the linkers described herein, {0318} Exemplary ADCs comprising a oernorebicin and linker include, hut are not limited to:
FNU-159682 maleimide acetai-Ab;
PN U-159682-val-cit-P AB-Ab;
PNU-159682-va l-cit- P AS-spacer-Ab;
PNU-159682-val-ctt-PAB-spacer(Ri RSj-Ab, wherein: R, and R2 are independently selected from H and C,-Cs alkyl: and
P N U-159682-ma lei m ί de -Ab.
[03191 The linker of PNU-159682 maieimide acetal-Ab is acid-labile, whiie the linkers of PhiU-159682-val-cii~PA8-Ab, PNU-159682~v3!-cit-PAS-sp3cer-Ab, and PNU-159682-val-cit4'JA8“Spacer(R1R;-i)-Ab are protease cleavabie. f6) Other Drug Moieties [9320] Drug moieties also include geldanamydn (Mandler et al (2000) J. Nat Cancer inst 92(19):1573-1581; Mandler et al (2000) Bioorganie &amp; Med, Chem. Letters 10:1025-1028; Mandler et al (2002) Biocanjugate Chem. 13:788-791); and enzymatically active toxins and fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, atpha-sarcin, Aieuritesfordii proteins, dianthia proteins, Phytofaca americaoa proteins (PAPI, PAP!!, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinaiis inhibitor, geionin, mitogeifin, restrictocin, pheno-mycin. enomyein and the tricothecenes. See, e.g., WO 93/21232.
[9321] Drug moieties also include compounds with nucteoiytic activity (e.g., a riboouctease or a DNA endonuciease), [9322] In certain embodiments, an irormmoconjugate may comprise a highiy radioactive atom, A variety of radioactive isotopes are avaiiabfe for the production of radtoconjugated antibodies. Examples incfude At211,1131,1125, Vs0, Re18e,
Re188, Sm153, 8i212, P32, Pb212 and radioactive isotopes of Lu. In some embodiments, when an imrounoconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example To" or i123, ora spin iahet for nuclear magnetic resonance (NOR) imaging (also known as magnetic resonance imaging, MR!), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 may be compiexed to various metai cheiatlng agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983). (0323) The radio- or other labels may be incorporated in the immuooconjugate in known ways. Forexampie, a peptide may be biosyniheslzed or chemically synthesized using suitable amino acid precursors comprising, for example, one or more fiuorine-19 atoms in piece of one or more hydrogens, tn some embodiments, fabels such as Tc*< P23, Re188, Re588 and in111 can be attached via a cysteine residue in the antibody, in some embodiments, yttrium-90 can be attached via a lysine residue of the antibody, in some embodiments, the iOOOGEN method (Fraker etal (1978) Biochem. Biophys. Res. Commun. 80:49-57 can be used to incorporate iodine-123. “Monocionai Antibodies in immunoscintigrapby" (Chatai, CRC Press 1989) describes certain other methods, (0324) In certain embodiments,, an immunoconjugate may comprise an antibody conjugated to a prodrug-activating enzyme, in some such embodiments, a prodrug-activating enzymeconvertsa prodrug (e,g„. a peptidyi chemotherapeutic agent, see WO 81/01145) to an active drug, such as an anti-cancer drug. Such immunoconjugates are useful, in some embodiments, in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"), Enzymes ihat may be conjugated to an antibody include, but are not limited to, aikaiine phosphatases, which are usefui for converting phosphate-containing prodrugs into free drugs; aryisuifatases, which are useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase, which is usefui for converting non-toxic. 5-fiuorocytosine into the anti-cancer drug, 5-fSuorauracii; proteases, such as serratia protease, thermolysin, subtiiisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are usefui for converting peptide-containing prodrugs into tree drugs; O-aianyicarboxypepiidases, which are useful for converting prodrugs that contain D-amtno acid substituents; carbohydrate-cleaving enzymes such as jf-gaiactosidase and neuraminidase, which are usefui for converting glycosylated prodrags into free drugs; jl-lactamase, which is useful for converting drugs dorivatized with p-factams into free drugs; and peniciiiin amidases, such as penicillin V amidase and penicillin G amidase, which are usefui for converting drugs derivaiized at their amine nitrogens with phenoxyaeetyl or phenyiacetyi groups, respectively, into free drugs, in some embodiments, enzymes may be covalently bound to antibodies by recombinant DNA techniques wei! known in the art. See, e.g., Neubergeretai,, Nature 312:604-808 (1984), c) Drug Loading [8325) Drug loading is represented by p, the average number of drug moieties par antibody in a molecule of Formula I Drug ioading may range from 1 tao 20 drug moieties (D) per antibody. ADCs of Formula i include collections of antibodies conjugated with a range of drug moieties, from 1 to 20. The average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy, EUSA assay, and HPLC. The quantitative distribution of ADC in terms of may also be determined, in some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
[0326) For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in certain exemplary embodiments above, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached, tn certain embodiments, higher drug ioading, e.g, p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an ADC ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. indeed, it has been shown that for certain ADCs, the optima! ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5 (US 7498298), [0327) In certain embodiments, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Generally, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; indeed most cysteine thiol residues in antibodies exist as disulfide bridges, in certain embodiments, an antibody may be reduced with a reducing agent such as dithiothreitoi (DTT) ortricarbonytethylphosphine(TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as Sysine or cysteine.
[0328) The ioading (drug/antibody ratio) of an ADC may be controlled in different ways, and for example, by; (i) limiting the molar excess of drug-inker intermediate or tinker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification. (0329] It is to be understood that where more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, then the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody. Tire average number of drugs per antibody may be cafcufated from the mixture by a dual EL ISA antibody assay, which is specific for antibody and specific for the drug, individual ADC moiecufes may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see. e g., McOonagh ei at (2006} Prot, Engr. Design &amp; Selection 19(7):299-307: Hambiett et ai (2004) Ciin. Cancer Res, 10:7063-7070; Hambiett, K. J., et af. "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate." Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31,2004. Proceedings of ihe AACR, Volume 45, March 2004, Aiiey, S.C., e! a! "controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 827, American Association for Cancer Research, 2004 Annual Meeting, March 27-31.2004. Proceedings of the AACR, Volume 45, March 2004). in certain embodiments, a homogeneous ADC with a singfe loading value may be isolated from the conjugation mixture by electrophoresis or chromatog-raphy, d) Certain Methods of Preparing immunoconjugates (03303 An ADC of Formula S may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skiited in the art, including. (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent to form Ab-L via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucieophiiic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, folfowed by reaction with a nucleophilic group of an antibody, Exempiary methods for preparing an ADC of Formula f via the tatter route are described in US 7498298. (03313 Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminaf amine groups, (a) side chain amine groups, e.g. lysine, {ίίί) side chain thioi groups, e.g. cysteine, and (iv) sugar hydroxy! or amino groups where the antibody is giycosyiated. Amine, thioi, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ti) alkyt and benzyl halides such as baloacetamides; and (lit) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges, Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreito!) or tricarbonyiethyiphosphine (TCEP), such that the antibody is fully or partially reduced. Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles, Additional nucleophilic groups can be introduced into antibodies through modification of tystne residues, e.g., by reacting lysine residues with 2-iminothiolane (Traut'sreagent}, resulting in conversion of an amine into a thiol. Reactive thiol groups may also be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.g., by preparing variant antibodies comprising one or more non-native cysteine amino acid residues). (03321 Antibody-drug conjugates provided Herein may also be produced by reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or drug. Useful nucieophiiic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicatbazone. hydrazine carboxytate, and aryihydrazide. in one embodiment, an antibody is modified to introduce electrophilic moieties that are capable of reacting with nucieophiiic substituenis on ihe finker reagent or drug, in another embodiment, the sugars of giycosyiated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resuming imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages, in one embodiment, reaction of the carbohydrate portion of a giycosyiated antibody with either gaiactose oxidase, or sodium meta-pedodate may yield carbonyl (aldehyde and ketone) groups in the antibody thai can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing N-terminal serine or threonine residues can react with sodium meta-pertodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan &amp; Stro, (1992) Biooonjugate Chem, 3:138-146; US 5362852). Such an aidehyde can be reacted with a drug moiety or linker nucleophile. (0333J Exempiary nucieophiiic groups on a drug moiety include, but are not limited to: amine, thioi, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxyfate, and aryihydrazide groups capable of reacting to form covalent bonds with electrophilic groups on tinker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid hafides; (it) alkyl and benzyl halides such as haioacetamides; (ίίί) aldehydes, ketones, carboxyl, and maleimide groups,
(0334] Nonlimiting exemplary cross-linker reagents that may be used to prepare ADC are described herein In the section titled "Exempiary Linkers." Methods of using such cross-linker reagents to link two moieties, including a proteinaceous moiety and a chemical moiety are known in the art, in some embodiments, a fusion protein comprising an antibody and a cytotoxic agent may be made, e.g.. by recombinant techniques or peptide synthesis, A recombinant DNA molecule may comprise regions encoding the antibody and cytotoxic portions of the conjugate either adjacent to one another or separated by a region encoding a linker pep tide which does not destroy the desired properties of the conjugate.
[0335] In yet another embodiment, an antibody may be conjugated to 3 "receptors” {such es streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a ligand" {e.g.. avldin) which is conjugated to a cytotoxic agent (e.g... a drug or radionucteotide). E. Methods and Compositions for Diagnostics and Detection [0336] in certain embodiments, any of the anti-FcRHS antibodies provided herein is useful for detecting the presence of FcRHS (e g,, FcRHS) in a biological sample. The term "defecting” as used herein encompasses quantitative or qualitative detection, in certain embodiments, a biological sample comprises a ceii or tissue, in certain embodiments, such tissues include normal and/or cancerous tissues that express FcRHS at higher Javels relative to other for example, B-eells and/or B-ce!i associated tissues, in some embodiments, the anti-FcRH5 antibody binds an isoform e-specific region of the extracellular domain of FcRHSc. In some embodiments, the anti-FcRHS antibodies binds lg-like domain 9 of FcRH5c.
[0337] In one aspect, provided herein are methods of detecting toe presence of FcRHS in a bioiogicai sample, in certain embodiments, the method comprises contacting the bioiogicai sample with an anti-FcRHS antibody under conditions permissive for binding of the anti-FcRHS antibody to FcRHS, and detecting whether a complex is formed between toe anti-FcRHS antibody and FcRHS. in one aspect, the invention provides a method of diagnosing a disorder associated with increased expression of FcRES. In certain embodiments, the method comprises contacting a test ceii with an anti-FcRHS antibody; determining the ievei of expression (either quantitatively or qualitatively) of FcRHS by toe test ceii by detecting binding of the anti-FcRHS antibody to FcRHS; and comparing the level of expression of FcRHS by the test ceii with toe level of expression of FcRHS by a confroi ceil (e.g., a normal ceii of the same tissue origin as the tesi cell or a cell that expresses FcRHS at levels comparable to such a norma! ceii), wherein a higher ievei of expression of FcRHS by toe test ceii as compared to the control ceii indicates the presence of a disorder associated with increased expression of FcRHS, in certain embodiments, the test cell is obtained from an individual suspected of having a disorder associated with increased expression of FcRHS, in certain embodiments, toe disorder is a ceii proliferative disorder, such as a cancer or a tumor, in some embodiments, the FcRHS is FcRHSc, in some embodiments, the anti-FcRHS antibody binds an isoform e-specific region of the extracellular domain of FcRHSc, in some embodiments, Ihe anti-FcRHS antibodies binds !g-like domain 9 of FcRHSc, [0338] Exemplary cell proliferative disorders that may be diagnosed using an antibody described herein include a 8-ceff disorder and/or a B-cell proliferative disorder including, but not limited to, lymphoma, multiple myeloma non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia. (CLL), small lymphocytic lymphoma, leukemia, hairy ceii leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cel! lymphoma.
[0339] In one embodiment, an anti-FcRHS antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of FcRHS in a biological sample is provided, In certai n embodiments, the method comprises contacting the bioiogicai sample with an anti-FcRHS antibody as described herein under conditions permissive for binding of the anti-FcRHS antibody to FcRHS, and detecting whether a complex is formed between the anti—FcRHS antibody and FcRHS in the biological sample. Such method may be an in vitro or in vivo method, in one embodiment, an anti-FcRHS antibody is used to select subjects eligible for therapy with an anti-FcRHS antibody, e.g, where FcRHS is a biomarker for selection of patients. In a further embodiment, the biological sample is a cell or tissue (e.g.. biopsy material), in some embodiments, the anti-FcRHS antibody binds an isoform e-specific region of the extracellular domain of FcRHSc, In some embodiments, the anti-FcRHS antibodies binds lg-like domain 9 of FcRHSc.
[0340] In a further embodiment, an antt-FcRH5 antibody is used in vivo to delect, e.g,, by in vivo imaging, an FcRHS-positive cancer in a subject, e.g.. for the purposes of diagnosing, prognosing, or staging cancer, determining the appropriate course of therapy, or monitoring response of a cancer to therapy. One method known in the art for in vivo detection is immuno-positron emission tomography (immuno-PET). as described, e.g,, in van Dangers et ai, The Oncologist 12:1379-1389 (2007) and Verel ai ai, J. Mac! Med. 44:1271-1281 (2003). in such embodiments, a method Is provided for detecting an FcRHS-posilive cancer in a subject, the method comprising administering a labeled anti-FcRHS antibody to a subject having or suspected of having an FcRHS-posifive cancer, and detecting the labeled antFFcRHS antibody in the subject, wherein detection of the labeled anti-FcRHS antibody indicates an FcRHS-posltive cancer in the subject. In certain of such embodiments, the labeled ami-FcRHS antibody comprises an snti-FcRH» antibody conjugated to a positron emitter, such as ^Ga, 1*F, s'4Cu, 86Y, T6Br, 89Z and 124l, In a particular embodiment, the positron emitter is ®%r. in some embodiments, the anti-FcRHIS antibody binds an isoform c-speclfie region of the extracellular domain of FcRHSc. In some embodiments, the anti-FcRH5 antibodies binds ig-iike domain 9 of FcRHSc, [0341] in further embodiments, a method of diagnosis or detection comprises contacting a first anti-FcRH5 antibody immobilized to a substrate with a biological sample to be tested for the presence of FcRHS. exposing the substrate to a second anti-FcRHS antibody, sod detecting whether the second enti-FcRHS is bound ίο a complex between the first anti-FcRHS antibody and FcRHS in the biological sample. A substrate may be any supportive medium., e.g., glass, metal, ceramic, polymeric beads, slides, chips, and other substrates. In certain embodiments, a biological sample comprises a ceii. blood, or tissue ie.g.. biopsy material} [0342] Exemplary disorders that may be diagnosed or detected according to any of the above embodiments include FcRHS-positive Cancers, such as FcRHS-positive 8-cefl proliferative disease, FcRH&amp;-positive plasma ceil neoplasm, and FcRHS-positive multiple myeloma, in some embodiments, an FcRH'i-positive cancer Is detected by anti-FcRBS immunohistochemistry (IHC) or in situ hybridization (iSH). In some embodiments, an FcRHS-positive cancer is a cancer that expresses FcRHS according to a reverse-transcriptase PCR (RT-PCR) assay that detects FcRHS mRNA. in some embodiments, the RT-PCR is quantitative RT-PCR, [0343] in certain embodiments, labeled antl-FcRHS antibodies are provided, In some embodiments, the anti-FcRH5 antibody binds an isoform c-specific region of the extracellular domain of FcRHSc, In some embod iments, the anti-FoRH5 antibodies binds lg-!ike domain 9 of FcRHSc. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as we!! as moieties, such as enzymes or ligands, that are detected indirectly, &amp;.g„ through an enzymatic reaction or molecular Interaction. Exemplary labels include, but are not limited to, the radioisotopes 32P, ,4C. ,25l; 3H, and 13il, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivative, dansyl, umbeffiferone, lueiferases. e.g,. firefly ludferase and bacteria! iuctferase (U.S, Patent No. 4,737,456), (uciferin, 2,3-dihydrophthaiazinediones, horseradish peroxidase (HRP), alkaline phosphatase, (f-gatactosidase, giucoamylase, lysozyme, saccharide oxidases, e.g.t glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that empfoys hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or mlcroperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the tike, in another embodiment, a label is a positron emitter. Positron emitters include but are not limited to 68Ga, rsF. t54Gu, soy rsgff 8¾. and 124l. In a particular embodiment, a positron emitter is S9Zr. F, Pharmaceutical Formulations [0344] Pharmaceutical formulations of an anti-FcRHS antibody or immunoconjugate as described herein are prepared by mixing such antibody or immunoconjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pbarmaceuticaf Sciences 16th edition, Osoi, A, Ed, (1980)), in tbe form of iyophized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbicacid end methionine; preservatives (such as octadecyidimefhyi benzyl ammonium chloride: hexamethonium chloride; beuzalkoniuro chloride; benzethonlum chloride; phenol, butyl or benzyl alcohol; alky! parabens such as methyl or propyl paraben; catechol; resorcinof; cyclohexane!; 3-pentanol; and m-cresol); iow molecular weight (iess than about 10 residues) polypeptides: proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poiyvinylpynOlidone; amino acids such as glycine, glutamine,, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including giucose, mannose, or dextrine; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes {e.g. Zn-protein compfexes); andtor non-ionic surfactants such as polyethylene glycol (PEG), Exemplary pharmaceutically acceptable carriers herein further include insterstitia! drug dispersion agents such as soluble neutral-active hyaiuronidase glycoproteins (sHASEGP), for example, human sofuble PH-20 hyaluroni-ciase glycoproteins, such as rHuPH2Q (HYLENEX©, Baxter International, Inc.}. Certain exemplary sHASEGPs and methods Of use, including rHuPHZO, are described in US Patent Publication Nos. 2005/0260186 and 2008/0104968. in one aspect, a sHASEGP is combined with one or more additional giycosaminoglycanases such as chondroitinases. in some embodiments, the anti-FcRH5 antibody binds an isoform e-specific region of the extracellular domain of FcRHSc, In some embodiments, the anti-FcRHS antibodies binds lg~iike domain 9 of FcRHSc.
[0345] Exemplary iyophized antibody or immunoconjugate formulations are described in US Patent No, 6,267,958, Aqueous antibody or immunoconjugate formulations include those described In US Patent No. 6.171,586 and WG20Q6/044908, the latter formulations including a histidine-acetate buffer.
[0346] The formulation herein may also contain more than one active ingredient as necessary for t he particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
[0347] Active ingredients may be entrapped in microcapsuies prepared, for example, by coacervation techniques or by inlerfaclai polymerization, for example, bydroxymefhyiceiiufose or geiatin-microcapsules and poiy-(methy!meth-acylate) microcapsuies, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16th edition, Gsol, A, Ed, (1980).
[0348} Sustained-release preparations may be prepared. Suitable examples of sustained-reiease preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody and/or immunoconjygate, which matrices are in the form of shaped arBcies, e.g. fiims, or mierocapsuies, [0349} The formulations to be used form vivo administration are generally sterile, Sterility may be readily accomplished, e.g., by filtration through sierife filtration membranes, G. Therapeutic Methods and Compositions [0350} Any of the anti· FcRHS antibodies {e,g,, FcRHS bispecific antibodies) and/or immunoconjugates provided herein may be used in methods, e.g., therapeutic methods. In some embodiments, the anti-FcRHS antibody binds an isoform c-spedfic region of tbe extracellular domain of FcRH5c. in some embodiments, the anti-FcRH5 antibodies binds fg-itke domain 9 of FcRHSc.
[0351} In one aspect, an anti-FcRH5 antibody (e.g., FcRHS bispecific antibody) and/or immunoconjugate provided herein is used in a method of inhibiting proliferation of an FcRHS-positive ceil, the method comprising exposing the ceil to the anti-FcRH5 antibody (e.g,, FcRHS bispecifie antibody) and/or immunoconjugate under conditions permissive for binding of the anti-FcRH5 antibody (e.g., FcRHS bispecific antibody) and/or immunoconjugate to FcRHS (e.g., FcRHSc) on the surface of the cell, thereby inhibiting the proliferation of the cell. In certain embodiments, the method is an in vitro or an in vivo method, in further embodiments, the ceil is a 8-cell proliferative disorder, in certain embodiments, the ceil proliferative disorder is associated with increased expression and/or activity of FcRHS (e.g., FcRHSc). For exampie, in certain embodiments, the B-ceii proliferative disorder is associated with increased expression erf FcRHS on the surface of a 8-ceii. in certain embodiments, the B-ceii proiiferative disorder is a tumor ora cancer, in some embodiments, tbe B-ceii proiiferative disorder is a plasma ceii neoplasm, in some embodiments, the piasma ceil neoplasm is multiple myeloma, plasmacytoma, and/or MGUS. Examples of 8-ceii proiiferative disorders to be treated by the antibodies and/or immunoconjugates Of the invention include, but are not limited to, lymphoma, multiple myefomanon-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL). small lymphocytic lymphoma, leukemia, hairy cel! leukemia (HCL), acute lymphocytic leukemia (ALL), and/or mantle cel! lymphoma.
[93521 Presence of various biomarkers in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skied artisan, including, but not limited to, immunohistochemistry ("IHC"}, Western blot analysis, im munoprecipitation, molecular binding assays, ELISA, ELiFA. fluorescence activated ceii sorting ("FACS”), MassARRAY, proteomies, quantitative biood based assays (as for exampie Serum ELfSA), biochemical enzymatic activity assays, in situ hybridization, Southern analysis. Northern analysis, whole genome sequencing, polymerase chain reaction f PCR") including quantitative real time PCR (“qRT-PCR") and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like, RNA-Seq, FISH, microarray analysis, gene expression profiling, and/or serial analysis of gene expression ("SAGE"), as well as any one of the wide variety of assays that can be performed by protein, gene, and/or tissue array analysis, Typieaf protocols for evaiusiing the status of genes and gene products are found, for exampie In Ausubei et al, eds.. 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotfing) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery f'MSD’’) may also be used.
[0353} Inhibition of ceil proliferation in vitro may be assayed using the CeSiTiter-Glo™ Luminescent Ceii Viability Assay, which is commercially available from Promega (Madison, Wl). That assay determines the number of viable ceils in culture based on quantitation ofATP present, which is an indication of metabolically active cells. See Crouch et al (1993) J. Immunol. Meth, 160:81-88, US Pat, No, 6502677. The assay may be conducted in 96- or 384-well format, making it amenable to automated high-throughput screening (HIS). See Cree et al, (1995) AntiCanoer Drugs 6:398-484. The assay procedure involves adding a single reagent (CeiiTiter-Gio® Reagent) directly to cultured cells. This resuits in ceil lysis and generation of a luminescent signal produced by a iuciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in culture. Data can be recorded by iuminometer or CCD camera imaging device. The luminescence output is expressed as relative light units (RLU).
[0354} In another aspect, an anti-FcRHS antibody (e g., FcRHS bispecific antibody) and/or immunoconjugate for use as a medicament is provided, in further aspects, an anti-FcRH5 antibody (e.g.,FcRHS bispecifie antibody) and/or immunoconjugate for use in a method of treatment is provided, in certain embodiments, an anti· FcRHS antibody (e.g., FcRHS bispecifie antibody) and/or immunoconjugate for use in treating FcRHS {e.g,, FcRB5c)-positive cancer is provided, in certain embodiments, provided herein the snt:-FcRH5 antibody (including FcRHS bispecifie antibody) and/or immuno-conjugate for use in a method of treating an individual having an FcRHS (e.g., FcRHScJ-positive cancer, the method comprising administering to the individual an effective amount of the anti-FcRHS antibody and/or immunoconjugate. in some embodiments, the anti-FcRHS antibody binds an isoform e-specific region of the extracellular domain of FcRHSc. In some embodiments, the anti-FcRHS antibodies binds Ig-like domain 9 of FcRHSc. in one such embodiment the method further comprises administering to the individual an effective amount of at feast one additional therapeutic agent, e.g., as described below.
[0355) !n a further aspect, provided herein are uses of an anti-FcRHS antibody {eg,, FcRHS bispecific antibody) and/or immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of FcRHS (e,g,, FcRH5c)-positive cancer. In a further embodiment, the medicament is for use in a method of treating FcRHS [e.g., FcRH5c)-posttive cancer, the method comprising administering to an individual having FcRHS (e.g., FcRHS expositive cancer an effective amount of the medicament, in one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below, in some embodiments, the anti· FcRHS antibody binds an isoform e-specifie region of the extracellular domain of FcRHSc. In some embodiments, the anti-FcRHS antibodies binds fg-iike domain 9 of FcRHSc, [0356) In a further aspect provided herein are methods for treating FcRHS (e.g,, FcRH5c)-positive cancer, in one embodiment, the method comprises administering to an individual having such FcRH35 (e.g., FcRH5c)-positive cancer an effective amount of an anti-FcRHS antibody (e.g., FcRH5 bispecific antibody) and/or immunoconjugate. in one such embodiment, the method further comprises administering to the individual an effective amount of at (east one additional therapeutic agent, as described below., in some embodiments, the anti-FcRHS antibody binds an isoform c-specific region of the extracellular domain of FcRHSc, In some embodiments, the anti-FcRHS antibodies binds lg-iike domain 9 of FcRHSc.
[0357) An FcRHS-positive cancer according to any of the above embodiments may be, e.g., FcRHS-positive 8-eeli proliferative disorder, FcRHS-positive plasma celi neoplasm, and/or FcRHS-positive muitipie myeloma, in some embodiments. an FcRHS-positive cancer is detected by anti-FcRHS immunohistochemisiry (iHC) or in situ hybridization (iSH), In some embodiments, an FcRHS-positive cancer is a cancer that expresses FcRHS according to a reverse-transcriptase PCR (RT-PCR) assay that detects FcRHS mRNA. in some embodiments, the RT-PCR is quantitative RT-PCR.
[0358} In some embodiments of any of the above embodiments, the individua! may be a human.
[0359) in a further aspect, provided herein are pharmaceutical form illations comprising any of the anti-FcRHS antibodies and/or immunoconjugate provided herein, e.g,, for use in any of the above therapeutic methods, in one embodiment, a pharmaceutical formulation comprises any of the anti-FcRHS antibodies (e.g., bispecific antibodies) and/or immunocon-jugates provided herei π and a pharmaceutically acceptable carrier, in another embodiment, a pharmaceutical formuiation comprises any of the anti-FcRHS antibod ies (e.g,, bispecific antibodies) and/or immunoconjugates provided herein and at least one additional therapeutic agent, e.g., as described below.
[0360) Antibodies {e.g., bispecific antibodies) and/or immunoconjugates provided herein can be used either atone or in combination with other agents in a therapy. Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody or immunoconjugate provided herein can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Antibodies and/or immunoconjugates provided herein can also be used in combination with radiation therapy.
[0361) An antibody (including bispecific antibody) and/or immunoconjugate provided herein (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, irrtrapulmonary, and intranasal, and, if desired for focal treatment, intralesional administration, Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitonsai, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic, Various dosing schedules including but not limited to single or multipie administrations over various time-points, bolus administration, and pulse infusion are conlem plated herein.
[0362) Antibodies (e.g,, bispecific antibodies) and/or immunoconjugates provided herein would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody (e.g., bispecific antibodies) and/or immunoconjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The affective amount of such other agents depends on the amount of antibody or immunoconjugate present in (ho formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empiricaliy/ciinically determined to be appropriate, [0363) For the prevention or treatment of disease, the appropriate dosage of an antibody (e.g., bispecific antibody) and/or immunoconjugate provided herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody or immunoconjugate, the severity and course of the disease, whether the antibody (e.g., bispecific antibody) and/or immunoconjugate is administered For preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody or immunoconjugate, and the discretion of the attending physician. The antibody {e.g., bispecific antibody) and/or immu- noconjugate are suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 ug/kg to 15 mg/kg (e.g, 0.1 mg/kg-tOmg/kg} of antibody or immunoconjugate can be an initia! candidate dosage for administration to the patient, whether,, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from: about Vg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over severe! days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody (e.g., bispecific antibody) and/or immunoconjugate would be in the range from about 0,05 mg/kg to about 10 mg/kg, Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g, such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initia! higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
[03641 it is understood that any of the above formulations or therapeutic methods may be carried out using both an immunoconjugate provided herein and an anti-FcRHS antibody, in some embodiments, the anti~FcRH5 antibody binds an isoform e-specific region of the extracellular domain of FcRHSc, In some embodiments, the anti-FcRHS antibodies binds Ig-iike domain 9 of FcRHSc. H. Articles of Manufacture [03651 In another aspect provided herein, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above Is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, battles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disorder and may have a sterile access port (for example the container may be an intravenous solution bag or a via! having a stopper pierceabie by a hypodermic injection needle). At least one active agent in the composition is an antibody or immunoconjugate provided herein. The label or package insert indicates that the composition is used for treating tee condition of choice, Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an FcRH5 antibody (e.g., bispecific antibody) and/or FcRHS immunoconjugate provided herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment, provided herein may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaeeutieaiiy-acceptable buffer, such as bacteriostatic water for injection (8WFI), phosphate-buffered saiine. Ringer's solution or dextrose solution. It may further indude other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the anti-FcRHS antibody binds an isoform c-specific region of the extracellular domain of FcRHSc. in some embodiments, tee anti-FcRH5 antibodies binds ig-iike domain 9 of FcRHSc,
I«. EXAMPLES
[0366} The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.
Materials and Methods
Immunogen {Ell-Sag) [036?! Amino acids 745-850 of human FcRHSc (SEQ ID N01) were cloned into mammalian expression vector pRK5.NT.Fiag using standard protocols and expressed transiently in CHO celts. The recombinant protein with N-terminal Flag-expression tag was purified using ansi-bag and size exclusion chromatography on an 8200 Superdex column.
Development and characterization of mouse anti-FcRHS E11 antibodies [03681 Bafb/c mice (Charles River, Hollister, CA) were immunized with 2 pg human FcRH5 E11 ECO protein {amino acid residues 743-850 of (SEQ ID NO; 1) (Genentech, South San Francisco, CA) mixed with MPl+TDM (Ribt) adjuvant via footpad injection. Mice received nine doses, followed by a prefusion boost in PBS atone via footpad and iV routes three days prior to fusion, [0368] Poplitea! lymph nodes were harvested and lymphocytes from these mice, all of whose sera demonstrated strong binding titers to the immunization protein by ELISA and showed strong FACS reactivity to SVT2 ceiis transfected with the human FcRHS E11 ECO, were fused withX63-Ag8.653 mouse myelomaceiis (American TypeCuiture Collection, Rockville, MO) via eiectrofusion (Harvard Apparatus, Holiiston, MA), Fused ceils were incubated at 37'C, 7% C02, overnight in Medium C (StemCeii Technoiogies, Vancouver, BC, Canada), before resuspension in semi-soiid Medium D (StemCeii Technoiogies) containing 0,01 mg/ml FITC labeled anti-mouse igG (Jackson immunoResearch, West Grove, PA) and plating into Omniweit trays (Thermo Fisher Scientific, Rochester, NY), Nine daysafter plating, fluorescent cefonies were selected and transferred into 96-weii plates conlatning Medium E (StemCeii Technoiogies) using a Clonepix FL (Genetix, New Milton, Hampshire, UK). Supernatants were screened by ELISA against anti-mouse igG (MP Biomed-icais, Santa Ana, CA) seven days after picking, [0370] Hybridomas demonstrating mouse IgG expression by ELISA were expanded and screened by FACS against SVT2 ceiis overexpressing fuii-length human FcRH5. cyno FcRH5, and human FcRHS E11 ECO. Strong FACS positive clones were subcloned by singie-celi sorting using a FACSAria (B0, Franklin Lakes, NJ), Final clones demonstrating the highest ELISA and FACS binding of interest after one or two rounds of subcloning were expanded for large-scale production in bioreactors (Integra Biosciences, Chur, Switzerland), Supernatants were then purified by Protein A affinity chromatography as previously described (Hongo ef a/, 2000).
Production of bis Fabs [0371] BisFabs were generated by crosslinking a Fab! of the anti-FcRHS Mab to a Fab’ of the anti-CD3 (UCHT1 ,v9) Mab at the hinge cysteine residues. To generate the Fab' 2 fragments from the hybridoma Abs different digestion conditions were used: Abs of the migG1 isotope were digested with 1:50 (w/w) pepsin at pH 3,5 for 1-2 H at 37“C.; mouse igG2a Abs were digested with Lysin C endopeptklase at 1 1:500 (w/w) ratio, pH 8, tor 2-4 h at 3?“C' and mouse igG2b Abs were digested with Lysin C at a 1:100 (w/w) ratio overnight at 37“C. In all cases the F(ab')? fragment was isolated from the reaction mixture by capture with a SP column and elution with 10 column voiumes of a Sineai gradient (0-100%) of 1M sodium ehforide. Under the digestion conditions mentioned above migGt and migG2b produced a F(ab*)a fragment, containing three Cysteine residues in the hinge, while the F(ab’)2 from mlgG2a showed two cysteine residues in the hinge. To generate Fab' with a single reactive Cys two different methods were used. For fragments containing an odd (3) number of hinge cysteines (rntgGI and m!gG2b) the isolated F(ab’)2s were reduced in 25 mM sodium acetate, pH 5,150 mM sodium chiorido, 2 mM EDTA. 2 mM TCEP for 2-6-H at RT, After tire reduction step was complete, the sample was diluted to 0.2 mg/mf, the pH was raised to 7.5 by adding Tris pH 8 and 5 mM dehydroascorbic acid (DH.AA) was added to drive re-oxidation of the cysteine. After an overnight incubation at room temperature the presence of reduced Thiols was evaluated by probing with an excess ofNEM end analyzing the MW shift by mass spectrometry, After confirming the presence of only one reactive Cysteine per molecule, the Fab' was purified by gel filtration to remove small amounts of homodimers.
[0372] For F(ab’)j fragments derived from m!gG2a and containing 2 Cysteine residues in the hinge, a single reactive Cysteine was produced by partial blocking with N-ethyl roaieimide (NEM) as described in Scheer ei al (in press). Briefly, the antibody was digested with pepsin (1% w/w) by treatment In sodium acetate buffer at pH 4.5. After digestion for 1 hour, the F(ab'}2 was isolated from the digestion mixture by capture on an SP-HP cation exchange resin and purified by a 10 CV salt gradient of 0-1 M NaCL The F(ab')p was then reduced with 1 mM TCEP in a buffer containing 25 mm MES, pH 5,8, 2 mM EDTA, and 300 mM Nad and the Fabs were oxidized by the addition of 5 mM dehydroacorbic add (DHAA) to reform the disulfide bond between the heavy chain and light chain, [0373] The effector arm of the hisfabs (UCHT1 ,v9) was generated by pepsin digestion, partial MEM blocking and conjugation to bismaleirnide as described before (Scheer et al; in press). Briefly, the two thiols (cys residues) at the hinge were then reacted with 1 equivalent of N-ethyimaieimide (NEM) (Sigma Aldrich), The different anti-FGRH5 Fab's containing a single reactive Cysteine were incubated with the anti-CD3 Fab’ conjugated to the bismaleirnide crosslinker overnight at room temperature. The - 100kDa cross!inked Fabs were separated from the unreacted species by ge! filtration and than characterized by SDS-PAGE, mass spectrometry and analytical size exclusion chromatography, PDB expression and purification [0374] TDBs were produced by two different approaches: co-culture of bacteria expressing each of the two antibody arms or by expressing each arm seoarateiy and then annealing them in vitro, The strategies have been described in Christoph Spiess et ai 2012 and described in PCT/US10/58958 filed on 31 May 2011. Breifiy for the coculture strategy E. coii expressing arrti-CD3 (hole) and Eeeii expressing anti-tumor target (knob) were grown together in shaker flasks at a predetermined ratio such that it produced similar amounts of each hemimer. The cocultured bacterial broth was then Harvested, the ceils disrupted in a microfiuidizer and the antibodies purified by Protein A affinity. It has been observed that during microfluidizing and protein A capture the two arms anneaied and formed the bing inter-chain disuifide bridges (Christoph S pi ess etai, 2012), Alternatively, the antibody hemimers were grown separately by high-cell density fermentation and independently isolated by Protein A chromatography. The purified hemimers were than combined at a 1:1 molar ration and indubated in 50 mM Tris, pH 8.5 in the presence of 2 mM OTT for 4 hours to allow annealing and the reduction of disulfides In the hing region. Dialysis against Ihe same buffer without DTT for 24-48 hours resuited in the formation of the inter-chain disuifide bonds. For both production strategies the Bispecific antibody was purified from contaminants by hydrophobic interaction chromatography (HIC) as described in Christoph Spiess et ai- 2012. The resuiting materiaf was analyzed tor endotoxin ieveis using an Endosafe potable etest system and when needed, the endotoxin content was reduced by washing the protein with 0.1% Triton X-f 14, TDB characterization [0375J The moiecuiar weight of the bispecific antibody was analyzed by mass spectrometry (LC-ESi/TCF) as described before (Jackman et ai, 2010), The antibodies were also analyzed by analytical size exclusion chromatography in Zenix SEC-300 column {Sepax Technologies URSA) using an Agilent 1:100 HPLC system. The presence of residual antibody fragments was quantified by electrophoresis using a 2100 Bioanaiyzer and a Protein 230 Chip.
Blood ceil fractionation [0376] PBMCs were separated from the blood of heaithy volunteers using lymphocyte separation medium (MP biomedicals, Solon, OH). CD8+ cells were extracted from PBMCS using human CD8+ Isolation Kit from Miltersyi (#130-094-156) by negative selection. in vitro cytotoxicity assays (T ceti kitting) [0377J For in vitro cytotoxicity assays IxlO4 target ceils were plated on 96 well piafes and incubated overnight. 3xf 04 CD8+ T-ceiis were added with or without TDB or BisFab and incubated 48 hours in +37¾. T ceiis were removed by washing twice with growth media, Cel viability was measured using CellTiter-Gle® Luminescent Cell Viability Assay (Promega, Madison, VVi).
[0378] Alternatively, in vitro cytotoxicity was monitored by flow cytometry. Target cells were labeled with carboxyfiu-orescein succioimidyl ester (CFSE) according to manufacturer's protocol (invifrogen, #C345S4), The CFSE -labefed target cells and purified CD8+ T cels from human P8MC were mixed in 3:1 E:T ratio and incubated with TD8 or BisFab for 48 hours. At the end of the incubation, the cells were lifted by trypsin and collected from the piate. The cells were resuspended in equal volume of PBS +2% F8S + 1 mM EDTA + propidium iodine (PI). Flow cytometry analysis was done on a FACSCaiibur in automation format. The number of five target cels was counted by gating on CFSE+/P1 negative cels. The percentage of cytotoxicity was calculated as follows: % cytotoxicity (live target cell number w/o TDB - live target ceil number w/TDB) / (live target ceil number w/b PDB) x 1QQ,
Analysis of T mil activation [0379] Target ceils and purified CD8+T cels were mixed in the presence or absence of TDB andTceil activation was analyzed by flow cytometry. At the end of the incubation, cels were stained with CDS-FiTC (BD Biosciences, 565634) and CD89-PE (80 Biosctences, 555631}.
Sinding ofsubdone supernatant, monoclonal antibodies, hisFabs and TDBs [0380] To test binding to endogenously FcRH5 expressing cancer cells or FeRHS transfected cancer ceiis, ceiis were lifted using EDTA/PBS. 1x10s cells were suspended in 100 ul and incubated h with primary antibodies (1 volume ofnon-igG quantitated subclone supernatant, 4 ug/mt IgG quantified subcione supernatant or 2ug/ui purified monoclonal antibodies). Ceiis were washed twice with FACS buffer (PBS 1%BSA 2mM EDTA) and incubated with 1:1000 dilution of goat anti-mouse secondary labeled with PE or 1:100 of goat: anti-mouse APC. Cels were washed twice with FACS buffer and Fiow cytometry analysis was done on a FACSCaiibur. Direct Xenon-labeling of antibodies was done according to manufacturer’s protocol (Invitragen), when indicated. To analyze binding to NK or B coils, 1 million human P8MC were incubated with 4 ug/mt IgG quantified subclone supernatants for 80 min, washed and incubated with 1:100 dilution of goat anti-mouse secondary labeled with APC. Ceils were then washed again twice and stained using amti-CDSS (PE; BID Biosaenees #555516) and anti-GDI 9 (PE; 80 Biosciences #340364) prior flow cytometry and analysis of binding to human CDC56+ and CD19+ cels.
Results [0381 j Initially to produce isoibrm specific antibodies for the membrane proximal Ig-domain. mice were immunized with recombinant bactiovirus produced E11 protein (amino acids 745-848 ofSEQ !0 NO:1) of human FcRHSc and C-termina! His-expression tag}. This immunization strategy did not resuit to significant immune response to FcRHS and failed to produce monoclonal antt-FcRHS antibodies. The second immunization strategy was DMA-immunization with plasmid encoding amino acids 745-977 of FcRHSc (SEQ iD NO:1} encoding membrane proximal ig-domain. transmembrane domain and intracellular domains of human FcRH5, This immunization strategy did not result to significant immune response to PcRH5 and failed to produce monoclonal anti-FcRHS antibodies. The third immunization strategy utilized peptides corresponding to membrane proximal Ig-domain of FcRHS, that were homologous to cyno FcRHS and non-homoiogous to human FcRHI, FcRH2, FcRH3, and FcRH4. This immunization strategy did not result to significant immune response to FcRHS and failed to produce monoclonal anti-FcRHS antibodies, [0382J For the fourth immunization strategy E11 protein was produced in CHO-eells consisting of the membrane proximal ig-domain of human FcRHS (amino acid residues 745-850 ofSEQ ID NO:1) with N-terminai Flag expression tag. The above recombinant protein was used to immunize mice, Immunization, development and characterization of mouse anti-FcRHS E11 antibodies was performed as described in detail above.
[03833 Afier 6 doses of the recombinant E11 (amino acid residues 745-850 of SEQ iD MO:1), serum was analyzed for FcRS binding antibodies using FACS. Significant reactivity was detected to SVT2 cells that express human full length FcRHS, cyno full length FcRHS. or the human El 1 domain transmembrane domain and cytoplasmic domains but not vector transfected SVT2 ceils indicating that FeRHS reactive antibodies were present in the sera of a!! 5 immunized mice, [0384} After 9 doses, lymphocytes from the immunized mice were eiectrofused with XS3-AgS.6S3 mouse myeloma cells, 323 igG positive hybrldoma subclones were selected for further screening. Clones were tested for binding to recombinane £11 protein (amino acid residues 745-850 of SEQ ID NO:1) by ELISA (not shown) and binding to SVT2 cells that express human full length FcRHS, cyno full length FcRHS or human E11 domain transmembrane domain and cytopiasmic domains of FcRHS by FACS. A total of 26 clones were identified that bound to ceiis that express human FcRHS and cells that express cyno FcRHS, indicative of cross-species reactivity (Iable 2), Subclone supernatants were further characterized for binding to A} multiple myeloma cells transfected with human FcRHS, B) cells that express human FcRHS endogenously (MQLP-2 myeloma ceiis, peripherai human CD19+ B-cells from healthy donors). C) SVT2 cells transfected to express human FcRHI, FcRH2, FcRHS or FcRH4, D) 293 ceils that express truncated version of human FcRHS (lacking 4 ig-domains including ET1; amino acids 464-850 of SEQ iD NQ.1) and E) NK-ceiis. in addition, binding of supernatants to soluble FcRHSa was analyzed by ELISA. Based on these analysis monoclonal antibodies were selected for purification.
[0385] Figure 2 shows the dose-range of binding of five purified E l 1 antibodies, non-isoform selective anti-FcRH5 antibody 10A8 (which binds ig-iike domains 4-5 ofFcRHSc) and a control antibody specific to the N-terminal gD-tag to the SVT2 cells expressing either human FcR5 {Figure 2A) or cyrto FcRS (Figure 2B). Antibodies in this assay were directly labeled with AFC-fiuoraphore according to manufacturer's protocol {lnvitrogen#z25G51,225151, z25251). Binding of representative £11 antibody 5A10 to human FeRHS transfected EJM (Figure 3A) and OPM2 {Figure 3B) multiple myeloma cel! lines was found to be similar or better compared previously described nan-isoform selective FeRHS antibodies 10A8 and ?D11 (both bind tg-like domains 4-5 of FcRHSc) (Elkins at si, 2012; Poison et a/., 2006). MOLP-2 ceiis are one of the very few known multiple myeloma eeif lines that express low feveis of FeRHS endogenously. 5A10, 5F1,3G7 and 6D2 subclone supernatants stained MOLP-2 ceiis with intensity similar to 7D11 (Figures 3C-F).
[0386] Two separate tests were designed to address dependency of binding on the presence of membrane proximal Ig-domain 9 (El 1}. First a truncated human FcRHSc mutant was generated that lacks Ig-domains 6-9 {amino acids 464-650 of SEQ ID NO:1) including the expected binding site for the antibodies derived from E11 immunisation. This construct with N-terminai gD-tag was expressed in 293 ceiis and subjected to 2.5 ug/mi subclone supernatants followed by PE labeled goal anti- mouse secondary antibody (1:1000 dilution) None of the tested subciones bound to 293 cells that express the truncated human FcRHSc (Figure 4A). In contrast binding was detected to 293 cells that express wild type human FeRHÆc. Binding of gO or non-isoform selective antibody done (10A8) was not altered by the mutation. This result demonstrates that binding site of the E11 antibodies was included in ig-domains 6-9.
[0387] Isoform selectivity was further demonstrated by testing binding to the soluble FcRHSa isoform. For this, 293 ceiis were transfected to express the soluble isofdrm with C-terminal HiS-expression tag. Expression of FcRHSa protein was confirmed with Western blot analysis using and-HiS antibody. A 65 kD band was detected in conditioned media from FcRHSa but not vector transfected cells (not shown). For the ELISA, plates were coated with anti-HiS capture antibody and incubated 1 hour with 1:10 diluted conditioned media inciuding the HIS-tagged soluble FcRHSa isoform. The E11 monoclonal antibodies were used for detection in 1 - 0.001 ug/mi concentration, incubated for 1 hour followed by incubation with goat anti-mouse HRP antibody and finally with TMB-substraie. While clones 2H7 and 5A10 demonstrate considerable reactivity to soluble FcRHSa, the other tested monoclonal antibodies do not show any detectable binding (Figure 5A). This result confirms that the ig-domain 9 (E11) is reguired for binding of the antibodies 1G7, 3A4, 3B12, 3G7 and SF1, and therefore these antibodies are selective for full the length FeRH5 isoform (FcRH5c), [0388] FeRHS is expressed endogenously in B-cells (Hatzivassilion et a/., 2001, Poison ef a/., 2006), To evaluate binding of subcione supernatants to B-cells, PBMCs were extracted from the blood of healthy donors, 1 million human PBfvtC were incubated with 4 ug/mi subcione supernatants for 60 min, washed and incubated with 1:100 dilution of goat anti-mouse secondary labeled with ARC. Celts were then washed again Iwtae and stained PE-labeled anii~CD19 {BD Biosciences #340364) prior flow cytometry and analysis of binding to CD19+ ceiis. Most of the supernatants induced a significant shift in the APC signal in 0019+ cells (Figure 58) over the controls (no primary antibody, anti-gD} indicative of binding to B ceils, [0389] Fc receptor homo log (FcRH) family molecules have a high degree of homology to one another (Miller et al-, 2002). The homology is especially high between the membrane proximal domains, which the E11 antibodies target {Miller e?a/„ 2002), investigated the cross reactivity to family members, FcRHi, FcRH2, FeRHS and FcRH4 (ail inciuding an N-terminal gD-expression tag) were expressed in SVT2 cells and cells were stained with subdone supernatants and goat and-mouse-PE secondary antibody. Expression of the transfected FcRH was confirmed by a signal: from anii-gD antibody in all cell lines. None of the supernatants bound significantly to FcRH2 expressing ceils as compared to staining with the gD antibody (Figure 8B), 188.1H11,3C10,4G8 and 6D2 demonstrated a low ievei of binding to FcRH1 (Figure 6A) and 1F4 bound to FcRH4 (Figure 6D), Overall, the signals from FcRHS-expressing SVT2 ceils were low, including the gD control antibody, indicative of low expression level. Low level of binding to FcRFD-expresstng SVT2 ceils was detected for 1F4 and 4H8 supernatants (Figure 6C). (8390! Since the overaii signal in the FeRHS-expressing SVT2 cells was low, further testing was done using PSMCs from healthy donors. PBfvICs were stained as described above, but instead of €019, CD56 (80 Biosciences #555518} was used to gate the investigated cell population to NK cells. NK-cells express endogenously FcRH3 (Poison ef 2006), and as expected, were stained by a previously described monoclonal anti-FcRHS antibody (Poison ef at, 2006). FcRHl expression was also detected in CD58+ ceils, but none of the E11 subdone supernatants significantly stained the NK cells (Figure 7) demonstrating lack of cross reactivity to endogenously expressed FcRH3, [0391| The cross reactivity of the family members were re-tested using the Identical protocol dseribed above tn SVT2 cells but using fresh reagents and re-transfecting SVT2 cells with FcRHl, FcRH2, FcRH3, and FcRH4, Re-testing: the purified antibodies as described: above resuited in significantly different results than the first series of experiments. These updated results are summarized in Tabie 4. Rather than showing little to no cross-reactivity with other FcRH family members, all but one antibody (1G7) showed significant binding to both FeRHS and at least one or more other family members. Without being bound by theory, this amount of antibody cross-reactivity Is what would be expected, given the sequence similarity of the last ig-like domain in the various FcRH family members, [0392J CD8+ T ceils are among the most potent immune effector cells. The activity of T ceils can be recruited to kill tumor cels by using bispeetfic antibodies (or antibody fragments) that simultaneously bind both T cel! and a tumor antigen. The dual binding can lead to a polyclonal activation ofT ceils and specific killing of tumor antigen expressing cells (Liu Of a!., 1985; Shafaby ef al, 1992). Several tumor targets and several bispecific antibody platforms have demonstrated general flexibility and precllnica! feasibility for this approach, Importantly, promising clinical activity has been demonstrated with a GDI 9 targeting, T caff activating bispecific scFv antibody fragment bfinatumomab (MX 103; Micro Met). Treatment with doses as low as 80 ug/m2'day results in prolonged responses in clinical trials for treatment of relapsed non-Hodgkin’s lymphoma and acute lymphoblastic leukemia (Bargou ef a/., 2008; Dreier ef a/., 2002) [0393$ The ability of the FeRHS antibodies to activate T cell and mediate killing in bispecific antibody format was investigated by generating bispeetfic blsFab molecules. In short, these bispeetfic moleculesare generated by proteoiyticai cleavage of the antibody, followed by reduction, re-oxidation reactions and conjugation of Fab-Jfagments using bis-maleamide (Seheer ef a/., 2012b and as described above). Anti-CD3 antibody done UCHT1 binds to human C03 that incorporates to T cell receptor. UCHTt.vS has previousfy been shown to be efficient T cel! binding arm (Junttila ef at., 2012 and as described above; Zhu ef a/., 1995) and therefore was used to the FcRH5 bisFabs, Nine anti-FeRH5 antibody clones (107,2H7, 3G7, 5A10,5F1,6D2,3B12,3C10,3F10} from the E11 immunization were chosen for the target arm and conjugated with UCHT1.v9 to result in CD3-FcRH5 bispeetfic bisFab molecules.
[0394J !rt addition to bisFab molecules, aiso full iength bispecific antibodies (T ceil dependent bispecific antibodies; TOBs) were produced using knobs-into-hoies technology (Merchant ef a/., 1998), which relies on a pair of complementary engineered Fc regions that drive bete rodi roe rization of antibody hemimers. As in the case ofbisFabs, the UCHT1,v9 (Zhu ef a/,, 1995} was used as the anti-CD3 {hole}. For the target arm (knob), antibody dones from the FeRHS Ell-immunization, a non-isoform selective anti-FcRHS clone (19A8) (Elkins etal, 2012} or anti-HER2clone405 (trastuzumab) (Carter ef a/., 1992} were used. Generation and purification of the TDBs has been described in detail (Junttila ef a/., 2012; Seheer ef a/., 2012a and as described above), [0395J The ability of the bispeetfic molecules to mediate killing of FcFHS transfected 293 target cells was investigated by incubating the targets with CDS* T ceiis (effector cells} for 48 hours and measuring the killing activity using Cell Titer Glo assay or FACS killing assay (assays described above). All nine bisFabs that incorporated an anti-FcRH5 E11 target arm were efficient in mediating target cel! killing (FIG, 8A-B), Killing activity was detected as low as 1-10 ng/mf concentrations and saturated at 10-100 ng/ml concentration. Maximal killing activity exceeded 80% for most of the ciones, The killing activity was similar compared to the HER2-TDB (FIG. 8A-B). Human HER2 is expressed in the 293 cells on low level (data not shown). In contrast, kitting activity far exceeded the non-isoform selective FcRH5-TD8 (10A8), which was capable in killing only approximately 20% of the targets (FfG. 8A-B), Similar robust activity was detected using a toil iength PDB format incorporating 2H7, 3G7 and 5A10 FcRH5-E11 clones as target arms (FIG, 8C-0). No significant difference was detected between TDB and bisfab versions of 2H? and 3G7 indicating that Fc is neither necessary for the activity nor inhibitory for the kiiiing activity. FeRHS bisFabs and toil iength TDBs incorporating 2H7 and 3G7 as target arm were aiso able to efficiently mediate kiiiing of MOLF2 cells, which express endogenously fow levels of FcR-H5 (FIG. 9A), T cell activation was followed in the reactions measuring the proportion of CD8+ cells that express CD69 on the cell membrane, T ceil activation corresponded the killing activity and was simitar for both bisFabs and TDBs (FtG. 98). A summary of the results are shown in Table 3.
References mm
Bargou, R. et al (ZOOS), Science 321,974-97?,
Carter, P, et al, (1992), Proc Natf Acad Set USA 89, 4285-4289.
Dreier, T. et al. (2002), Snt J Cancer 100, 690-697.
Elkins, K. et al. (2012). Mol Cancer Ther 11,2222-2232.
Hatzivassiou, G. et ai. {2001}, immunity 14,277-289,
Liu, M, A. et ai. (1985). Proc Natl Acad Sci U S A 82,8648-8652.
Merchant, A, M, et al. (1998). Nat Biotechno! 16,677-681.
Mitter, 1 et al. (2002). Blood 99, 2662-2669.
Poison, A, G. etat. (2008). Expression pattern of the human FcRH/IRTA receptors in normal tissue and in 8-ehronic lymphocytic leukemia. International immunology 18,1363-1373,
Shaiaby, M. R. et al. (1992). J Exp Med 175,217-225.
Zhu, Z. et ai, (1995). Int J Cancer 62,319-324, [0397} Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention which is defined in the claims.
Variable Light Chain Domain [0398} 1C8.1
DfVMTQSQRFM5T$LGDRV5VTCKA5GNVfTNVAWyQO.KPGG.SPKAUY$ASVRY$GVP0t1FiG$GSGTDFTLTt$N VaSEDUEVFCQAVTNYPWWTfGGGTRlEmmrVA {Sm ID NO:JlQ) 1G7.2
DiVMTQSHK!iylSTSVGDRVSfTCKASQDVSNiV‘VWFQaKP<3aSPMUjY$A5YfiyTt3V9Dftn‘6$GS<5TDFTFnSSV O.AEDf..AVVvX:QOjivSSPY'rR5G6Tlt.EtKRlVAA(SEa ID NO: 112} 2H7,3 flVLTQSf'ATLSVT PODSVSLSCRASQ Ni R NN LHWYQO.KS Si E$ PRLUI<|^QSiS6IPSRFTGSSSSfDfSSI^SVf.T:E 9:pMyFCO.Q.SNNWPQYTR56CTKLf{K«TVAA i$EO. ID NO:114} 3A4.2 D?afvr!QSFATiSVTP6DSVStSCBAS05!SNNLHVWOAKSH£SPRLLMfASQSSGPSRFSGSGSGTDFTtSsN$Ve£ DFGMYFCQQSNNW^YTFGSGmELKRTVAA (SEQ ID ΝΟ:116} 3B12.1.1
DiaMTaSPA$lSASV60VTn'C8A^N}YSNlAWYaUCaSKSPQU.VYSAANlMSVPSRfS(sSGS6TaYSLKIMSia SEDFGTWCClHFWSIPWTR3€S<3TKl£iK^TVAA {SSCMD NO;118) 3C10 D^QMTCYiPlSLPWLSGQA$iS€RSSQSEVHRNSNTYtHWYtQRPG€SPNLUYN¥$NRf$GypDRF5GS6$GTDFTt IOSBYEA£OL6VYFCSaSTHVPPTfGGGTKlElKSTVAA {SEQ IO NQ:12G} 3F10
DsVsv1TaSPASlSASVSFTVTn'CRASFFPYSHlAWYatKaG5<$PQU.V¥6AA?4LAF6VPSR;S6$6SGTQYSLK^SLa S£DFGTYYCQHFWGtP^YF<sGGTKp|KMYAA|SFQ ID 80:2¾¾} 3G3 DI¥MTQ$PA$l$A$V6 E7VTITC FtASENiYSNlA\.WQLKQGSSPQlLVYGMNlAE6VPSBiS6$GSGTU¥$IXiNS,Q $E0FGTYYCQHF WGIP WTFGGGTKIEIKR.7YA A (SEQ ID 80:124} 307,1,5 OIVIiaSPAltSWlGOSY$l$CMASa$!SNNl,BYmsai<SH£SPRiyKFASaSISGsPSBFB6S6«$TOFriJINSVETED F6?YFCQQ$8NWFQYTFG6GTKt!:I.KRTYAA (SEOJD NO: 126) 5A1Q.1.3
DftÆlX»PANLSVIPeDSVSt^RAWlSMs«iRI«YS#røESP»tlli^^MSSTl^WS6^^^^|:; TE0FGMYFCQQ5NN WPQnFGGGmEI KRTVAA {SEQ ID NQ;128) 5F1.1.5 QAVVTQ£SALTTSP<5£TVTtTCRSSTGTVTTSNFANWVQEKPDHLFTGU6GTSNRAPGVPARFSGSU6DKAALTIT GAQTEDEAiYFCVLWCSNtWVFGGGTKtTVLGQPKAA {SEQ ID NO :130} 6D2
DiVfVtTQ5HKFMSTS¥GPRVSrrCKASQDVGTSVAWYQQKPGQSPKlliFWPSTRHT6V?Df?n'<3SGS6TDF!Tn6 NVQ$EGLADYFCQOFS$lPHTFGGGTKL£!KRTVAA iSUl 10 NO:132) 1G7‘
Da%4TaSHKfMSTSVG0R¥SfTCSCASaDVS«iVVWFaaKS>GaSPMLMYSASV'RV7CA^BRFLT<5.S6SCjrDFTFTBSV QAMLAWCQaHYSSmTGGSTKLEIK (SEQ ID NG:134)
Variable Heavy Chain Domain [0399} 1C8.1 EVataOSG^ElVKPSASMKlSCEASSYSiRTAYIMNWVKQSRSKfil^lGUNPYNGETTyNaKfKeKAUTVDQSSS TAYMEtWtTSED^WF^^^I^^nVlMS^^PS^iAP {SEG <D NQ:U3j IG7.2
SVQtaeSG^LVaPSC^lSrrCTVSGiFiiTBfSVHWVR^GKGLEm^W^SSliyNMEMSRlTTOI^IS
QVFPKL^SL^øgmiYYCSNHYYGSSømEPlYWG^TSVIVSSASrKGPSVKPl#511¾ IS 2H7.3
EVQLQQSGPELWKPGASVKMSCKASGYTRPYYMKVWKQTHGKStfWIGDINPNNGETFYSQKFKGKATLTVDKS STTAYMQlNSLTSEDSAVVYCAHGLYRFOYWGaGfTLTVSSASTKGPSVFPLAPtSEQ ID NQ:115) 3Ά4.2 EVCU.QQSGPELVKSGASVKMSCKASGYTPTDYYMKWVKQSHGsiSL£WIGblNPYhiGETF¥Md}feLk<ikATLT¥0jt3S NTWMftlNSLTSE^VYYCARGlYFFAYWGQGmTVSSASTKGPSVFRAP [SEQ ID 140:11?) 3812.1,1 EVQLQ,QS6PFLVKP6ASVK!SCKTSGYTFrEYTfHWVK<iSHGKSL£{i}GGlNPNNDAVSYMORF8GKATlTVDKS3ST AYMELRSlT$£DSAVWCAKiG8GYYFOYWGQGTTLTVS$ASTKGPSVFPLAP (SEQ. ID NO:X19) 3C10 Q.VQLQQP6AELVRPGASVKLSCKTSGYTFiSYW!NWVKClRP6Q6L£W}GN!YPSDSYTNYNCyiFKDKATtTVDTSSS TAYMQlTSPTSEDSAVYYCTRSlYGYDASYFDYWGQGmTVSSASTKGPSVFPUP {SEQ ID NQ;12X| 3F10 avataaSGPSlVKPGASVKSCKTSGYTFTEYTIHWVKQSHGICSlEBIGGINPNNaAlSYNaKFflGKATlWD^^TA 't^gfca^g|^Ym.li|^WFDY«<Stf^WSASrKGPSVFPUP (SEQ 10 M0:1B) 3G3 SVQlC^O^tVKP^VKISCK'f-SSyrFTmiHWVKaSHGKSlERiGSINPN^DAlSYNaKFRSKAUTVOKSSSTA YMEU^tTS£DSAVVYCAKiGI?6YyH>¥W6røTtWSSASTKeP$VF?y^CS£a 10 μο&amp;25) 307.1,5
eyataaSGPEtVKP<5ASVKf«SC8AS(3YTFTOYYM:KWVRaNH<5KRl£W!GOINPYNGDTFYKQKFKDKATLTVOKS SSTAYMQFNStl^EDSAWYCARGlYFFHYVVGd.GWL!t\iS^ASTKGFiSVFPi.AP (S£Q. ID NQ:127) 5A10.1.3 ^UKfcGP? L WKPGASVKM5CK ASGYI ί-TDY rM KWYKaSH6KSl£Wi<30iNPNNGETfY?jaKFK«KAmfVOiSS. TSI AYMtimimQSAVYYCAHGlyrFOYWGQGTTEWSSAA.VniGPSVFRAf* iSEQ ID NO:129) 5F1.1.5
avaiaaSGAOtVKPSTSVKVSCKASi3YArrNYi|gVYVKaRI»GaeLEWIøVlMPSS6GTMYf€KFICGKATlTAOKSS
Sm¥MatSSL»DSAVYfCARTRNVGVViDYWaesirTLTVSSASTKi3PSVFFLAf> {SEQ ID 80:131} 6D2
QVQLaQS6P£lVKP6ASVKSCKASG?SFrAYFMNW¥KaSHGK$ffWlGmMPY«eETPfNaNFK0KATlTVDKSS fiTAHMELLSLT^DDSAVYYCGRGLYYLNYWGQG’TTOVSSASTKGPSVPPlAP (56010 80::13¾
1GF avatKaSSPGlVOFSmLSSTCTVSSFSlTIlFGVHWyROSPSKGtgWiGVlWRGOSiDYNAAFMSRlTTTKDNSIS QVFEKLNStKVODTAlYYCSNHYYO^DYAIDNWOaGISVTVSS (SEQ ID N0;13S)
Ksba; CIXR LI (HV.R-IJ)
Kiibat CD.R Hi (HVR-H1)
MLt,WVEl,VlAPVSGQFÅRTFRPjrFL0PF^rrV|^CSETOTtKGFRITSFQKTO^lMliCi •O.iLROT0NfiEVQi^HYRCQAQGSH^PVm.riFiiSA$LUJ?APLSVFBGD$VVL&amp;CRAKM'
VTLKMIlYKN'DNVLAFL.NKR'i'.DFHIPMAC:.lKJ>NGAV.RCri'GYKFS(X'PVS$NTVKIGVQEPFTE
PVLRASSFQP^SONFYTLTCE'rQLSIJKR SDV PiJRFR FFRDDQTXXSLØWSiSFNFQFf AM WSKDS GFY\¥CKAÅTMPYSVlSiMFRSWiQV^IFAFHP%'LTLSPEKALNFEGlXYTLMCHTQfct1$LRTi
YlFYHia\TLRHKSVRCmOASISFSLTrENSON¥YCTADHGLGAKPSKAVSLSVrvP¥SHPV
LNLSSPEDLIFEØAKVILHCEAQRGSIPILYQFIIIIEGAALERRSANSACIGVAISFSLTaEHSGN
YYCTADNGFGPQRSKAVSLSVTVPVSHPVLTLSSAEAL1FEGATVTUICEVQRGSPQBLYQFY
HBDMPLWSSSIPSYGE.VSFSFSIA'EGHSGNyYCTABNGFGPQRSEVVSLFVTVPVSRPn/ILRV
PRAQAWGDLLELHCEAPRGSPPILYWFYH.EDVTLGSS8APSGGEASFMLSLTAe-ISGNYSCE
ANNGLVAQHSI>TISLSVIVPVSRPrLTFRAP.RAQAVVGØLLEmCEALRGSSPILYWFYHEDVT
'LGKISAPSGGGASFNLSLTTEHSGIYSCEAONGLEAQRSEMY'rOv.VAVPYSRPVLTLRAPGTH
AAVGDLLELHCEALRGSPIILYRFFHEDVTLGNRSSPSCIGASiJNtSLTAEHSGNYSCEADNOL
GAQRSEI'VTLYITGLTANRSGPFATG'VAOGLLSIAGLAAGALLLYCWLSRKåGR'KPåSDPåRS
FSDSDSQBFTYHNVPAWEELQPVYTNANPRGEHVVYSEVEIlQElilCiaiAVASDPRHLRNKGS
PilYSEVKVASIPVSGSLHASSAPHR (SEQ ID NO;!} SEQUENCE LISTING [0400] <110> GENENTECH, INC.
<120> AMTI-FCRH5 ANTIBODIES
<130> 6ΝΕΌ413 WO <140> <141> <150> 61/838.534 <151 >2013-06-24 <180> 136 <170> Patentlo version 3.5 <21G> 1 <211 >977
<212> PRT <213> Homo sapiens <400> 1
Met Leu Leu Trp Val Ile Leu Leu Val Le« Ala Fro Val Ser Gly Gin
1 5 10 IS
Phe Ala Arg Thr Pro Arg Pro lie lie Phe lieu G1« Pro Pro Trp Thr 20 25 30
Thr Val Phe Gin Gly GI« Arg Val Thr Leu Thr Cys Lys Gly Phe Arg 35 40 45
Phe Tyr Ser Pro Gin Lys Thr Lys Trp Tyr His Arg Tyr Leu Gly Lys 50 55 60
Glu lie Leu Arg Glu Thr Pro Asp Asa He Leu Glu Val Gin Glu Ser 65 70 75 80
Gly Glu Tyr Arg Cys Gin Ala Gin Gly Ser Pro Leu Ser Ser Pro Val 85 90 35
His Leu Asp Phe Ser Ser Ala Ser Le« Ile Le« Gin Ala Pro Leu Ser 100 105 110
Val Phe G1« Gly Asp Ser Val Val Leu Arg Cys Arg Ala Lys Ala Glu 115 120 125
Val Thr Leu Asn Asn Thr He Tyr Lys As« Asp As« Val Leu Ala Phe 130 135 140
Leu Asn Lys Arg Thr Asp Phe His He Pro Sis Ala Cys Leu Lys Asp 145 150 155 160
Asn Gly Ala Tyr Arg Cys Thr Gly Tyr Lys Glu Ser Cys Gys Pro Val 3,65 170 175
Ser Ser Asn Thr Val Lys Ile Gin Val Gin Glu Pro Phe Thr Arg Pro 180 1S5 190
Val Le« Arg Ala Ser Ser Phe Gin Pro Ile Ser Gly Asn Pro Val Thr 195 200 205
Leu Thr Gys Glu Thr Gin Len Ser Leu Glu Arg Ser Asp Val Pro Leu 210 215 220
Arg Fhe Arg Phe Phe Arg Asp Asp Gin Thr Leu Gly Leu Gly Trp Ser 225 230 235 240
Leu Ser Pro Asn Phe Gin Ile Thr Ala Met Trp Ser Lys Asp Ser Gly 245 250 255
Phe Tyr Trp Cys Lys Ala Ala Thr Met Pro Tyr Ser Val Ile Ser Asp 260 265 270
Ser Pro Arg Ser Trp tle Gin Val Gin Ile Pro Ala Ser Eis Pro Val 275 280 285
Leu Thr Leu Ser Pro Gin Lys Ala Leu Asn Phe Glu Gly Thr Lys Val 290 295 300
Thr Leu His Cys Glu Thr Gin Glu Asp Ser Leu Arg Thr Leu Tyr Arg 305 310 315 320
Phe Tyr His Glu Gly Val Pro Leu Arg His Lys Ser Val Arg Cys Glu 325 330 335
Arg Gly Ala Ser Ile Ser Phe Ser Leu Thr Thr Glu Asn Ser Gly Asn 340 345 350
Tyr Tyr Cys Thr Ala Asp Asn Gly Leu Gly Ala Lys Pro Ser Lys Ala 355 360 365
Val Ser Leu Ser Val Thr Val Pro Val Ser His Pro Val Leu Asn Leu 370 375 380
Ser Ser Pro Glu Asp Leu Ile Phe Glu Gly Ala Lys Val Thr Leu His 385 390 395 400
Cys Glu Ala Gin Arg Gly Ser Leu Pro ile Leu Tyr Gin Phe His His 405 410 «15
Glu Gly Ala Ala leu Glu Arg Arg Ser Ala Asn Ser Ala Gly Gly Val 420 425 430
Ala Ile Ser Phe Ser leu Thr Ala Glu Bis Ser Gly Asn Tyr Tyr Cys 435 440 445
Thr Ala Asp Asn Gly Phe Gly Pro Gin Arg Ser lys Ala Val Ser leu 450 455 460
Ser Val Thr Val Pro Val Ser His Pro Val leu Thr leu Ser Ser Ala 465 470 475 480
Glu Ala leu Thr Phe Glu Gly Ala Thr Val Thr leu His Cys Glu Val 485 490 495
Gin Arg Gly Ser Pro Gin lie leu Tyr Gin Phe Tyr His Glu Asp Met 500 505 510
Pro leu Trp Ser Ser Ser Thr Pro Ser Val Gly Arg Val Ser Phe Ser 515 520 525
Phe Ser leu Thr Glu Gly His Ser Gly Asn Tyr Tyr Cys Thr Ala Asp 530 535 540
Asa Gly Phe Gly Pro Gin Arg Ser Glu val val Ser leu Phe val Thr 545 550 555 560
Val Pro Val Ser Arg Pro He leu Thr leu Arg Val Pro Arg Ala Gin 565 570 575
Ala Val Val Gly Asp leu leu Glu leu His Cys Glu Ala Pro Arg Gly 580 585 590
Ser Pro Pro He leu Tyr Trp Phe Tyr His Glu Asp Val Thr leu Gly 595 600 605
Ser Ser Ser Ala Pro Ser Gly Gly Glu Ala Ser Phe Asn leu Ser leu 610 615 620
Thr Ala Glu His Ser Gly Asn Tyr Ser Cys Glu Ala Asn Asn Gly leu 625 630 635 640
Val Ala Gin His Ser Asp Thr He Ser leu Ser Val He Val Pro Val 645 650 655
Ser Arg Pro lie leu Thr Phe Arg Ala Pro Arg Ala Gin Ala Val Val 660 665 670
Gly Asp lieti Leu Glu ken His Cys Glu Ala Leu Arg Gly Ser Ser Pro 675 680 685 lie Leu Tyr Trp Phe Tyr His Glu Asp Val Thr Leu Gly Lys lie Ser 690 695 700
Ala Pro Ser Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Thr Glu 705 710 715 720
His Ser Gly Ile Tyr Ser Cys Glu Ala Asp Asn Gly Leu Glu Ala Gin 725 730 735
Arg Ser Glu Met Val Thr Leu Lys Val Ala Val Pro Val Ser Arg Pro 740 745 750
Val Leu Thr Leu Arg Ala Pro Gly Thr His Ala Ala Val Gly Asp Leu 755 760 765
Leu Glu Leu His Cys Glu Ala Leu Arg Giy Ser Pro Leu He Leu Tyr 770 775 780
Arg Phe Phe His Glu Asp Val Thr Leu Gly Asa Arg Ser Ser Pro Ser 785 790 795 800
Gly Gly Ala Ser Leu Asn Leu Ser Leu Thr Ala Glu His Ser Gly Asn 805 810 815
Tyr Ser Cys Glu Ala Asp Asn Gly Leu Gly Ala Gin Arg Ser Glu Thr 820 825 830
Val Thr Leu Tyr He Thr Gly Leu Thr Ala Asn Arg Ser Gly Pro Phe 835 840 845
Ala Thr Gly Val Ala Gly Gly Leu Leu Ser Xle Ala Gly Leu Ala Ala 850 855 860
Sly Ala Leu Leu Leu Tyr Cys Trp Leu Ser Arg Lys Ala Gly Arg Lys 865 870 875 880
Pro Ala Ser Asp Pro Ala Arg Ser Pro Ser Asp Ser Asp Ser Gin Glu 885 890 895
Pro Thr Tyr His Asn Val Pro Ala Trp Glu Glu Leu Gin Pro Val Tyr 900 305 910
Thr Asn Ala Asn Pro Arg Gly Glu Asn Val Val Tyr Ser Glu Val Arg 915 920 925
Ile ile Gin Glu Lys Lys Lys His Ala Val Ala Ser Asp Pro Arg His 930 935 940
Leu Arg Asn Lys Gly Ser Pro Ile Ile Tyr Ser Glu Val Lys Val Ala 945 950 955 960
Ser Tiir Pro val Ser Gly Ser Leu Phe Leu Ala Ser Ser Ala Pro His 965 970 975
Arg
<210» 2 <211» 11 <212> PRT <213> Artificial Sequence <220» <221» source <223> /'note—Description of Artificial Sequence: Synthetic peptide” <400» 2
Lys Ala Ser Gin Asn Val Ile Thr Asn Val Ala l 5 10
<210»3 <211> 11 <212» PRT <213» Ariifidai Sequence <220» <221» source <223» /note-‘Description of Artificial Sequence: Synthetic peptide” <400» 3
Lys Ala Ser Gin Asp Val Ser Asn Ile Val Val 1 5 10
<210>4 <211» 11 <212» PRT <213» Artificial Sequence <220» <221 > source <223» /note-‘Description of Artificial Sequence: Synthetic peptide" <400»4
Arg Ala Ser Gin Asn Ile Arg Asn Asn Le« His 15 10
<210» 5 <211> 11 <212> PRT «213» Artificiai Sequence <220» <221> source <223> /note="Description of Artificial Sequence; Synthetic peptide" <40Q>5
Arg Ala Ser Gin Ser Ile Ser Asn Asn leu His 15 10
<210> 6 <211» 11 «212» PRT «213» Artiliciai Sequence «220» <221> source <223» /note="Description of Artificial Sequence: Synthetic peptide" «400» 6
Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 15 10
«210 7 <211> 16 «212» PRT «213» Artiliciai Sequence <220» <221» source <223> /note—Description of Artificial Sequence: Synthetic peptide" <400 7
Arg Ser Ser Gin Ser Leu Val His Arg Asn Gly Asn Thr Tyr Le« His 15 10 15
<210> 8 <211> 11 <212> PRT <213> Artificiai Sequence <220» <221» source <223» /note-'Description of Artificial Sequence: Synthetic peptide" «400» 8
Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10
<210>9 <211> 11 <212> PRT <213> Artificiai Sequence <220 <221 > source <223> /note^'Description of Artificial Sequence: Synthetic peptide" <400 9
Arg Ala Ser Glu Asn ile Tyr Ser Asn Lau Ala 1 5 10
<21 G> 10 <21T> 11 <212> PRT <2T3> Artificial Sequence <220> <221 > source <223> /note=nDescription of Artificial Sequence: Synthetic peptide" <400 10
Arg Ala Ser Gin Ser Ile Ser Asn Asn Leu His 1 5 10
<210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> /oofe-'Desaipiion of Artificial Sequence: Synthetic peptide” <400> 11
Arg Ala Ser Gin Asn Ile Arg Asn Asn Leu His 1 5 10
<210> 12 <211» 14 <212> PRT <213> Artificiai Sequence <220> <221> source <223> /note -"Descri p ti on of Artificiai Sequence: Synthetic peptide” <400> 12
Arg Ser Ser Thr Giv Thr Val Thr Thr Ser Asn Phe Ala Asn 15 10 <210 13 <211> 11
<212> PRT <213> Artifidai Sequence <220> <221 > source <223> /note»'‘Description of Artificial Sequence: Synthefic peptide4’ <4Q0> 13 lys Ala Sar Gin Asp Val Gly Thr Ala Val Ala 1 5 10 <21Q> 14 <211> 7
<212> PRT <213> Artifidai Sequence <220> <221 > source <223> /note="Descripiion of Artifidai Sequence: Synthetic peptide” <400> 14
Sar Ala Sar Tyr Arg Tyr Sar 1 5
<210> 15 <211>7 <212> PRT <213> Artifidai Sequence <220> <221> source <223> toots-‘Description of Artifidai Sequence: Synthefic peptide" <400> 15
Sar Ala Sar Tyr Arg Tyr Thr 1 5
<21Q> 16 <211> 7 <212> PRT <213> Artifidai Sequence <22Q> <221 > source <223> toots-‘Desaiption of Artifidai Sequence: Synthetic peptide4’ <400> 16
Pha Thr Sar Gin Sar lie Sar 1 5
<210> 17 <211>7 <212> PRT <213> Artifidai Sequence <220> <221 > source <223> /note="Descripiioo of Artificial Sequencer Synthetic peptide" <4QG> 17
Pfee Ala Ser Gin Ser Ile Ser 1 5
<210> 18 <211> 7 <212» PRT <213» Artificiai Sequence <220» <22t> source <223> /nof8="DescripSioo of Artificial Sequence* Synthetic peptide" <40G> 18
Gly Ala Ala Asn Leu Ala Glu 1 5
<210> 19 <211> 7 <212» PRT <213> Artificiai Sequence <220> <221> source <223> /note="Oescription of Artificial Sequence: Synthetic peptide" <400» 19
Lys Val Ser Asn Arg She Ser 1 S
<21Q> 20 <211> 7 <212> PRT <213> Artificiai Sequence <220> <221» source <223> /nofe«"Qeserlpiion of Artificial Sequence: Synthetic peptide" <40Q> 20
Gly Ala Ala Asn Leu Ala Glu 1 5
<210> 21 <211> 7 <212> PRT <213> Artificiai Sequence <220> <221 > source <223> /ooie-Oescripiion of Artificial Sequence: Synthetic peptide" <400 21
Gly Ala Ala Asn leu Ala Glu 1 5
<210> 22 <211» 7 <212> PRT <213> Artiticiai Sequence <22Q> <221 > source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400 22
Phe Ala Ser Gin Ser tie Ser 1 5
<21G> 23 <211» 7 <212> PRT <213> Artiticiai Sequence <220> <221 > source <223» /ηο·<»:: Description of Artificial Sequence: Synthetic peptide" <400 23
Phe Ala Ser Gin Ser Met Ser 1 5
<210» 24 <211» 7 <212> PRT <213» Artiticiai Sequence <220» <221» source <223» /note-'Description of Artificial Sequence: Synthetic peptide" <400 24
Gly Thr Ser Asn Arg Ala Pro 1 5
<210» 25 <211» 7 <212» PRT <213» Artificial Sequence <220» <221» source <223» /note-'Description of Artificial Sequence: Synthetic peptide" <400> 25
Trp Fro Ser Thr Arg His Thr i s
<210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <221 > source <223> toots-‘Description of Artificia! Sequence: Synthetic peptide" <400> 28
Gin Gin Tyr Xhr Asn Tyr Fro Met Trp Xhr 15 10
<210> 27 <211>9 <212> PRT <213> Artificial Sequence <220> <221 > source <223> toote-‘Description of Artificial Sequence: Synthetic peptide” <400> 27
Gin Gin His Tyr Ser Ser Pro Tyr Thr 1 5
<210> 28 <211> 10 <212> PRT <213> Artificia! Sequence <220> <221 > source <223> toote-'Description of Artificial Sequence: Synthetic peptide” <400 > 28
Gin Gin Ser asn Asn Trp Pro Gin Tyr Thr 1 5 10
<210> 29 <211> 10 <212> PRT <213> Artificia! Sequence <220> <221 > source <223> /note="Description of Artificia! Sequence: Synthetic peptide” <4Q0> 29
Gin Gin Ser Asn Asn Trp Fro Gin Tyr Thr 15 10
<210> 30 <211>9 <212> PRT <213> Artificiai Sequence <220> <221> source <223> /note=KOescription of Artificial Sequence: Synthetic peptide" <400> 30
Gin His Phe Trp Giy Ile Fro Trp Thr 1 5
<21 Q> 31 <211> 9 <212> PRT <213> Artificiai Sequence <220> <221» source <223> /note—Description of Artificial Sequence: Synthetic peptide" <400> 31
Ser Gin Ser Thr His Val Fro Pro Thr 1 5
<21Q> 32 <211> 9 <212> PRT <213> Artificiai Sequence <220> <221> source <223> rnote^-Desc-ription of Artificial Sequence: Synthetic peptide” <400> 32
Gin His Phe Trp Gly Ile Pro Trp Thr 1 5
<210> 33 <211> 9 <212> PRT <213> Artificiai Sequence <220> <221> source <223> /note="Description øf Artificial Sequence: Synthetic peptide" <400> 33
Gin His Phe Trp Gly Ile Pro Trp Thr 1 5
<210> 34 <211> 10 <212> PRT <213> Artificia! Sequence <220> <22t> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <400> 34
Gin Gin Ser Asn Asn Trp Pro Gin Tyr Thr 15 10
<210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence. Synthetic peptide" <400> 35
Gin Gin Ser Asn Asn Trp Pro Gin Tyr Thr 15 10 <210> 36
<211> 9 <212> PRT <213> Artificial Sequence <229> <221 > source <223> /note-Oeseription of Artificial Sequence: Synthetic peptide" <400> 36
Val leu Trp Cys Ser Asn leu Trp Val 1 5
<210> 37 <211> 9 <212> PRT <213> Artificial Sequence <22G> <221> source <223* /note-’Description of Artificial Sequence: Synthetic peptide" <400> 37
Gin Gin Phe Ser Ser leu Pro His Thr 1 5 <210* 38 <211> 5
<2!2> PRT <213> Artificial Sequence <220» <221 > source <223> Mote~*Descrlpfion of Artificial Sequence: Synthetic peptide” <40Q> 38
Ala Tyr He Met Asn 1 5
<210> 39 <211> 5 <212» PRT <213> Artificial Sequence <220» <221 > source <223» Mote-Description of Artificial Sequence: Synthetic peptide” <400> 39
Arg Phe sly vai His X 5
<210> 40 <211>5 <212> PRT <213> Artificial Sequence <220» <221 > source <223> Mote-Description of Artificial Sequence: Synthetic peptide” <400» 40
Asp Tyr Tyr Met Lys 1 5
<210> 41 <211» 5 <212» PRT <213> Artificial Sequence <220> <221 > source <223> Mote-Description of Artificial Sequence: Synthetic peptide” <400> 41
Asp Tyr Tyr Met Ays 1 5
<210» 42 «211» 5 <212» PRT <213» Artificial Sequence <220> <221> source <223> /nofe«"Descripiion of Artificial Sequence· Synthetic peptide" <4C0> 42
Gin Tyr Thr lie His 1 5
<210> 43 <211> 5 <212> PRT <213> Artificiai Sequence <220> <221> source <223> /nofe="Descripf!on of Artificial Sequence: Synihefic peptide" <400> 43
Ser Tyr Trp lie Asn
1 S <210> 44 <211> 5
<212> PRT <213> Artificiai Sequence <220> <221> source <223> /not8~"Description of Artificial Sequence: Synihefic peptide" <40G> 44
Glu Tyr Thr lie His 1 5
<210> 45 <211> 5 <212> PRT <213> Artificiai Sequence <220> <221 > source <223> /nof8«’'Description of Artificial Sequence: Synihefic peptide" <4Q0> 45
Gin Tyr Thr He His 1 5
<210> 46 <211> 5 <212> PRT <213> Artificiai Sequence <220> <221 > source <223> /note-’Description of Artificial Sequence: Synthetic peptide" <400 46
Asp Tyr Tyr Met Ays 1 5
<21G> 47 <211» 5 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /nofe="Description of Artificial Sequence: Synthetic peptide" <40O> 47
Asp Tyr Tyr Met Ays 1 5
<21G> 48 <210 5 <212> PRT <213» Artificial Sequence <220» <221» source <223» /note-’Description of Artificial Sequence: Synthetic peptide" <400» 48
Asn Ayr Leu lie Glu 1 5
<210» 49 <211» S <212> PRT <213» Artificial Sequence <220» <221» source <223> /note-'Description of Artificial Sequence: Synthetic peptide" <400 49
Ala Tyr Phe Met Asn 1 5
<210» 50 <211» 10 <212» PRT <2T3> Artificial Sequence <220» <221 > source <223> /nofe="Description of Artificial Sequence: Synthetic peptide" <4Q0> 50
Gly Tyr Ser Phe Thr Ala Tyr He Met Asn 15 10
<210> 51 <211> 10 <212> PRT <213> Artificiai Sequence <220> <221» source <223> Mote=:“Descrip}ion of Artificial Sequence: Synthetic peptide" <400> 51
Gly Phe Ser leu Thr Arg Phe Gly Val His 1 5 10
<210> 52 <211> 10 <212> PRT <213» Artificiai Sequence <22G> <221 > source <223> /note-‘Description of Artificial Sequence: Synthetic peptide” <400> 52
Gly Tyr Thr Phe Thr Asp Tyr Tyr Met irys 15 10
<210> 53 <211> 10 <212> PRT <213> Arliftciai Sequence <22G> <221 > source <223> /cote“'‘Description of Artificial Sequence: Synthetic peptide” <400> 53
Gly Tyr Thr phe Thr Asp Tyr Tyr Met Lys 15 10
<210> 54 <211» 10 <212> PRT <213» Artificiai Sequence <22Q> <221 > source <223» /note="Description of Artificial Sequence: Synthetic peptide” <4QG> 54
Gly Tyr Thr Phe Thr Glu Tyr Thr lie His 15 10
<210» 55 <211>10 <212> PRT <213> Artificiai Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic peptide" <4Q0> 55
Gly Tyr Thr Phe Tie Ser Tyr Trp Tie Asn 15 10
<210> 58 <211> 10 <212> PRT <213> Adificiaf Sequence <220> <221> source <223» /not&amp;«"Descnpiion of Artificial Sequence: Synthetic peptide" <400> 56
Gly Tyr Thr Phe Thr Glu Tyr Thr lie His 1 5 10 <21Q> 57
<211 > 10 <212» PRT <213> Aftifidai Sequence <220» <221» source <223> /noteæ"Description of Artificiai Sequence: Synthetic peptide" <400> 57
Gly Tyr Thr Phe Thr Glu Tyr Thr He His 1 5 10
<210> 58 <211> 10 <212> PRT <213> Artificial Sequence <220» <221» source <223» /note-'Descripiion of Artificial Sequence: Synthetic peptide"
<409> SB
Gly Tyr Thr Phe Thr Asp Tyr Tyr Met T>ys 1 5 10
<210 > 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note-'Description of Artificia! Sequence: Synthetic peptide" <400> 59
Gly Tyr Thr Phe Thr Asp Tyr Tyr Met I>ys 1 5 10
<21 G> 60 <211> 10 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note—Description of Artificial Sequence: Synthetic peptide" <40G> 60
Sly Tyr Ala Phe Thr Asn Tyr Leu IT© Glu 1 5 10
<210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note-'Description of Artificial Sequence: Synthetic peptide” <400 61
Gly Phe Ser Phe Thr Ala Tyr Phe Met Asn 1 5 10
<210> 62 <211> 17 <212> PRT <213> Artificia! Sequence <220> <221> source <223> /ooie-'Oescriplion of Artificial Sequence: Synthetic peptide" <400> 62 leu He Asn Pro Tyr Asn Gly Glu Thr Thr Tyr Asn Gin I»ys Phe Ays 1 5 10 15
Gly
<210> 63 <211> 16 <212> PRT <213> Artificial Sequence <220> <221 > source <223> toote*Oescription of Artificial Sequence: Synthetic peptide" <400 63
Val lie Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met Ser
1 S 10 IS
<210> 64 <211> 17 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note="DescriptiOfi of Artificial Sequence: Synthetic peptide" <400> 64
Asp He Asn Pro Asn Asn Gly Glu Thr Phe Tyr Ser Gin Lye Phe Lys 15 10 15
Gly
<210 65 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note-’Oescription of Artificial Sequence: Synthetic peptide" <400> 65
Asp He Asn Pro Tyr Asn Gly Glu Thr Phe Tyr Asn Gin Lys Leu Lys 15 10 15
Gly <21G> 66 <211> 17
<212> PRT <213> Artificial Sequence <220 <221 > source <223> /note-’Description of Artificial Sequence: Synthetic peptide” <4Q0> 66
Gly Ile Asn Øro Asn Asn Asp Ala Val Ser Tyr Asn Gin Arg Phe Arg 15 10 15
Gly <210> 67 <211> 17
<212> PRT <213> Artificial Sequence <220> <221 > source <223> /nøfe="Description of Artificial Sequence: Synthetic peptide" <400> 67
Asn ile Tyr Pro Ser Asp Ser Tyr Tfer Asn Tyr Asn Gin Lys Pfee Lys 15 10 15
Asp
<21Q> 68 <211>17 <212> PRT <213> Artificial Sequence <22G> <221 > source <223> /note-'Description erf Artificial Sequence: Synthetic peptide" <400> 68
Gly Ile Asn Pro Asn Asn Asp Ala Ile Ser Tyr Asn Gin Lys Phe Arg 1 5 10 15
Gly
<219> 69 <211> 17 <212> PRT <213» Artificial Sequence <220> <221 > source <223> /noie-''Descripiion of Artificial Sequence: Synthetic peptide” <400> 69
Gly Ile Asn Pro Asn Asn Asp Ala Ile Ser Tyr Asn Gin Lys Phe Arg 1 5 10 15
Gly
<210> 70 <211» 17 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /no1e="Descripiion of Artificial Sequence: Synthetic peptide11 <400 70
Asp lie Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Glo Lys Phe Lys 1 5 10 15
Asp <210» 71 <211> 17
<212» PRT <213» Artificiai Sequence <220» <221> source <223» /note="Descriptfon of Artificial Sequence: Synthetic peptide" <400» 71
Asp lie Asn Pro Asn Asn Gly Gin Thr Phe Tyr Asn Gin Lys Phe Lys 15 10 15
Gly
<210» 72 <211» 17 <212» PRT <213> Artificial Sequent® <220» <221» source <223» /note~'Deseription of Artificial Sequence: Synthetic peptide” <400» 72
Val lie Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Gin Lys Phe Lys 15 10 15
Gly
<210» 73 <211> 17 <212» PRT <213» Artificial Sequence <220» <221» source <223> /ooie-'Descripiton of Artificial Sequence: Synthetic peptide" <400> 73
Arg lie Asa Pro Tyr Asn Gly Glu Thr Phe Phe Asn Gin Asn Phe Lys 1 5 IP 15
Asp
<210> 74 <211» 18 <212> PRT <213» Artifieiaf Sequence <22G> <221> source <223> /note=“Description of Artificial Sequence: Synthetic peptide" <400> 74
Gly Leu lie Asn Pro Tyr Asn Gly Glu Thr Thr Tyr Asn Gin Lys Phe 1 5 10 15 lys Gly
«210» 75 «211» 17 <212» PRT <213> Adificiai Sequence <220 <221 > source <223> /note-'Description of Artificial Sequence: Synthetic peptide” <4G0> 75
Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met 15 10 15
Ser
<210> 76 <211> 18 <210 PRT <213> Artificiai Sequence <220» <221» source <223> /note -"Description of Artificial Sequence: Synthetic peptide" <400» 76
Gly Asp lie Asn Pro Asn Asn Gly Glu Thr Phe Tyr Ser Gin Lys Phe 15 10 15
Lys Gly <21Q> 77 <211> 18
<212> PRT <213> Artificiai Sequence <220» <221> source <223> /røte-'Description of Artificial Sequence: Synthetic peptide" <4QQ> 77
Gly Asp Ile Asn Pro Tyr Asn Gly Glu Thr Pha Tyr Asn Gin Lys Leu 1 5 10 15
Lys Gly
<21Q> 78 <211> 18 <212> PRT <213> Artificiai Sequence <220> <221 > source <223» /note-'Description of Artificiai Sequence: Synthetic peptide” <400> 78
Gly Gly ile Asn Pro Asn Asn Asp Ala Val Ser Tyr Asn Gin Arg Phe 15 10 15
Arg Gly
<210> 79 <211» 18 <212> PRT <213» Artificiai Sequence <220» <221> source <223» /note«"Descripiion of Artificial Sequence: Synthetic peptide" <400 79
Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gin Lys Phe 15 10 15
Lys Asp
<210» 80 <211> 18 <212> PRT <213> Artificiai Sequence <220» <221» source <223» /note="Descr i pf i on of Artificial Sequencer Synthetic peptide" <4GQ> 80
Gly Gly He Asn Pro Asn Asn Asp Ala Ile Ser Tyr Asn Gin Lys Phe 1 5 10 15
Arg Gly
<210> 81 <211> 18 <212» PRT <213» Artificial Sequence <220» <221» source <223» /note="Description of Artificiel Sequence: Synthetic peptide" <400» 61
Gly Gly II« Asn Pro Asn Asn Asp Ala II® S®r Tyr Asn Gin Lys PA© 15 10 15
Arg Gly
<210» 62 <211» 18 <212» PRT <213» Artificial Sequence <220» <221» source <223» /note^’'Description of Artificial Sequence: Synthetic peptide" <40Q> 82
Gly Asp He Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gin Lys Phe 15 10 15 lys Asp
<210» 83 <211» 16 <212» PRT <213» Artificial Sequence <220» <221» source <223» /note-‘Description of Artificial Sequence: Synthetic peptide" <400> 63
Gly Asp Ile Asn Pro Asn Asn Gly Glu Thr Phe Tyr Asn Gin Lys Phe 15 10 15
Lys Gly
<210> 84 <211> 18 <212> PRT <213> Arfificiai Sequence <220> <221> source <223> /note-’Description of Artificial Sequence: Synthetic peptide” <400> 84
Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 15 10 15
Lys Gly
<210> 85 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note="Descriptioo of Artificial Sequence: Synthetic peptide" <400> 85
Gly Arg Ile Asn Pro Tyr Asn Gly Glu Thr phe Phe Asn Gin Asn Phe 15 10 15
Lys Asp
<210> 86 <211>7 <212> PRT <213> ArtifraaS Sequence <220> <221 > source <223> /note="Descriptron of Artificial Sequence: Synthetic peptide" <400> 86
Gly Leu Tyr Trp Phe Pro Tyr 1 5
<210> 87 <21f> 12 <212> PRT <213> Artificial Sequence <220» <221 > source <223> /note="Description of Artificial Sequence: Synthetic peptide" <4QQ> 87
His Tyr Tyr Gly Ser Ser Asp Tyr Ala, Leu Asp Asn IS 10
<210> 88 <211> 7 <212» PRT <213» Artificial Sequence <220» <221» source <223> /nofe="Descripfion of Artificial Sequence: Synthetic peptide"
<400» 8B
Gly Leu Tyr Arg Phe Asp Tyr 1 5
<210» 89 <211» 7 <212» PRT <213» Artificial Sequence <220» <221» source <223> /nofe~"Descripiion of Artificial Sequence: Synthetic peptide" <400» 89
Gly Leu Tyr Phe Phe Ala Tyr 1 S <210> 90
<211» 9 <212» PRT <213» Artificial Sequence <220» <221» source <223> /note«"Description of Artificial Sequence: Synthetic peptide" <4GQ> 90
Leu Gly Arg Gly Tyr Tyr Phe Asp Tyr 1 5
<210» 91 <211> 12 <212» PRT <213» Artificial Sequence <220» <221» source <223» /nots-’Description of Artificial Sequence: Synthetic peptide" <400» 91
Ser leu Tyr Gly Tyr Asp Ala Ser Tyr 9he Asp Tyr 1 5 10
<21G> 92 <211» 9 <212> PRT <213» Artificial Sequence <220> <221» source <223» /note=’'Description of Artificial Sequence: Synthetic peptide" <400» 92 lieu Gly Arg Gly Tyr Tyr Phe Asp Tyr 1 5
<210» 93 <211» 9 <212» PRT <213» Artificial Sequence <220» <221» source <223» /note-’Description of Artificial Sequence: Synthetic peptide" <400» 93
Aeu Gly Arg Gly Tyr Tyr Phe Asp Tyr 1 5
<210» 94 <211» 7 <212» PRT <213» Artificial Sequence <220» <221» source <223» /note-'Description of Artificial Sequence: Synthetic peptide11 <400» 94
Gly lieu Tyr Phe Phe His Tyr 1 S
<210» 95 <211» 7 <212» PRT <213» Artificiai Sequence <220» <221» source <223» /noie^’Description of Artificiai Sequence. Synthetic peptide" <4Q0> 95
Gly Leu Tyr hrg Phe Asp Tyr 1 5
<210> 96 <211> 10 <212> PRT <213> Artificial Sequence <220> <221 > source <223> toots-‘Description of Artificial Sequence: Synthetic peptide" <400> 96
Thr Arg Asn Tyr Gly Tyr Val lie Asp Tyr IS 10
<210> 97 <211>7 <212> PRT <213> Artificiai Sequence <220> <221 > source <223> toote-‘Description of Artificial Sequence: Synthetic peptide" <400> 97
Gly Leu Tyr Tyr Leu Asn Tyr 1 5
<210> 98 <211> 9 <212> PRT <213> Artificiai Sequence <220> <221 > source <223> toote-'Descriplion of Artificiai Sequence: Synthetic peptide" <400> 98
Ala Arg Gly Leu Tyr Trp Phe Pro Tyr 1 5
<210> 99 <211 > 14 <212> PRT <213> Artificiai Sequence <220> <221 > source <223> toole="Deschpiion of Artificiai Sequence: Synthetic peptide” <4Q0> 99
Ser Asn His Tyr Tyr Gly ser Ser Asp Tyr Ala len Asp Asn 15 10
<210> 100 <211>9 <212> PRT <213> Artificial Sequence <220» <221> source <223> /note="Oescription of Artificial Sequence: Synthetic peptide" <40Q> 100
Ala Arg Gly Leu Tyr Arg Phe Asp Tyr 1 5
<210> 101 <211> 9 <212» PRT «213» Artificiai Sequence <220» <221> source <223> /note=KDescription of Artificial Sequence: Synthetic peptide" <4QQ> 101
Ala Arg Gly Leu Tyr Phe Phe Ala Tyr 1 5
<210> 102 <211> 11 <212» PRT <213» Artificiai Sequence <220> <221> source <223> /note="Descripiion of Artificiai Sequence: Synthetic peptide" <4Q0> 102
Ala Lys Leu Gly Arg Gly Tyr Tyr Phe Asp Tyr 1 5 10
<210> 103 <211> 14 <212» PRT <213> Artificiai Sequence <220» <221» source <223» /note=''Description of Artificiai Sequence: Synthetic peptide" <400» 103
Thr Arg Ser Le« Tyr Gly Tyr Asp Ala Ser Tyr Phe Asp Tyr 1 5 10
<210> 104 <211» 11 <212> PRT <213» Artificial Sequence <220> <221 > source <223> /note-'Oescription of Artificial Sequence: Synthetic peptide" <400> 104
Ala Lys Leu Gly Arg Gly Tyr Tyr Phe Asp Tyr 1 5 10
<210> 105 <211> 11 <212> PRT <2T3> Artificial Sequence <220» <221 > source <223> /note-'Description of Artificial Sequence: Synthetic peptide" <400» 105
Ala Lys Leu Gly Arg Gly Tyr Tyr Phe Asp Tyr 1 5 10 <210> 106
<211> 9 <212» PRT <213> Artificial Sequence <220> <221 > source <223» /note-’Description of Artificial Sequence: Synthetic peptide" <40Q> 106
Ala Arg Gly Leu Tyr Phe Phe His Tyr 1 5
<210» 107 «211» 9 <212» PRT <213» Artificial Sequence <220> <221> source <223> /note-’Description of Artificial Sequence: Synthetic peptide" <400» 107
Ala Arg Gly Leu Tyr Arg Phe Asp Tyr 1 5 <210> 108 <211» 12
<212> PRT <213> Artificial Sequence <22G> <221 > source <223> /oote^Descnpiion of Artificial Sequence: Synthetic peptide” <4Q0> 108
Ala Arg Thr Arg Asn Tyr Gly Tyr Val lie Asp Tyr 15 18
<210> 109 <211> 9 <212> PRT <213> Artificiai Sequence <220> <221 > source <223> /'ncte="DescripSon of Artificial Sequence: Synthetic peptide” <400> 109
Gly Arg Gly leu Tyr Tyr leu Asn Tyr 1 5
<210> 110 <211» 112 <212> PRT <213> Artificial Sequence <220> <221 > source <223> Mcte='Oescripfion of Artificial Sequence: Synthetic polypeptide" <4QG> 110
Asp He Val Met Thr Gin Ser Gin Arg Phe Met Ser Thr Ser leu Gly 15 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gin Asn Val He Thr Asn 20 25 30
Val Ala Trp Tyr Gin Gin lys Pro Gly Gin Ser Pro lys Ala leu Xle 35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr leu Thr He Ser Asn Val Gin Ser 65 70 75 80
Glu Asp leu Ala Glu Tyr Phe Cys Gin Gin Tyr Thr Asn Tyr Pro Met 85 90 95
Trp Thr Phe Gly Gly Sly Thr Arg Leu Glu Xle Lys Arg Thr Val Ala 100 105 110
<210» 111 «211» 129 <212» PRT <213» Artificial Sequence <22G> <221 > source «223» /oote**Deseripfion of Arttficia! Sequence: Synthetic polypeptide" <400> 111
Glu Val Gin Leu Gin Gin Sen Gly Pro Glu Leu Val Lys Pro Gly Ala 15 10 15
Sen Met Lys ile Ser Cys Glu Ala Ser Gly Tyr Ser Phe Thr Ala Tyr 20 25 30 lie Met Asn Trp Val Lys Gin Ser Arg Gly Lys Asn Leu Glu Trp lie 35 40 45
Gly Leu Xle Asn Pro Tyr Asn Gly Glu Thr Thr Tyr Asn Gin Lys Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Gin Ser Ser Ser Thr Ala Tyr fiS 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95
Ala Arg Gly Leu Tyr Trp Phe Pro Tyr Trp Gly Gin Gly Thr Leu Val 100 105 110
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125
Pro
<210» 112 <211» 112 <212> PRT <213-· Artificial Sequence <220» <221» source <223» /rtote^'Description of Artificial Sequence: Synthetic polypeptide" <400> 112
Asp Ile Val Met Thr Gin Ser His Lys Ile Met Ser Thr Ser Val Gly 1 S 10 X5
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gin Asp Val Ser Asn Ile 20 25 30
Val Val Trp Phe Gin Gin Lys Pro Gly Gin Ser Pro Asn Lau Leti Ile 35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr phe Thr Ile Ser Ser Val Gin Ala 65 70 75 §0
Glu Asp Leu Ala Val Tyr Tyr Cys Gin Gin His Tyr Ser Ser Pro Tyr 85 90 S5
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110
<210> 113 <211 > 133 <212> PRT <213> Artificial Sequence <229> <221 > source <223> /noie-'Description of Artificial Sequence: Synthetic polypeptide" <40Q> 113
Slu Val Gin lew Gin GIu Ser Gly Pro Gly lew Val Gin Pro Ser Gin 1 5 10 15
Ser leu Ser lie Thr Cys Thr Val Ser Gly Phe Ser leu Thr Arg Phe 20 25 30
Gly Val His Trp Val Arg Gin Ser Pro Gly lys Gly leu Glu Trp leu 35 40 45
Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60
Ser Arg Leu Thr He Thr Lys Asp Asn Ser Lys Ser Gin Val Phe Phe 65 70 75 80
Lys leu Asn Ser Leu lys Val Asp Asp Thr Ala lie Tyr Tyr Cys Ser 85 90 95
Asn His Tyr Tyr Gly Ser Ser Asp Tyr Ala Leu Asp Asn Trp Gly Gin 100 105 110
Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125
Phe Pro Leu Ala Pro 130
<219> 114 <211> 113 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note="Descripiion of Artificial Sequence: Synthetic polypeptide'* <400> 114
Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Val Thr Pro Gly IS 10 15
Asp Ser val ser leu Ser Cys Arg Ala Ser Glu Asn ile Arg Asn Asn 20 25 30
Leu His Trp Tyr Gin Gin Lys Ser His Glu Ser Pro Arg Leu Leu lie 35 40 45
Lys Phe Thr Ser Gin Ser lie Ser Giy lie Pro Ser Arg Phe Thr Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser lie Asn Ser Val Glu Thr 65 70 75 80
Glu Asp Phe Gly Met Tyr Phe Cys Gin Gin Ser Asn Asn Trp Pro Gin 85 90 35
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg Thr Val Ala 100 105 110
Ala
<21 G> 115 <211> 129 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note-"Description of Artificial Sequence: Synthetic polypeptide” <400> 115
Olu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Trp lys Pro Gly Ala 15 IS 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met Lys Trp Val Lys Gin Thr His Gly Lys Ser Leu Glu Trp Ile 35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Glu Thr Phe Tyr Ser Gin Lys Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80
Met Gin Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 SO 95
Ala Arg Gly Leu Tyr Arg Phe Asp Tyr Trp Gly Gin Gly Thr Thr Leu 100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125
Pro
<21Q> 116 <211>113 <212> PRT <213> Artificiai Sequence <220> <221> source <223> /note='‘Descripiion of Artificial Sequence: Synthetic polypeptide" <40θ> 116
Asp Ile Gin Met Thr Gin Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15
Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gin Ser Ile Ser Asn Asn 20 25 30
Leu His Trp Tyr Gin Gin Lys Ser Sis Glu Ser Pro Arg Leu Leu Ile 35 40 45
Lys Phe Ala Ser Gin Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Plia Thr Leu Ser Ile Asn Ser Val Glu Thr 65 75 80
Glu Asp Phe Gly Met Tyr Phe Cys Gin Gin Ser Asn Asn Trp Pro Gin 85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala 100 105 110
Ala
<210» 117 <211» 129 <212» PRT <213» Artificiaf Sequence <220» <221» source <223» ,;not9=KDescription of Artificiaf Sequence: Synthetic polypeptide" <400»117
Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Ser Gly Ala 15 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met Lys Trp Val Lys Gin Ser Sis Gly Lys Ser Leu Glu Trp Ile 35 40 45
Gly Asp Ile Asn Pro Tyr Asn Gly Glu Thr Phe Tyr Asn Gin Lys Leu 50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Val Phe S5 70 75 80
Met Gin Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Gly Leu Tyr Phe Phe Ala Tyr Trp Gly Gin Gly Thr Thr Leu 100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125
Pro
<210 118 <211>112 <212> PRT <213> Adificiaf Sequence <220> <221> source <223> /note»"Oescription of Artificial Sequence: Synthetic poiypepfide" <400> 118
Asp He Gin Met Thr Gin Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 28 25 38
Leu Ala Trp Tyr Gin Lett Lys Gin Gly Lys Ser Pro Girt Lett Leu Val 35 48 45
Tyr Gly Ala Ala Asn Leu Ala Glu Gly Val Pro Ser Arg He Ser Gly 58 55 60
Ser Gly Ser Gly Thr Gin Tyr Ser Leu Lys He Asn Ser Leu Gin Ser 65 70 75 30
Glu Asp Phe Gly Thr Tyr Tyr Cys Gin His Phe Trp Gly He Pro Trp 85 SO 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg Thr Val Ala Ala 100 105 110
<210 119 <211> 131 <212> PRT <213> Artificial Sequence <220 <221 > source <223> /note^Descripfion of Artificial Sequence: Synthetic polypeptide* <400> 119
Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys He Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30
Thr Ile His Trp Val lys Gin Ser His Gly Lys Ser Leu Glu Arg Ile 35 40 45
Gly Gly ile Asn Fro Asn Asn Asp Ala Val Ser Tyr Asn Gin Arg Phe 50 55 60
Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 SO
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Als Val Tyr Tyr Cys 85 90 95
Ala Lys Leu Gly Arg Gly Tyr Tyr Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 UO
Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Fro Ser Val Phe Fro 115 120 125
Leu Ala Pro 130
<210> 120 <211> 117 <212> PRT <213> Artificial Sequence «220> <221 > source <223> toots-‘Description of Artificial Sequence: Synthetic polypeptide" <4Q0> 120
Asp Ile Gin Met Thr Gin Thr Pro Leu Ser Leu Fro Val Thr Leu Gly 1 5 10 15
Asp Gin Ala Ser Ile Ser Cys Arg Ser Ser Gin Ser Leu Val His Arg 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pra 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gin Ser 85 SO 95
Thr His Val Pro Pro Thr Phe Sly Gly Sly Thr Lys lieu Slu Leu Lys 100 105 110
Arg Thr Val Ala Ala 115
<210> 121 <211> 134 <212> PRT <213> Artificial Sequence <22G> <221 > source <223> /note="Descriptton of Artificial Sequence: Synthetic poiypeptide" <490> 121
Glii Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 15 10 15
Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30
Trp He Asn Trp val Lys Gin Arg Pro Sly Gin Sly Leu Slu Trp He 35 40 45
Gly Asn He Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Sin Lys Phe 50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Sin Leu Thr Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95
Thr Arg Ser Leu Tyr Gly Tyr Asp Ala Ser Tyr Phe Asp Tyr Trp Gly 100 105 110
Sin Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125
Val Phe Pro Leu Ala Pro 130
<210> 122 <211» 112 <212> PRT <213> Artificiai Sequence <22G> <221> source <223> /ooie- Oescription of Artificial Sequence: Synthetic polypeptide" <400> 122
Asp Ile Val Met Thr Gin Ser Pro Ala Ser leu Ser Ala Ser Val Gly 1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 leu Ala Trp Tyr Gin leu lys Gin Gly lys Ser Pro Gin Leu Leu Val 35 40 45
Tyr Gly Ala Ala Asn leu Ala Glu Gly Val Pro Ser Arg lie Ser Gly 50 55 60
Ser Gly Ser Gly Thr Gin Tyr Ser leu lys He Asn Ser leu Gin Ser
65 70 75 SO
Glu Asp Phe Gly Thr Tyr Tyr Cys Gin His Phe Trp Gly He Pro Trp 85 90 95
Thr Phe Gly Gly Gly Thr lys leu Glu He lys Arg Thr Val Ala Ala 100 105 110
<21 Q> 123 <211> 131 <212> PRT <213> Artificial Sequence <220> <22 1> source <223> /note="Oescripfion of Artificial Sequence: Synthetic polypeptide” <4GG> 123
Gin Val Gin Leu Gin Gin Ser Gly Pro Giu leu Val Lys Pro Gly Ala 1 S 10 15
Ser Val Lys lie Ser Cys Lye Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30
Thr Ile His Trp Val Lys Gin Ser His Gly Lys Ser Leu Glu Arg He 35 40 45
Gly Gly lie Asn Pro Asn Asn Asp Ala Ile Ser Tyr Asn Gin Lys Phe 50 55 60
Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
«5 70 75 SO
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 30 95
Ala Lys Leu Gly Arg Gly Tyr Tyr Phe Asp Tyr Trp Gly Arg Gly Thr 100 105 110
Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125
Leu Ala Pro 130
<21G> 124 <211> 112 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note^'Description of Artificial Sequence: Synthetic polypeptide* <400> 124
Asp Ile Val Met Thr Gin Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 GI« Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30
Leu Ala Trp Tyr Gin Leu Tys Gin Gly Tys Ser Pro Gin Leu Leu Val 35 40 45
Tyr Gly Ala Ala Asn Le« Ala GI« Gly Val Pro Ser Arg Ile Ser Gly 50 55 SO
Ser Gly Ser Gly Thr Gin Tyr Ser Leu Lys Ile Asa Ser Le« Gin Ser 65 70 75 80
Glu Asp Phe Gly Thr Tyr Tyr Cys Gin His Phe Trp Gly Ile Pro Trp 85 90 95
Thr Phe Gly Gly Gly Thr Lys Le« Glu Ile Lys Arg Thr Val Ala Ala 100 105 110
<210» 125 <211»131 <212» PRT <213» Artificial Sequence <220» <221» source <223» toots-‘Description of Artificial Sequence: Synthetic polypeptide" <400» 125
Glu Val Gin Lau Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys xle Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30
Thr Xle His Trp Val Lys Gin Ser His Gly Lys Ser Leu Glu Arg Xle 35 40 45
Gly Gly Xle Asn Pro Asn Asn Asp Ala Ile Ser Tyr Asn Gin Lvs Phe 50 55 60
Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Leu Gly Arg Gly Tyr Tyr Phe Asp Tyr Trp Gly Arg Gly Thr 100 105 110
Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125
Leu Ala Pro 130
<21Q> 126 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> source <223> /note- 'Descripiion of Artificial Sequence: Synthetic polypeptide" <400> 126
Asp xle Val Leu Ile Gin Ser Pro Ala Thr Leu Ser Val Thr Leu Gly 15 10 15
Gly Ser Val Ser Leu Ser Cys Arg Ala Ser Gin Ser Ile Ser Agn Asn 20 25 30
Leu His Trp Tyr Gin Gin Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45
Lys Phe Ala Ser Gin Ser Ile Ser Gly Xle Pro Ser Arg Phe Arg Gly 50 55 60
Ser GXy Ser GXy Thr Asp Phe Thr Leu Thr Ile Asn Ser VaX ©lu Thr 65 70 75 80 ©lu Asp Phe ©Xy rie Tyr Phe Cys ©In GXn Ser Asn Asn Trp Pro Gin 85 90 95
Tyr Thr Phe Gly ©Xy Gly Thr lys leu Glu Xeu lys Arg Thr Val Als 100 105 110
Als
<210> 127 <211> 129 <212> PRT <213> Artificial Sequence <22ø> <221> source <223> /ooie="Descripiion of Artificial Sequence: Synthetic polypeptide" <400> 127 GXu Val GXn leu GXn Gin Ser Gly Pro Glu leu Val lys Pro Gly Ala 15 10 15
Ser Val lys Met Ser Cys lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met lys Trp Val Arg Gin Asn His ©ly lys Arg leu ©lu Trp Ile 35 40 45
Gly Asp Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gin lys Phe 50 55 60 lys Asp lys Aia Thr leu Thr Val Asp lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gin Phe Asn Ser leu Thr Ser Glu Asp Ser AXa Val Tyr Tyr Cys 85 90 95
Ala Arg Gly leu Tyr Phe Phe His Tyr Trp Gly Gin Gly Thr Thr leu 100 105 110
Thr Val Ser Ser Ala Ser Thr lys ©ly Pro Ser Val Phe Pro Leu Ala 115 120 125
Pro <21Q> 128
<211> 113 <212> PRT <213> Ariifraa! Sequence <220> <221 > source <223> /note~“Description of Artificial Sequence: Synthetic polypeptide" <400» 128
Asp Ile Val Leu Thr Sin Ser Pro Ala Asn lie« Ser Val lie Pro Gly 15 10 15
Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gin Asn Ile Axg Asn Asn 20 25 30
Leu His Trp Tyr Gin Sin Lys Ser Gin Slu Ser Pro Arg Leu Leu lie 35 40 45
Lys Phe Ala Ser Gin Ser Met Ser Gly Thr Pro Ser Arg Phe Thr Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Asn Thr Val Glu Thr 65 70 75 80
Glu Asp Phe Gly Met Tyr Phe Cys Gin Gin Ser Asn Asn Trp Pro Gin 85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg Thr Val Ala 100 105 110
Ala
<21Q> 128 <211> 130 <212» PRT <213» Artiflciai Sequence <220» <221> source <223> /note-'Description of Artificial Sequence; Synthetic poiypeptide" <4QG> 129
Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Trp Lys Pro Gly Ala 15 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met Lys Trp Val Lys Gin Ser His Gly Lys Ser Len Glu Trp Ile 35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Glu Thr Phe Tyr Asn Gin Lys Phe
50 55 gO
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 SO
Met Glu Leu Asn Ser Leu Thr Thr Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Gly Leu Tyr Arg Phe Asp Tyr Trp Gly Gin Gly Thr Thr Leu 100 105 110
Thr Val Ser Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro 130
<210> 130 <211> 115 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note^'Oescripfion of Artificial Sequence: Synthetic polypeptide" <4QG> 130
Gin Ala Val Val Thr Gin Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Thr Val Thr Thr Ser 20 25 30
Asn Phe Ala Asn Trp Val Gin Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45
Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60
Ser Gly Ser Leu tle Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80
Gin Thr Glu Asp Glu Ala Ile Tyr Phe Cys Val Leu Trp Cys Ser Asn 85 90 95
Le« Trp val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gin Pro 100 105 110
Lys Ala Ala 115
<210> 131 <211> 132 <212> PRT <213> Artificial Sequence <22Q> <221 > source <223> /note="Desaiptiort of Artificial Sequence: Synthetic polypeptide" <4ø0> 131
Gi« Val Gin Le« Gin Gin Ser Gly Ala Asp Le« Val Arg Pro Gly Thr 15 10 15
Ser Val Lys Val Ser Gys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Le« Ile GI« Trp Val Lys Gin Arg Pro Gly Gin Gly Le« Glu Trp Ile 35 40 45
Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Gi« Lys Phe 50 55 60
Lys Gly Lys Ala Thr Le« Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gin Leu Ser Ser Le« Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 30 35
Ala Arg Thr Arg Asn Tyr Gly Tyr Val Ile Asp Tyr Trp Gly Gin Gly 100 105 110
Thr Thr Le« Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125
Pro Le« Ala Pro 130
<210> 132 <211> 112 <212> PRT <213> Artificiai Sequence <220> <221> source <223> /note-’Description of Artificial Sequence: Synthetic polypeptide" <490» 132
Asp lie val Met Thr Gin Ser His Ays Phe Met Ser Thr ser vai Giy 1 S 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gin Asp Val Giy Thr Ala 20 25 30
Val Ala Trp Tyr Gin Gin Lys Pro Giy Gin Ser Pro Lys Leu Leu He 35 40 45
Phe Trp Pro Ser Thr Arg His Thr Giy Val Pro Asp Arg Phe Thr Giy 50 55 60
Ser Giy Ser Giy Thr Asp Phe Thr Leu Thr lie Giy Asn Val Gin Ser 65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gin Gin Phe Ser Ser Leu Pro His 85 90 95
Thr Phe Giy Giy Giy Thr Lys Leu Gi« Ile Lys Arg Thr Val Ala Ala 100 105 110
<210> 133 <211»129 <212> PRT <2I3> Artificial Sequence <220> <221> source <223> /not e="Description of Artificial Sequence: Synthetic polypeptide” <40Q> 133
Gin Val Gin Leu Gin Gin Ser Giy Pro Glu Leu Val Lys Pro Giy Ala 15 10 15
Ser Val Lys He Ser Cys Lys Ala Ser Giy Phe Ser Phe Thr Ala Tyr 20 25 30
Phe Met Asn Trp Val Lys Gin Ser His Giy Lys Ser Pro Glu Trp He 35 40 45
Giy Arg He Asn Pro Tyr Asn Giy Glu Thr Phe Phe Asn Gin Asn Phe 50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His 65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95
Gly Arg Gly Leu Tyr Tyr Leu Asn Tyr Trp Gly Gin Gly Thr Thr Leu 100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125
Pro
<210> 134 <211> 107 <212> PRT <213> Artificial Sequence <220> <221 > source <223> /note="Description of Artificial Sequence: Synthetic polypeptide" <400> 134
Asp lie Val Met Thr Gin Ser His Lys lie Met Ser Thr Ser Val Gly 1 5 10 15
Asp Arg val Ser lie Thr Cys Lys Ala Ser Gin Asp Val Ser Asa lie 20 25 30
Val Val Trp Phe Gin Gin Lys Pro Gly Gin Ser Pro Asn Leu Leu lie 35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Val Gin Ala 65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gin Gin His Tyr Ser Ser Pro Tyr 85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 100 105
<210> 135 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> source <223> /nois=”Descripiion of Artificial Sequence: Synthetic polypeptide" <400 135
Gin Val Gin leu Lys Gin Ser Gly Pro Gly leu val Gin Pro Ser Gin 15 10 15
Ser leu Ser lie Thr Cys Thr val Ser Gly Phe Ser leu Thr Arg Phe 20 25 30
Gly Val His Trp Val Arg Gin Ser Pro Gly lys Gly Leu Glu Trp leu 35 40 45
Gly Val lie Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60
Ser Arg leu Thr He Thr lys Asp Asn Ser lys Ser Glu Val Phe Phe 65 70 75 80 lys leu Asn Ser leu lys Val Asp Asp Thr Ala He Tyr Tyr Cys Ser 85 30 35
Asn His Tyr Tyr Gly Ser Ser Asp Tyr Ala leu Asp Asn Trp Gly Gin 100 105 110
Gly He Ser Val Thr Val Ser Ser 115 120 <210> 138 <211 >233
<212> PRT <213> Homo sapiens <400> 136
Val Ala Val Pro Val Ser Arg Pro Val Leo Thr Leu Arg Als Pro Gly 15 10 15
Thr His Ala Ala Val Gly Asp Leu Leu Glu Leu His Gys Glu Ala Leu 20 25 30
Arg Gly Ser Pro Leu Ile Leu Tyr Arg Phe Phe His Glu Asp Val Thr 35 40 45
Leu Gly Asn Arg Ser Ser Pro Ser Gly Gly Ala Ser Leu Asn Leu Ser 50 55 60
Leu Thr Ala Glu His Ser Gly Asn Tyr Ser Cys Glu Ala Asp Asn Gly 65 70 75 80
Leu Gly Ala Gin Arg Ser Glu Thr Val Thr Leu Tyr Ile Thr Gly Leu 85 90 95
Thr Ala Agn Arg Ser Gly Pro Phe Ala Thr Gly Val Ala Gly Gly Leu 100 105 110
Leu Ser Ile Ala Gly Leu Ala Ala Gly Ala Leu Leu Leu Tyr Cys Trp 115 120 125
Leu Ser Arg Lys Ala Gly Arg Lys Pro Ala Ser Asp Pro Ala Arg Ser 130 135 140
Pro Ser Asp Ser Asp Ser Gin Glu Pro Thr Tyr His Asn val Pro Ala 145 150 155 160
Trp Glu Glu Leu Gin Pro Val Tyr Thr Asn Ala Asn Pro Arg Gly Glu 165 170 175
Asn Val Val Tyr Ser Glu Val Arg Xle Ile Gin Glu Lys Lys Lys His 180 185 190 AXa Val Ala Ser Asp Pro Arg His Leu Arg Asn Lys Gly Ser Pro Ile 195 200 205
Ile Tyr Ser Glu Val Lys Val Ala Ser Thr Pro Val Ser Gly Ser Leu 210 215 220
Phe Leu Ala Ser Ser Ala Pro His Arg 225 230
REFERENCES CiTEO iN THE DESCRiPTiON
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Claims (16)

1. Isoleret anti-FcRH5-antistof, som binder en isoform-c-specifik region af det ekstracellulære domæne af FcRH5c vist som aminosyrer 743-850 af SEQ ID NO:1, hvor antistoffet ikke signifikant binder et andet lg-lignende domæne af FcRH5.
2. Anti-FcRH5-antistof ifølge krav 1, hvor anti-FcRH5-antistoffet omfatter: (a) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:39, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:63 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:87 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:3, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:15 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:27; (b) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:38, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:62 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:86 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:2, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:14 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:26; (c) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:40, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:64 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:88 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:4, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:16 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:28; (d) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:41, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:65 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:89 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:5, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:17 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:29; (e) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:42, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:66 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:90 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID N0:6, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:18 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:30; (f) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:43, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:67 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:91 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:7, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:19 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:31; (g) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:44, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:68 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:92 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:8, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:20 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:32; (h) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:45, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:69 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:93 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:9, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:21 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:33; (i) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:46, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:70 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:94 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:10, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:22 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:34; (j) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:47, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:71 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:95 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:11, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:23 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:35; (k) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:48, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:72 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:96 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID N0:12, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:24 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:36; eller (I) en tungkæde omfattende et HVR-H1 omfattende aminosyresekvensen af SEQ ID NO:49, et HVR-H2 omfattende aminosyresekvensen af SEQ ID NO:73 og et HVR-H3 omfattende aminosyresekvensen af SEQ ID NO:97 og en letkæde omfattende et HVR-L1 omfattende aminosyresekvensen af SEQ ID NO:13, et HVR-L2 omfattende aminosyresekvensen af SEQ ID NO:25 og et HVR-L3 omfattende aminosyresekvensen af SEQ ID NO:37.
3. Anti-FcRH5-antistof ifølge krav 1 eller 2, hvor anti-FcRH5-antistoffet omfatter: (a) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:113 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:112; (b) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:135 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:134; (c) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:111 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:110; (d) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:115 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:114; (e) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:117 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:116; (f) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:119 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:118; (g) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:121 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:120; (h) en VFI-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:123 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:122; (i) en VH-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:125 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:124; (j) en VH-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:127 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:126; (k) en VH-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:129 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:128; (l) en VH-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:131 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:130; eller (m) en VH-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:133 og en VL-sekvens med mindst 95 % sekvensidentitet til aminosyresekvensen af SEQ ID NO:132.
4. Anti-FcRH5-antistof ifølge et af kravene 1-3, hvor anti-FcRH5-antistoffet omfatter: (a) en VH-sekvens af SEQ ID NO:111 og en VL-sekvens af SEQ ID NO:110; (b) en VH-sekvens af SEQ ID NO:135 og en VL-sekvens af SEQ ID NO:134; (c) en VH-sekvens af SEQ ID NO:113 og en VL-sekvens af SEQ ID NO:112; (d) en VH-sekvens af SEQ ID NO:115 og en VL-sekvens af SEQ ID NO:114; (e) en VH-sekvens af SEQ ID NO:117 og en VL-sekvens af SEQ ID NO:116; (f) en VH-sekvens af SEQ ID NO:119 og en VL-sekvens af SEQ ID NO:118; (g) en VH-sekvens af SEQ ID NO:121 og en VL-sekvens af SEQ ID NO:120; (h) en VH-sekvens af SEQ ID NO:123 og en VL-sekvens af SEQ ID NO:122; (i) en VH-sekvens af SEQ ID NO:125 og en VL-sekvens af SEQ ID NO:124; (j) en VH-sekvens af SEQ ID NO:127 og en VL-sekvens af SEQ ID NO:126; (k) en VH-sekvens af SEQ ID NO:129 og en VL-sekvens af SEQ ID NO:128; (l) en VH-sekvens af SEQ ID NO:131 og en VL-sekvens af SEQ ID NO:130; eller (m) en VH-sekvens af SEQ ID NO:133 og en VL-sekvens af SEQ ID NO:132.
5. Anti-FcRH5-antistof ifølge et af kravene 1-4, hvor anti-FcRH5-antistoffet er: (a) et monoklonalt antistof; (b) et humant, humaniseret eller kimærisk antistof; (c) et antistoffragment, som binder FcRH5; (d) et lgG1-, lgG2a- eller lgG2b-antistof; (e) et bispecifikt antistof, eventuelt hvor det bispecifikke antistof binder FcRFI5 og CD3; og/eller (f) konjugeret til en mærkning, hvor mærkningen eventuelt er en positronemitter, og positronemitteren eventuelt er 89Zr.
6. Anti-FcRFI5-antistof ifølge et af kravene 1-5, hvor anti-FcRFI5-antistoffet har en eller flere af de følgende egenskaber: (a) krydsreaktiv med humant og cyno FcRH5 med fuldlængde, (b) krydsreagerer ikke med FcRH1, FcRH2, FcRFI3 og/eller FcRFI4, (c) binder til endogent FcRFI5 og (d) krydsreagerer ikke med FcRH5a.
7. Isoleret nukleinsyre, der koder anti-FcRFI5-antistoffet ifølge et af kravene 1-6.
8. Værtscelle, der omfatter nukleinsyren ifølge krav 7.
9. Fremgangsmåde til produktion af et anti-FcRFI5-antistof omfattende dyrkning af værtscellen ifølge krav 8, således at anti-FcRFI5-antistoffet produceres.
10. Immunkonjugat, som omfatter anti-FcRFI5-antistoffet ifølge et af kravene 1-6 og et cytotoksisk middel, eventuelt hvor immunkonjugatet har formlen Ab-(L-D)p, hvor: (a) Ab er anti-FcRFI5-antistoffet ifølge et af kravene 1-6; (b) L er en linker; (c) D er et lægemiddel udvalgt blandt et maytansinoid, et auristatin, et cali-cheamicin, et pyrrolobenzodiazepin og et nemorubicin derivat; og (d) p ligger i området fra 1 -8 og fortrinsvis fra 2-5.
11. Immunkonjugat ifølge krav 10, hvor linkeren er spaltbar ved en protease eller er syrelabil.
12. Farmaceutisk formulering omfattende anti-FcRH5-antistoffet ifølge et af kravene 1-6 og/eller immunkonjugatet ifølge krav 10 eller 11 og et farmaceutisk acceptabelt bærestof og eventuelt endvidere omfattende et yderligere terapeutisk middel.
13. Anti-FcRFI5-antistof ifølge et af kravene 1-6 og/eller immunkonjugat ifølge krav 10 eller 11 til anvendelse i en fremgangsmåde til behandling af et individ, som har en FcRH5-positiv cancer, hvilken fremgangsmåde omfatter indgivelse af en virksom mængde af anti-FcRH5-antistoffet og/eller immunkonjugatet til individet, hvor den FcRFI5-positive cancer eventuelt er en B-celle-proliferativ lidelse, og fremgangsmåden eventuelt endvidere omfatter indgivelse af et yderligere terapeutisk middel til individet.
14. Anti-FcRFI5-antistof ifølge et af kravene 1-6 og/eller immunkonjugat ifølge krav 10 eller 11 til anvendelse i en fremgangsmåde til inhibering af proliferation af en FcRFI5-positiv celle, hvor fremgangsmåden omfatter eksponering af den FcRH5-positive celle for anti-FcRH5-antistoffet og/eller immunkonjugatet under betingelser, som tillader binding af anti-FcRFI5-antistoffet og/eller immunkonjugatet til FcRH5 på overfladen af den FcRH5-positive celle, hvorved proliferation af den FcRH5-positive celle inhiberes, hvor den FcRH5-positive celle eventuelt er en B-celle.
15. Anti-FcRH5-antistof ifølge et af kravene 1-6 til anvendelse i en fremgangsmåde til detektering af humant FcRFI5 i en biologisk prøve, hvor fremgangsmåden omfatter at bringe den biologiske prøve i kontakt med anti-FcRFI5-antistoffet under betingelser, som tillader binding af anti-FcRFI5-antistoffet til et naturligt forekommende humant FcRFI5, og detektering af, om der er dannet et kompleks mellem anti-FcRFI5-antistoffet og et naturligt forekommende humant FcRH5 i den biologiske prøve, hvor den biologiske prøve eventuelt er en blodprøve.
16. Anti-FcRH5-antistof ifølge et af kravene 1-6 til anvendelse i en fremgangsmåde til detektering af en FcRH5-positiv cancer, hvor fremgangsmåden omfatter (i) indgivelse af anti-FcRH5-antistoffet til et individ, som har eller mistænkes for at have en FcRH5-positiv cancer, hvor anti-FcRH5-antistoffet er et mærket anti-FcRFI5-antistof, og (ii) detektering af det mærkede anti-FcRH5-antistof i individet, hvor detektering af det mærkede anti-FcRH5-antistof viser en FcRH5-positiv cancer i individet, og hvor det mærkede anti-FcRFI5-antistof eventuelt omfatter et anti-FcRFI5-antistof, som er konjugeret til en positronemitter, og positronemitteren eventuelt er 89Zr.
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