WO2023196903A1

WO2023196903A1 - Bispecific antigen-binding molecules that bind and cd3 and tumor associated antigens (taas) and uses thereof

Info

Publication number: WO2023196903A1
Application number: PCT/US2023/065437
Authority: WO
Inventors: Eric Smith; Tong Zhang
Original assignee: Regeneron Pharmaceuticals, Inc.
Priority date: 2022-04-06
Filing date: 2023-04-06
Publication date: 2023-10-12

Abstract

The present disclosure provides bispecific antibodies that bind to both CDS and tumor associated antigens (TAAs) that activate T cells via the CDS complex, as well as populations of T cells comprising yδ T cells. The bispecific antigen-binding proteins of the disclosure are useful for the treatment of diseases and disorders, such as cancer, and may be administered in combination with the population of T cells comprising yδ T cells.

Description

BISPECIFIC ANTIGEN-BINDING MOLECULES THAT BIND CD3 AND TUMOR ASSOCIATED ANTIGENS (TAAS) AND USES THEREOF

RELATED APPLICATIONS

[0001] The instant application claims priority to U.S. Provisional Application No. 63/328,157, filed on April 6, 2022. The entire contents of the foregoing application are expressly incorporated herein by reference.

FIELD

[0002] The present invention relates to bispecific antigen-binding molecules that bind CD3 and tumor associated antigens (TAAs) and methods of use thereof, including coadministration with a population of T cells comprising gamma-delta (yd) T cells.

BACKGROUND

[0003] CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four different chains: epsilon, zeta, delta and gamma. The CD3 dimeric arrangements include gamma/epsilon, delta/epsilon, and zeta/zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide- loaded MHC molecules. However, CD3 antibodies alone are not sufficient to meet the needs of every cancer patient. Bispecific antigen-binding molecules that bind both CD3 and tumor associated antigens, such as CD20, would be useful in therapeutic settings, including coadministration with a population of T cells comprising gamma-delta (yd) T cells.

BRIEF SUMMARY

[0001] In one aspect, disclosed herein is a method for treating a cancer in a subject, said method comprising administering to the subject: a) an antigen-binding protein, wherein said antigen-binding protein binds to CD3 and to a tumor-associated antigen; and b) a population of T cells comprising yd T cells.

[0002] In some embodiments, the population of yd T cells is an ex vivo expanded population of yd T-cells. In some embodiments, the population of yd T cells is expanded with an aminobisphosphonate. In some embodiments, the aminobisphosphonate is zoledronate. In some embodiments, the aminobisphosphonate is combined with IL-2 to induce expansion. In some embodiments, the population of yd T cells is expanded with one or more antibodies.

1

SUBSTITUTE SHEET ( RULE 26) [0003] In some embodiments, the antigen-binding protein is an antibody or antigen-binding fragment thereof. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody.

[0004] In some embodiments, the antigen-binding protein is administered concurrently with the population of T cells. In other embodiments, the antigen-binding protein is administered prior to, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours prior to the population of T cells. In some embodiments, the population of T cells is administered prior to, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours prior to the antigen-binding protein.

[0005] In some embodiments, the tumor-associated antigen is CD20. In some embodiments, the tumor-associated antigen is CD20, EpCam, GD2, and GD3, mesothelin, NY-ESO-1 , Folate Binding Protein (FBP), human epidermal growth factor receptor 2 (HER- 2/neu), IL-13 receptor a2, melanoma-associated antigen 1 (MAGE-A1 ), EPH receptor A2 (EphA2). carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), survivin, tyrosine related protein 1 (tyrpl), tyrosine related protein 1 (tyrp2), brachyury, 5 alpha reductase, alphafetoprotein, AM-1 , APC, April, BAGE, beta-catenin, Bc112, bcr-abl, CA-125, CASP-8/FLICE, cathepsins, CD19, CD21 , CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a, FLK-1/KDR, folic acid receptor, G250, GAGE- family, gastrin 17, gastrin-releasing hormone, GD2/GD3/GM2, GnRH, GnTV, GP1 , gp100/Pme117, gp-100-in4, gp15, gp75/TRP-1 , hCG, heparanse, HMTV, Hsp70, hTERT, IGFR1 , IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, MAGE-family, mammaglobin, MAPI 7, melan-A/MART-1 , mesothelin, MIC A/B, MT-MMPs, mucin, NY- ESO-1 , osteonectin, p15, P170/MDR1 , p53, p97/melanotransferrin, PAI-1 , PDGF, uPA, PRAME, probasin, progenipoientin, PSA, PSM, RAGE-1 , Rb, RCAS1 , SART-1 , SSX-family, STAT3, STn, TAG-72, TGF-alpha, TGF-beta, Thymosin-beta-15, TNF-alpha, TYRP-, TYRP- 2, tyrosinase, VEGF, ZAG, p16INK4, or glutathione-S-transferase.

[0006] In some embodiments, the population of T cells comprises >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%, or 100% yd T cells. In some embodiments, the population of T cells is autologous to said subject. In some embodiments, the population of T cells is allogeneic to said subject. In some embodiments, the population of yd T cells has been modified to express a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor binds a tumor-associated antigen.

[0007] In another aspect, disclosed herein is a composition comprising: a) an antigenbinding protein, wherein said antigen-binding protein binds to CD3 and to a tumor- associated antigen; and b) a population of T cells comprising yd T cells. In some embodiments, the population of yd T cells is an ex vivo expanded population of yd T-cells. In some embodiments, the population of yd T cells has been expanded with an aminobisphosphonate. In some embodiments, the aminobisphosphonate is zoledronate. In some embodiment, the aminobisphosphonate is combined with IL-2 to induce expansion. In some embodiments, the population of yd T cells is expanded with one or more antibodies. [0008] In some embodiments, the antigen-binding protein is an antibody or antigen-binding fragment thereof. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody.

[0009] In some embodiments, the tumor-associated antigen is CD20. In some embodiments, the tumor-associated antigen is CD20, EpCam, GD2, and GD3, mesothelin, NY-ESO-1 , Folate Binding Protein (FBP), human epidermal growth factor receptor 2 (HER- 2/neu), IL-13 receptor a2, melanoma-associated antigen 1 (MAGE-A1 ), EPH receptor A2 (EphA2). carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), survivin, tyrosine related protein 1 (tyrpl), tyrosine related protein 1 (tyrp2), brachyury, 5 alpha reductase, alphafetoprotein, AM-1 , APC, April, BAGE, beta-catenin, Bc112, bcr-abl, CA-125, CASP-8/FLICE, cathepsins, CD19, CD21 , CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a, FLK-1/KDR, folic acid receptor, G250, GAGE- family, gastrin 17, gastrin-releasing hormone, GD2/GD3/GM2, GnRH, GnTV, GP1 , gp100/Pme117, gp-100-in4, gp15, gp75/TRP-1 , hCG, heparanse, HMTV, Hsp70, hTERT, IGFR1 , IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, MAGE-family, mammaglobin, MAPI 7, melan-A/MART-1 , mesothelin, MIC A/B, MT-MMPs, mucin, NY- ESO-1 , osteonectin, p15, P170/MDR1 , p53, p97/melanotransferrin, PAI-1 , PDGF, uPA, PRAME, probasin, progenipoientin, PSA, PSM, RAGE-1 , Rb, RCAS1 , SART-1 , SSX-family, STAT3, STn, TAG-72, TGF-alpha, TGF-beta, Thymosin-beta-15, TNF-alpha, TYRP-, TYRP- 2, tyrosinase, VEGF, ZAG, p16INK4, or glutathione-S-transferase.

[0010] In some embodiments, the population of T cells comprises >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%, or 100% yd T cells. In some embodiments, the population of T cells is autologous to said subject. In some embodiments, the population of T cells is allogeneic to said subject. In some embodiments, the population of yd T cells has been modified to express a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor binds a tumor-associated antigen.

[0011] Other embodiments will become apparent from a review of the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 depicts cytotoxicity data for anti-CD3 x TAA bispecific antibodies in combination with yb T cells. These bispecific antibodies triggered strong cytotoxicity by ex- vivo expanded Vb2 yb T cells in four different cell types.

[0013] FIG. 2 depicts cytotoxicity data for an anti-CD20 x CD3 bispecific antibody. Both Vb1 and Vb2 yb T cells mediated effective Raji cell killing when combined with the anti-CD20 x CD3 bispecific Ab (BsAb).

[0014] FIG. 3 depicts cytotoxicity data for an anti-STEAP2 x CD3 bispecific antibody, yb T cells mediated effective 22RV1 cell killing when combined with the anti-STEAP2 x CD3 bispecific Ab (BsAb).

[0015] FIG. 4 depicts cytotoxicity data for an anti-PSMA x CD3 bispecific antibody, yb T cells mediated rapid C4-2 target cell killing when combined with the anti-PSMA x CD3 bispecific antibody (BsAb).

[0016] FIG. 5 depicts cytotoxicity data for an anti-MUC16 x CD3 bispecific antibody. yb2 yb T cells mediated effective killing of OVCAR3 cells when combined with MUC16 x CD3 bispecific Ab (BsAb).

DETAILED DESCRIPTION

[0017] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1 , 99.2, 99.3, 99.4, etc.).

[0019] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties.

Definitions

[0020] The expression "CD3," as used herein, refers to an antigen which is expressed on

T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3- epsilon, CD3-delta, CD3-zeta, and CD3-gamma. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a nonhuman species. Thus, the expression "CD3" means human CD3 unless specified as being from a non-human species, e.g., "mouse CD3," "monkey CD3," etc.

[0021] As used herein, "an antibody that binds CD3" or an "anti-CD3 antibody" (which can be used interchangeably herein) includes antibodies and antigen-binding fragments thereof that specifically recognize a CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The antibodies and antigen-binding fragments of the present disclosure may bind soluble CD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants such as, e.g., monomeric and dimeric CD3 constructs, that lack a transmembrane domain or are otherwise unassociated with a cell membrane.

[0022] As used herein, the expression "cell surface-expressed CD3" means one or more CD3 protein(s) that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes CD3 proteins contained within the context of a functional T cell receptor in the membrane of a cell. The expression "cell surface-expressed CD3" includes CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The expression, "cell surface- expressed CD3" also includes a CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma) that is expressed by itself, without other CD3 chain types, on the surface of a cell. A "cell surface-expressed CD3" can comprise or consist of a CD3 protein expressed on the surface of a cell which normally expresses CD3 protein. Alternatively, "cell surface-expressed CD3" can comprise or consist of CD3 protein expressed on the surface of a cell that normally does not express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0023] As used herein, "tumor associated antigens" or “TAA” are antigens expressed or present on a human tumor cell (e.g., antigens expressed on the cell surface and antigens presented by a major histocompatibility complex such as a human leukocyte antigen). Exemplary TAAs include CD20, EpCam, GD2, and GD3, mesothelin, NY-ESO-1 , Folate Binding Protein (FBP), human epidermal growth factor receptor 2 (HER-2/neu), IL-13 receptor a2, melanoma-associated antigen 1 (MAGE-A1), melanoma-associated antigen 1 (MAGE-A4), EPH receptor A2 (EphA2). carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), survivin, tyrosine related protein 1 (tyrpl), tyrosine related protein 1 (tyrp2), brachyury, 5 alpha reductase, alpha-fetoprotein, AM-1 , APC, April, BAGE, beta-catenin, Bc112, bcr-abl, CA-125, CASP-8/FLICE, cathepsins, CD19, CD21 , CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a, FLK- 1/KDR, folic acid receptor, G250, GAGE-family, gastrin 17, gastrin-releasing hormone, GD2/GD3/GM2, GnRH, GnTV, GP1 , gp100/Pme117, gp-100-in4, gp15, gp75/TRP-1 , hCG, heparanse, HMTV, Hsp70, hTERT, IGFR1 , IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N- ras, KSA, LKLR-FUT, MAGE-family, mammaglobin, MAPI 7, melan-A/MART-1 , mesothelin, MIC A/B, MT-MMPs, mucin, NY-ESO-1 , osteonectin, p15, P170/MDR1 , p53, p97/melanotransferrin, PAI-1 , PDGF, uPA, PRAME, probasin, progenipoientin, PSA, PSM, RAGE-1 , Rb, RCAS1 , SART-1 , SSX-family, STAT3, STn, TAG-72, TGF-alpha, TGF-beta, Thymosin-beta-15, TNF-alpha, TYRP-, TYRP-2, tyrosinase, VEGF, ZAG, p16INK4, glutathione-S-transferase, and combinations thereof. Other non-limiting examples of specific tumor-associated antigens include, e.g., AFP, ALK, BAGE proteins, p-catenin, brc-abl, BRCA1 , BORIS, CA9, carbonic anhydrase IX, caspase-8, CCR5, CD19, CD20, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1 , CYP1 B1 , EGFR, EGFRvlll, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1 , FOLR1 , GAGE proteins (e.g., GAGE-1 , -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1 , -2, -3, -4, -6, and -12), MART-1 , mesothelin, ML-IAP, Muc1 , Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1 , NA17, NY-BR1 , NY- BR62, NY-BR85, NY-ESO1 , 0X40, p15, p53, PAP, PAX3, PAX5, PCTA-1 , PLAC1 , PRLR, PRAME, PSMA (F0LH1), RAGE proteins, Ras, RGS5, Rho, SART-1 , SART-3, Steap-1 (STEAP1), Steap-2 (STEAP2), survivin, TAG-72, TGF-p, TMPRSS2, Tn, TRP-1 , TRP-2, tyrosinase, and uroplakin-3.

[0024] As used herein, "an antibody that binds TAA" or an "anti-TAA antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize antigens on tumors or tumor-associated antigens.

[0025] In the context of bispecific antibodies of the present disclosure wherein one arm of the antibody binds CD3 and the other arm binds a target antigen, the target antigen can be a cancer-associated antigen, e.g., a tumor associated antigen. As used herein, the terms “bispecific antibody,” “bsAb,” and BsAb” may be used interchangeably. Without wishing to be bound by theory, an antigen-binding protein that binds to CD3 and to a TAA such as CD20 (e.g., a CD3 x CD20 bispecific antibody) can provide substantial benefits in the context of cancer treatment by targeting T cells (e.g., gamma delta T cells) to cells expressing the TAA (e.g., a tumor). When administered in combination with a population of T cells comprising gamma delta T cells, those cells then will preferentially target the TAA- expressing tumor cells, resulting in enhanced cytotoxicity as compared to treatment with either the antigen-binding protein or the T cells alone.

[0026] According to certain exemplary embodiments, the present disclosure includes bispecific antigen-binding molecules that specifically bind CD3 and CD20. Such molecules may be referred to herein as, e.g., "anti-CD3/anti-CD20," or "anti-CD3xCD20" or "CD3xCD20" bispecific molecules, or other similar terminology.

[0027] The term "CD20," as used herein, refers to the human CD20 protein unless specified as being from a non-human species (e.g., "mouse CD20," "monkey CD20," etc.). The human CD20 protein has the amino acid sequence shown in SEQ ID NO:1369.

[0028] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, e.g., bispecific antibodies.

[0029] The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., TAA or CD3). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V_H) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1 , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V ) and a light chain constant region. The light chain constant region comprises one domain (Ci_1 )- The V and V regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the disclosure, the FRs of the anti-TAA antibody or anti-CD3 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0030] The term "antibody", as used herein, also includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0031] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody {e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigenbinding fragment," as used herein.

[0032] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V domain associated with a V_L domain, the V and V domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V -V , V -V or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0033] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) V -CH1 ; (ii) V - C_H2; (iii) V_H-C_H3; (iv) V_H-C_H1 -C_H2; (v) VH-C_H1 -C_H2-CH3; (vi) V_H-C_H2-C_H3; (vii) V_H-C_L; (viii) V_L- C_H1 ; (ix) V_L-CH2; (X) V_L-C_H3; (xi) V_L-C_H1 -C_H2; (xii) VL-C_H1 -C_H2-CH3; (xiii) V_L-C_H2-C_H3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non- covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0034] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0035] The antibodies of the present disclosure may function through complementdependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to lysis of antigen-expressing cells by an antibody of the disclosure in the presence of complement. "Antibody-dependent cell- mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Patent Nos 5,500,362 and 5,821 ,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.

[0036] In certain embodiments, the antibodies (e.g., CD3 bispecific antibodies) are human antibodies. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0037] The antibodies may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) NucL Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V and V regions of the recombinant antibodies are sequences that, while derived from and related to human germline V and V sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0038] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via interchain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

[0039] The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human lgG1 hinge. The instant disclosure encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[0040] The antibodies may be isolated antibodies. An "isolated antibody," as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for purposes of the present disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[0041] The antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The some embodiments, the antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V_H and/or V domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (/.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).

Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0042] Some embodiments of the disclosure also include anti-CD3 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-TAA or anti-TAA/anti-CD3 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences. [0043] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0044] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0045] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331 , herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0046] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1 . Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1 . FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.

Germline Mutations

[0047] The antibodies disclosed herein (e.g., CD3 bispecific antibodies) comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. [0048] The present disclosure also includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"), and having desired binding properties to an CD3 antigen or tumor associated antigens, for example, weak or no detectable binding of anti-CD3 antibodies to CD3.

[0049] Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be tested for one or more desired properties such as, improved binding specificity, weak or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner given the guidance of the present disclosure are encompassed within the present disclosure.

[0050] The present disclosure also includes anti-CD3 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-CD3 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences. The antibodies and bispecific antigen-binding molecules of the present disclosure comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived, while maintaining or improving the desired binding to TAA or CD3, for example, weak or no detectable binding of anti-CD3 antibodies to CD3 antigen. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein, i.e. the amino acid substitution maintains or improves the desired binding affinity in the case of anti-TAA and/or anti-CD3 binding molecules, for example, weak to no detectable binding or anti-CD3 antibodies to CD3 antigen. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0051] The present disclosure also includes antigen-binding molecules comprising an antigen-binding domain with an HCVR and/or CDR amino acid sequence that is substantially identical to any of the HCVR and/or CDR amino acid sequences disclosed herein, while maintaining or improving the desired property to TAA and/or CD3 antigen. The term "substantial identity" or "substantially identical," when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331.

[0052] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1 . Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1 . FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0053] Once obtained, antigen-binding domains that contain one or more germline mutations are tested for decreased binding affinity utilizing one or more in vitro assays. Although antibodies that recognize a particular antigen are typically screened for their purpose by testing for high (/.e. strong) binding affinity to the antigen, the antibodies of the present disclosure exhibit weak binding or no detectable binding. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are also encompassed within the present disclosure and are found to be advantageous as cancer therapies.

[0054] Unexpected benefits, for example, improved pharmacokinetic properties and low toxicity to the patient may be realized from the methods described herein.

Binding Properties of the Antibodies

[0055] As used herein, the term "binding" in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to either, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.

[0056] For instance, binding affinity typically corresponds to a K_D value of about 10^-6 M or less, such as about 10^-7 M or less, such as about 10^-8 M or less, such as about 10^-9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody, Ig, antibodybinding fragment, or Fc-containing protein as the analyte (or antiligand). Cell-based binding strategies, such as fluorescent-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201 (2):223-31 ; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1 -2):68-77).

[0057] Accordingly, the antibody or antigen-binding protein of the disclosure binds to the predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to a K_D value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the present disclosure, the affinity of an antibody corresponding to a K_D value that is equal to or less than ten-fold lower than a non-specific antigen may be considered non-detectable binding, however such an antibody may be paired with a second antigen binding arm for the production of a bispecific antibody of the disclosure.

[0058] The term "K_D" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. There is an inverse relationship between K_D and binding affinity, therefore the smaller the K_D value, the higher, i.e. stronger, the affinity. Thus, the terms “higher affinity” or “stronger affinity” relate to a greater ability to form an interaction and therefore a smaller K_D value, and conversely the terms “lower affinity” or “weaker affinity” relate to a lesser ability to form an interaction and therefore a larger K_D value. In some circumstances, a higher binding affinity (or K_D) of a particular molecule (e.g. antibody) to its interactive partner molecule (e.g. antigen X) compared to the binding affinity of the molecule (e.g. antibody) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding affinity ratio determined by dividing the larger K_D value (lower, or weaker, affinity) by the smaller K_D (higher, or stronger, affinity), for example expressed as 5- fold or 10-fold greater binding affinity, as the case may be.

[0059] The term "k_d" (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibodybinding fragment. Said value is also referred to as the k_Oft value.

[0060] The term "k_a" (M-1 x sec-1 or 1/M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0061] The term "K_A" (M-1 or 1/M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing the k_a by the k_d.

[0062] The term “EC50” or “EC50” refers to the half maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between the baseline and maximum after a specified exposure time. The EC50 essentially represents the concentration of an antibody where 50% of its maximal effect is observed. In certain embodiments, the EC50 value equals the concentration of an antibody of the disclosure that gives half-maximal binding to cells expressing CD3 or tumor associated antigen, as determined by e.g. a FACS binding assay. Thus, reduced or weaker binding is observed with an increased EC50, or half maximal effective concentration value.

[0063] In some embodiments, decreased binding can be defined as an increased EC50 antibody concentration which enables binding to the half-maximal amount of target cells. [0064] In some embodiments, the EC50 value represents the concentration of an antibody of the disclosure that elicits half-maximal depletion of target cells by T cell cytotoxic activity. Thus, increased cytotoxic activity {e.g. T cell-mediated basophils killing) is observed with a decreased EC₅o, or half maximal effective concentration value.

Bispecific Antigen-Binding Molecules

[0065] The antibodies of the present disclosure to be administered with the population of T cells may be monospecific, bi-specific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., T utt et aL, 1991 , J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-CD3 bispecific antibodies can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked {e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second or additional binding specificity.

[0066] Use of the expression "anti-CD3 antibody" herein is intended to include both monospecific anti-CD3 or bispecific antibodies comprising a CD3-binding arm and a binding arm to tumor associated antigens (TAAs). Thus, the present disclosure includes bispecific antibodies wherein one arm of an immunoglobulin binds human CD3, and the other arm of the immunoglobulin is specific for human tumor associated antigen. The CD3-binding arm can comprise any of the HCVR/LCVR or CDR amino acid sequences known in the art.

[0067] In certain embodiments, the CD3-binding arm binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3-binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm binds weakly to human CD3 and induces ablation of mast cells and/or basophils in the context of a bispecific or multispecific antibody. In other embodiments, the CD3-binding arm binds or is associated weakly with human CD3, yet the binding interaction is not detectable by in vitro assays known in the art.

[0068] According to certain exemplary embodiments, the present disclosure includes bispecific antigen-binding molecules that specifically bind CD3 and any tumor associated antigen. Such molecules may be referred to herein as, e.g., "anti-CD3/anti-TAA," or "anti- CD3xTAA," or “anti-TAA/anti-CD3,” or “anti-TAAxCD3,” or "CD3xTAA" bispecific molecules, or “TAAxCD3” bispecific molecules, or other similar terminology e.g., anti-TAA x anti-CD3). [0069] The aforementioned bispecific antigen-binding molecules that specifically bind CD3 and a TAA may comprise an anti-CD3 antigen-binding molecule which binds to CD3 with a weak binding affinity such as exhibiting a K_D of greater than about 40 nM, as measured by an in vitro affinity binding assay. [0070] As used herein, the expression "antigen-binding molecule" means a protein, polypeptide or molecular complex comprising or consisting of at least one complementarity determining region (CDR) that alone, or in combination with one or more additional CDRs and/or framework regions (FRs), specifically binds to a particular antigen. In certain embodiments, an antigen-binding molecule is an antibody or a fragment of an antibody, as those terms are defined elsewhere herein.

[0071] As used herein, the expression "bispecific antigen-binding molecule" means a protein, polypeptide or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the bispecific antigen-binding molecule comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen (e.g. TAAs). In the context of the present disclosure, the first antigen-binding domain specifically binds a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds a second, distinct antigen (e.g., TAA).

[0072] In certain exemplary embodiments of the present disclosure, the bispecific antigenbinding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule comprising a first and a second antigen-binding domain (e.g., a bispecific antibody), the CDRs of the first antigen-binding domain may be designated with the prefix "A1" and the CDRs of the second antigen-binding domain may be designated with the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1 , A1 -HCDR2, and A1- HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1 , A2-HCDR2, and A2-HCDR3.

[0073] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule of the present disclosure. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigenbinding molecule. As used herein, a "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgG 1 , lgG2, lgG3, and lgG4, as well as any allotype within each isotype group. [0074] Bispecific antigen-binding molecules will typically comprise two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., lgG1/lgG1 , lgG2/lgG2, and lgG4/lgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., lgG1/lgG2, lgG1/lgG4, lgG2/lgG4, etc.

[0075] In certain embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0076] Any bispecific antibody format or technology may be used to make the bispecific antigen-binding molecules of the present disclosure. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked {e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigenbinding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgG1/lgG2, dual acting Fab (DAF)-lgG, and Mab² bispecific formats {see, e.g., Klein et al. 2012, mAbs 4:6, 1 -11 , and references cited therein, for a review of the foregoing formats).

[0077] In the context of bispecific antigen-binding molecules of the present disclosure, the multimerizing domains, e.g., Fc domains, may comprise one or more amino acid changes {e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain. For example, the disclosure includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction {e.g., enhanced or diminished) between Fc and FcRn. In some embodiments, the bispecific antigen-binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment {e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 {e.g., E or Q); 250 and 428 {e.g., L or F); 252 {e.g., U /F/W or T), 254 {e.g., S or T), and 256 {e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 {e.g., L/R/S/P/Q or K) and/or 434 {e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., F or P), and 434. In some embodiments, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).

[0078] In some embodiments of the present disclosure includes bispecific antigen-binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In some embodiments, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by Ell numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). See, for example, US Patent No. 8,586,713. Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of lgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of lgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of lgG4 antibodies.

[0079] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise part or all of a CH2 sequence derived from a human IgG 1 , human lgG2 or human lgG4 CH2 region, and part or all of a CH3 sequence derived from a human IgG 1 , human lgG2 or human lgG4. A chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" sequence, derived from a human IgG 1 , a human lgG2 or a human lgG4 hinge region, combined with a "lower hinge" sequence, derived from a human IgG 1 , a human lgG2 or a human lgG4 hinge region. A particular example of a chimeric Fc domain that can be included in any of the antigenbinding molecules set forth herein comprises, from N- to C-terminus: [lgG4 CH1 ] - [lgG4 upper hinge] - [lgG2 lower hinge] - [lgG4 CH2] - [lgG4 CH3]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [lgG1 CH1 ] - [lgG1 upper hinge] - [lgG2 lower hinge] - [lgG4 CH2] - [IgG 1 CH3]. These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present disclosure are described in US Publication 2014/0243504, published August 28, 2014, which is herein incorporated in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, which in turn affects Fc effector function.

Sequence Variants

[0080] The antibodies and bispecific antigen-binding molecules of the present disclosure to be administered with the population of T cells may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The antigenbinding molecules of the present disclosure may comprise antigen-binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V_H and/or V domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (/.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigenbinding domains obtained in this general manner are encompassed within the present disclosure.

[0081] The present disclosure also includes antigen-binding molecules wherein one or both antigen-binding domains comprise variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes antigen-binding molecules comprising an antigenbinding domain having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 loglikelihood matrix.

[0082] The present disclosure also includes antigen-binding molecules comprising an antigen-binding domain with an HCVR, LCVR, and/or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. The term "substantial identity" or "substantially identical," when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331 , herein incorporated by reference.

[0083] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1 . Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1 . FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference. pH-Dependent Binding

[0084] The present disclosure includes anti-CD3 antibodies, and anti-CD3/anti-TAA bispecific antigen-binding molecules, with pH-dependent binding characteristics, to be administered with the population of T cells. For example, an anti-TAA antibody of the present disclosure may exhibit reduced binding to a TAA at acidic pH as compared to neutral pH. Alternatively, anti-TAA antibodies of the disclosure may exhibit enhanced binding to a TAA at acidic pH as compared to neutral pH. The expression "acidic pH" includes pH values less than about 6.2, e.g., about 6.0, 5.95, 5,9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1 , 5.05, 5.0, or less. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1 , 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0085] In certain instances, "reduced binding ... at acidic pH as compared to neutral pH" is expressed in terms of a ratio of the K_D value of the antibody binding to its antigen at acidic pH to the K_D value of the antibody binding to its antigen at neutral pH (or vice versa). For example, an antibody or antigen-binding fragment thereof may be regarded as exhibiting "reduced binding to TAA at acidic pH as compared to neutral pH" for purposes of the present disclosure if the antibody or antigen-binding fragment thereof exhibits an acidic/neutral K_D ratio of about 3.0 or greater. In certain exemplary embodiments, the acidic/neutral K_D ratio for an antibody or antigen-binding fragment of the present disclosure can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.

[0086] Antibodies with pH-dependent binding characteristics may be obtained, e.g., by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH as compared to neutral pH. Additionally, modifications of the antigenbinding domain at the amino acid level may yield antibodies with pH-dependent characteristics. For example, by substituting one or more amino acids of an antigen-binding domain (e.g., within a CDR) with a histidine residue, an antibody with reduced antigenbinding at acidic pH relative to neutral pH may be obtained.

Antibodies Comprising Fc Variants

[0087] According to certain embodiments of the present disclosure, anti-CD3/TAA bispecific antigen-binding molecules, are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present disclosure includes antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 and/or 308 (e.g., F or P), and 434. In some embodiments, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); a 307 modification and/or a 308 modification (e.g., 308F or 308P). [0088] For example, the present disclosure includes anti-CD3/TAA bispecific antigenbinding molecules, comprising an Fc domain comprising one or more pairs or groups of mutations. All possible combinations of Fc domain mutations, and other mutations within the antibody variable domains are contemplated within the scope of the present disclosure. Biological Characteristics of the Antibodies and Bispecific Antigen-Binding Molecules [0089] The present disclosure includes antibodies and antigen-binding fragments thereof that bind human CD3 and induce T cell proliferation, in combination with administering a population of T cells. For example, the present disclosure includes anti-CD3 antibodies that induce human T cell proliferation with an EC₅o value of less than about 1 pM, as measured by an in vitro cell proliferation assay (e.g., assessing the proliferation of Jurkat cells or human PBMCs in the presence of anti-CD3 antibodies), or a substantially similar assay. [0090] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind human CD3 and induce T cell-mediated killing of tumor cells. For example, the present disclosure includes anti-CD3 antibodies that induce T cell-mediated killing of tumor cells with an EC₅o of less than about 2.3 pM, as measured in an in vitro T cell- mediated tumor cell killing assay (e.g., assessing the extent of U937 tumor cell killing by human PBMCs in the presence of anti-CD3 antibodies), or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure induce T cell-mediated tumor cell killing (e.g., PBMC-mediated killing of U937 cells) with an EC₅O value of less than about 2.3 pM, less than about 2.2 pM, less than about 2.1 pM, less than about 2.0 pM, less than about 1 .8 pM, less than about 1 .6 pM, less than about 1 .4 pM, less than about 1 .2 pM, less than about 1 .0 pM, less than about 0.8 pM, less than about 0.6 pM, or less than about 0.5 pM, as measured by an in vitro T cell-mediated tumor cell killing assay.

[0091] The present disclosure includes antibodies and antigen-binding fragments thereof that bind human CD3 with high affinity. The present disclosure also includes antibodies and antigen-binding fragments thereof that bind human CD3 with medium or low affinity, depending on the therapeutic context and particular targeting properties that are desired. For example, in the context of a bispecific antigen-binding molecule, wherein one arm binds CD3 and another arm binds a tumor associated antigen (e.g., CD20), it may be desirable for the target antigen-binding arm to bind the target antigen with high affinity while the anti-CD3 arm binds CD3 with only moderate or low affinity. In this manner, preferential targeting of the antigen-binding molecule to cells expressing the target antigen may be achieved while avoiding general/untargeted CD3 binding and the consequent adverse side effects associated therewith.

[0092] According to certain embodiments, the present disclosure includes antibodies and antigen-binding fragments of antibodies that bind human CD3 (e.g., at 25^eC) with a K_D of less than about 15 nM as measured by surface plasmon resonance. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure bind CD3 with a K_D of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 180 pM, less than about 160 pM, less than about 140 pM, less than about 120 pM, less than about 100 pM, less than about 80 pM, less than about 60 pM, less than about 40 pM, less than about 20 pM, or less than about 10 pM, as measured by surface plasmon resonance (e.g., mAb- capture or antigen-capture format), or a substantially similar assay.

[0093] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind CD3 with a dissociative half-life (t¹/z) of greater than about 10 minutes as measured by surface plasmon resonance at 25^eC or 37^eC. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure bind CD3 with a t¹/z of greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes, as measured by surface plasmon resonance at 25^eC or 37^eC (e.g., mAb-capture or antigen-capture format).

[0094] The present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies) which are capable of simultaneously binding to human CD3 and human CD20. According to certain embodiments, the bispecific antigen-binding molecules of the disclosure specifically interact with cells that express CD3 and/or CD20. The extent to which a bispecific antigen-binding molecule binds cells that express CD3 and/or CD20 can be assessed by fluorescence activated cell sorting (FACS). For example, the present disclosure includes bispecific antigen-binding molecules which specifically bind human T-cell lines which express CD3 but not CD20 (e.g., Jurkat), human B-cell lines which express CD20 but not CD3 (e.g., Raji), and/or primate T-cells (e.g., cynomolgus peripheral blood mononuclear cells [PBMCs]). The present disclosure includes bispecific antigen-binding molecules which bind any of the aforementioned cells and cell lines with an EC50 value of from about 9.0x10^-6 to about 2.0x10^-9, or less, as determined using a FACS assay.

[0095] The present disclosure also includes anti-CD3/anti-CD20 bispecific antigen-binding molecules which bind to CD3-expressing human T-cells (e.g., Jurkat) with an EC50 value of between 1.0 pM and 1000 nM. In certain embodiments, the anti-CD3/anti-CD20 bispecific antigen-binding molecules bind to CD3-expressing human T-cells with an EC50 value of between 1 nM and 60 nM. For example, the present disclosure includes anti-CD3/anti-CD20 bispecific antigen-binding molecules which bind to CD3-expressing human T-cells (e.g., Jurkat) with an EC50 value of about 1 pM. about 10 pM, about 100 pM, about 500 pM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1000 nM, or more.

[0096] The present disclosure also includes anti-CD3/anti-CD20 bispecific antigen-binding molecules which exhibit one or more characteristics selected from the group consisting of: (a) inducing PBMC proliferation in vitro; (b) activating T-cells, inducing IFN-gamma release and CD25 up-regulation in human whole blood; (c) inducing T-cell mediated cytotoxicity on anti-CD20-resistant cell lines; (d) inducing cytotoxicity to human B-cells (e.g., Raji); (e) depleting B-cells (e.g., CD19+ B-cells) in mice reconstituted with human immune cells; and (f) decreasing B-cell tumor volume (e.g., Raji tumor volume) in mouse xenografts.

[0097] The present disclosure includes anti-CD3/anti-CD20 bispecific antigen-binding molecules which are capable of depleting B cells in a subject. For example, according to certain embodiments, anti-CD3/anti-CD20 bispecific antigen-binding molecules are provided, wherein a single administration of the bispecific antigen-binding molecule to a subject (e.g., at a dose of about 0.1 mg/kg, about 0.08 mg/kg, about 0.06 mg/kg about 0.04 mg/kg, about 0.04 mg/kg, about 0.02 mg/kg, about 0.01 mg/kg, or less) causes a reduction in the number of B cells in the subject (e.g., in a blood sample taken from the subject) below detectable levels. In certain embodiments, a single administration of the anti-CD3/anti-CD20 bispecific antigen-binding molecule at a dose of about 0.1 mg/kg causes a reduction in the number of B cells in the subject below detectable levels by about day 7, about day 6, about day 5, about day 4, about day 3, about day 2, or about day 1 after administration of the bispecific antigen-binding molecule to the subject. According to certain embodiments, a single administration of an anti-CD3/anti-CD20 bispecific antigen-binding molecule of the disclosure, at a dose of about 0.01 mg/kg, causes the number of B-cells to remain below detectable levels until at least about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days or more, following the administration. As used herein, the expression "below detectable levels" means that no B cells can be directly or indirectly detected in a blood sample drawn from a subject using standard B-cell detection assays, e.g., a FACS assay for B-cell markers.

[0098] In related embodiments, an anti-CD3/anti-CD20 bispecific antigen-binding molecule is provided, wherein the number of B-cells per microliter of blood drawn from a subject at about day 1 through about day 28 after administration of a single dose of about 0.01 mg/kg of the antigen-binding molecule to the subject is less than 25% the number of B-cells per microliter of blood drawn from the subject prior to the administration. In certain other embodiments, an anti-CD3/anti-CD20 bispecific antigen-binding molecule is provided, wherein the number of B-cells per microliter of blood drawn from a subject at about day 1 through about day 56 after administration of a single dose of about 0.01 mg/kg of the antigen-binding molecule to the subject is less than 50% the number of B-cells per microliter of blood drawn from the subject prior to the administration.

[0099] The present disclosure also provides anti-CD3/anti-TAA bispecific antigen-binding molecules that, when administered to a subject, cause no more than a transient decrease in T cells. For example, anti-CD3/anti-TAA bispecific antigen-binding molecules are provided that, when administered to a subject at a dose of about 0.01 mg/kg cause the number of T cells to decline at day 1 following administration, but wherein the number of T cells per microliter of blood rebounds at timepoints thereafter (e.g., by about day 2, day 7, day 14, day 28, day 42, day 56 or later following the administration). For example the present disclosure provides an anti-CD3/anti-TAA bispecific antigen-binding molecule, wherein the number of T cells per microliter of blood drawn from the subject at about day 14 through about day 56 after administration of the antigen binding molecule to the subject at a dose of about 0.01 mg/kg is equal to or greater than the number of T cells per microliter of blood drawn from the subject prior to administration of the bispecific antigen-binding molecule.

[00100] The present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies) which are capable of simultaneously binding to human CD3 and a human TAA. The extent to which a bispecific antigen-binding molecule binds cells that express CD3 and/or TAA can be assessed by fluorescence activated cell sorting (FACS). [00101] For example, the present disclosure includes antibodies, antigen-binding fragments, and bispecific antibodies thereof which specifically bind human T-cell lines which express CD3 but do not express TAA and/or TAA-expressing cells.

[00102] The present disclosure includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind human CD3 and induce T cell activation.

[00103] The present disclosure includes anti-CD3/anti-TAA bispecific antigen-binding molecules which are capable of depleting TAA-expressing cells in a subject.

Epitope Mapping and Related Technologies

[00104] The epitope on CD3 and/or TAA to which the antigen-binding molecules of the present disclosure bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a CD3 or TAA protein. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of CD3 or a TAA. The antibodies o may interact with amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma), or may interact with amino acids on two or more different CD3 chains. The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen. [00105] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, e.g., routine cross-blocking assay such as that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY), alanine scanning mutational analysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding domain of an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium- labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267 (2):252-259; Engen and Smith (2001) Anal. Chem. 73256A-265A. X-ray crystallography of the antigen/antibody complex may also be used for epitope mapping purposes.

[00106] The present disclosure also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3 with low or detectable binding affinity, and a second antigen binding domain that specifically binds a TAA, wherein the first antigen-binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen-binding domains described herein, and/or wherein the second antigen-binding domain binds to the same epitope on TAA as any of the specific exemplary TAA specific antigen-binding domains described herein.

[00107] Likewise, the present disclosure also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3, and a second antigen binding domain that specifically binds human TAAs wherein the first antigen-binding domain competes for binding to CD3 with any of the specific exemplary CD3-specific antigen-binding domains described herein, and/or wherein the second antigen-binding domain competes for binding to a TAA with any of the specific exemplary TAA-specific antigen-binding domains described herein.

[00108] One can easily determine whether a particular antigen-binding molecule (e.g., antibody) or antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule of the present disclosure by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on TAA (or CD3) as a reference bispecific antigen-binding molecule of the present disclosure, the reference bispecific molecule is first allowed to bind to TAA protein (or CD3 protein). Next, the ability of a test antibody to bind to the TAA (or CD3) molecule is assessed. If the test antibody is able to bind to TAA (or CD3) following saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of TAA (or CD3) than the reference bispecific antigenbinding molecule. On the other hand, if the test antibody is not able to bind to the TAA (or CD3) molecule following saturation binding with the reference bispecific antigen-binding molecule, then the test antibody may bind to the same epitope of TAA (or CD3) as the epitope bound by the reference bispecific antigen-binding molecule of the disclosure.

Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference bispecific antigen-binding molecule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with certain embodiments of the present disclosure, two antigen-binding proteins bind to the same (or overlapping) epitope if, e.g., a 1 -, 5-, 10-, 20- or 100-fold excess of one antigenbinding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et aL, Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. Two antigenbinding proteins are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other.

[00109] To determine if an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antigen-binding molecule is allowed to bind to an TAA protein (or CD3 protein) under saturating conditions followed by assessment of binding of the test antibody to the TAA (or CD3) molecule. In a second orientation, the test antibody is allowed to bind to an TAA (or CD3) molecule under saturating conditions followed by assessment of binding of the reference antigen-binding molecule to the TAA (or CD3) molecule. If, in both orientations, only the first (saturating) antigen-binding molecule is capable of binding to the TAA (or CD3) molecule, then it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to TAA (or CD3). As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Preparation of Antigen-Binding Domains and Construction of Bispecific Molecules [00110] Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigenbinding domains, specific for two different antigens (e.g., CD3 and TAA), can be appropriately arranged relative to one another to produce a bispecific antigen-binding molecule of the present disclosure using routine methods. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigenbinding molecules of the disclosure are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and/or light chains of the bispecific antigen-binding molecules of the present disclosure can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generating technology), high affinity chimeric antibodies to a particular antigen (e.g., CD3 or TAA ) are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the bispecific antigen-binding molecules of the present disclosure.

[00111] Genetically engineered animals may be used to make human bispecific antigenbinding molecules. For example, a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US 2011/0195454). Fully human refers to an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain thereof. In some instances, the fully human sequence is derived from a protein endogenous to a human. In other instances, the fully human protein or protein sequence comprises a chimeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes in the junctions of component sequences, e.g. compared to any wild-type human immunoglobulin regions or domains.

Bioequivalents

[00112] The present disclosure encompasses antigen-binding molecules having amino acid sequences that vary from those of the exemplary molecules disclosed herein but that retain the ability to bind CD3 and/or TAA. Such variant molecules may comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen-binding molecules.

[00113] The present disclosure includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antigen-binding proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[00114] In some embodiments, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[00115] In some embodiments, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching. [00116] In some embodiments, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[00117] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in v/vo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.

[00118] Bioequivalent variants of the exemplary bispecific antigen-binding molecules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigenbinding proteins may include variants of the exemplary bispecific antigen-binding molecules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the molecules, e.g., mutations which eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

[00119] According to certain embodiments of the disclosure, antigen-binding molecules are provided which bind to human CD3 but not to CD3 from other species. Also provided are antigen-binding molecules which bind to a human TAA but not to a TAA from other species. The present disclosure also includes antigen-binding molecules that bind to human CD3 and to CD3 from one or more non-human species; and/or antigen-binding molecules that bind to a human TAA and to a TAA from one or more non-human species, e.g., cynomolgus.

[00120] According to certain exemplary embodiments of the disclosure, antigen-binding molecules are provided which bind to human CD3 and/or a human TAA and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus, cynomolgus or chimpanzee CD3 and/or TAA. For example, in a particular exemplary embodiment of the present disclosure bispecific antigen-binding molecules are provided comprising a first antigen-binding domain that binds human CD3, and a second antigen-binding domain that binds human or cynomolgus TAA. Therapeutic Formulation and Administration

[00121] The present disclosure provides pharmaceutical compositions comprising the antigen-binding molecules of the present disclosure, as well as pharmaceutical compositions comprising the population of T cells. Such pharmaceutical compositions may be combined, administered at the same time, or administered sequentially.. The pharmaceutical compositions of the disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[00122] The dose of antigen-binding molecule and population of T cells administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When a bispecific antigen-binding molecule of the present disclosure is used for therapeutic purposes in an adult patient, it may be advantageous to intravenously administer the bispecific antigen-binding molecule of the present disclosure normally at a single dose of about 0.01 to about 50 mg/kg body weight, more preferably about 0.1 to about 25, about 1 to about 25, or about 5 to about 25 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a bispecific antigen-binding molecule may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 , Pharmaceut. Res. 8:1351).

[00123] Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et aL, 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[00124] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[00125] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present disclosure. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

[00126] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In some embodiments, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 ). In some embodiments, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In some embodiments, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[00127] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[00128] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antigen-Binding Molecules

[00129] The present disclosure includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-CD3 antibody or a bispecific antigen-binding molecule that specifically binds CD3 and a target antigen (e.g., a tumor-associated antigen, e.g., CD20), in combination with a population of T cells comprising yb T cells. The therapeutic composition can comprise any of the antibodies or bispecific antigen-binding molecules as disclosed herein and a pharmaceutically acceptable carrier or diluent. The population of T cells comprising yb T cells can be administered prior to, concurrent with, or after administration of an antigen-binding protein provided herein. In some embodiments, the population of T cells comprising yb T cells is co-administered in a single composition with the antigen-binding protein. In some embodiments, the population of T cells comprising yb T cells is administered in a separate composition as the antigen- binding protein. As used herein, the expression "a subject in need thereof" means a human or non-human animal that exhibits one or more symptoms or indicia of cancer {e.g., a subject expressing a tumor or suffering from any of the cancers mentioned herein below), or who otherwise would benefit from an inhibition or reduction in tumor associated antigen activity, e.g., CD20 activity or a depletion of CD20+ B cells.

[00130] The antibodies and bispecific antigen-binding molecules of the disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulation, activation and/or targeting of an immune response would be beneficial. In particular, the anti-CD3/anti-TAA bispecific antigen-binding molecules of the present disclosure may be used for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by a TAA, e.g., CD20, expression or activity or the proliferation of CD20+ B cells. The mechanism of action by which the therapeutic methods of the disclosure are achieved include killing of the cells expressing a TAA, e.g., CD20, in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing a TAA, e.g., CD20, which can be inhibited or killed using the bispecific antigen-binding molecules of the disclosure include, for example, tumorigenic B cells. [00131] The antigen-binding molecules of the present disclosure may be used to treat, e.g., primary and/or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the bispecific antigen-binding molecules of the disclosure are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, breast cancer, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer {e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma. According to certain exemplary embodiments, the bispecific antigen-binding molecules of the present disclosure are used to treat a B cell cancer e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], precursor B cell lymphoblastic leukemia/lymphoma, mature B cell neoplasms, B cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicle center lymphoma, marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma). [00132] According to certain embodiments of the present disclosure, the antigenbinding molecules are useful for treating a patient afflicted with a B-cell lymphoma (e.g., NHL) that is resistant to, or incompletely responsive to anti-TAA, e.g., anti-CD20, therapy alone (e.g., resistant to rituximab therapy). According to other related embodiments of the disclosure, methods are provided comprising administering an anti-CD3/anti-TAA, e.g., anti- CD20, bispecific antigen-binding molecule as disclosed herein to a patient who is afflicted with a B-cell lymphoma (e.g., NHL) that is refractory to anti-CD20 therapy (e.g., a patient with a rituximab-refractory tumor or with relapsed or refractory B-cell lymphoma). Analytic/diagnostic methods known in the art, such as tumor scanning, etc., may be used to ascertain whether a patient harbors as tumor that is resistant to, incompletely responsive to, or refractory to anti-TAA, e.g., anti-CD20, therapy alone.

[00133] The present disclosure also includes methods for treating residual cancer in a subject. As used herein, the term "residual cancer" means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy. [00134] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with a TAA, e.g., CD20, expression (e.g., B cell lymphoma) comprising administering one or more of the bispecific antigen-binding molecules described elsewhere herein to a subject after the subject has received anti- AA, e.g., anti- CD20, mono-therapy (e.g., after administration of a pharmaceutical composition comprising an anti-CD20 antibody such as rituximab). For example, the present disclosure includes methods for treating B cell lymphoma comprising administering an anti-CD3/anti-TAA bispecific antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received anti-CD20 mono-therapy (e.g., rituximab treatment or an equivalent treatment thereof). In other aspects, a bispecific antigen-binding molecule of the disclosure (an anti-CD3/anti-TAA bispecific antigen-binding molecule) comprising an lgG4 Fc domain is initially administered to a subject at one or more time points (e.g., to provide robust initial depletion of B cells), followed by administration of an equivalent bispecific antigen-binding molecule comprising a different IgG domain, such as an IgG 1 Fc domain, at subsequent time points.

Combination Therapies and Formulations

[00135] The present disclosure provides methods which comprise administering a pharmaceutical composition comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined with or administered in combination with an antigen-binding molecules of the present disclosure include, for example, gamma delta (yd)-T cells.

[00136] yd-T cells are thymus-derived lymphocytes that differ from ap-T cells in their anatomical distributions and mechanisms of activation and function. They could be found as resident lymphocytes in human tissues or as circulating lymphocytes in peripheral blood. Unlike ap-T cells that function exclusively in adaptive immunity, yd-T cells are innate-like immune cells that recognize malignant cells through their repertoire of activating receptors in a MHC-independent manner, which is similar to natural killer cells. Any known method may be used to expand or activate the population of y5-T cells ex vivo. For example, the yd T cells can be expanded or activated using bisphosphophonates such as nitrogen containing bisphosphonates. Exemplary bisphosphonates include zoledronate (ZOL). In other examples, the yd T cells can be expanded or activated using a DOT (“delta one T cells”) two-step process with selected cytokines including IL-15 in the second step. Other protocols for expanding highly cytolytic yd T cells mainly of the Vd1 variety can use mitogen phytohemagglutinin (PHA) plus IL-7 stimulation or artificial antigen-presenting cells (APCs) expressing costimulatory molecules and CMV-pp65 antigens. In other methods, polyclonal yd T cells expressing various TOR VyVd elements and broad cytotoxic activity against various tumor cells have also been generated in the presence of CD137L-expressing artificial APC and IL-2 plus IL-21 . The most widely used protocol for selectively expanding Vd2T cells relies on ZOL stimulation of PBMCs in the presence of IL-2,100 but similarly efficient Vd2T cell activation in vitro can be achieved with synthetic pAgs such as BrHPP20 and HMBPP. For further discussion on the expansion of yd T, see Kabelitz, D., Serrano, R., Kouakanou, L. et al. Cancer immunotherapy with yd T cells: Many paths ahead of us. Cell Mol Immunol 17, 925-939 (2020). https://doi.org/10.1038/s41423-020-0504-x. For further discussions of expanding or activating yd T cells see also, for example, U.S. Patent Application Publication No. 20050196385A1 and U.S. Patent No. 8012949B2, each of which are herein incorporated by reference in their entirety.

[00137] In some embodiments, the population of T cells comprises >50%, >55%, >60%, >65%, >70%, >75%, >80%, >81%, >82%, >83%, >84%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9%, or 100% yd T cells. In some embodiments, the population of T cells is autologous to said subject. In some embodiments, the population of T cells is allogeneic to said subject. In some embodiments, the population of yd T cells has been modified to express a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor binds a TAA. This TAA can be either the same TAA that the antigen-binding protein binds to, or a different TAA. [00138] The present disclosure further provides methods which comprise administering a pharmaceutical composition comprising any of the exemplary antigenbinding proteins and T cells described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined with or administered in combination with an antigen-binding protein and population of T cells of the present disclosure include, e.g., an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvlll (e.g., an antibody that specifically binds EGFRvlll), a cMET antagonist (e.g., an anti-cMET antibody), an IGF1 R antagonist (e.g., an anti-IGF1 R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-a inhibitor (e.g., an anti-PDGFR-a antibody), a PDGFR-p inhibitor (e.g., an anti- PDGFR-p antibody), a VEGF antagonist (e.g., a VEGF-Trap, see, e.g., US 7,087,411 (also referred to herein as a "VEG F-in hibiting fusion protein"), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody disclosed in US 2011/0027286 such as H1 H685P), a FOLH1 antagonist (e.g., an anti- FOLH1 antibody), a PRLR antagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), etc. Other agents that may be beneficially administered in combination with the antigen-binding molecules of the disclosure include cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL- 1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11 , IL-12, IL-13, IL-17, IL-18, or to their respective receptors. The pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising an anti-CD3/anti-CD20 bispecific antigen-binding molecule as disclosed herein) may also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara- C), cisplatin (e.g., Platinol®-AQ); and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ). [00139] The present disclosure also includes therapeutic combinations comprising any of the antigen-binding proteins and populations of T cells mentioned herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvlll, cMet, IGF1 R, B-raf, PDGFR-a, PDGFR- , FOLH1 , PRLR, STEAP1 , STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies and minimal recognition units). The antigen-binding molecules of the disclosure may also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. The antigen-binding molecules of the disclosure may also be administered as part of a treatment regimen that also includes radiation treatment and/or conventional chemotherapy.

[00140] The additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of an antigen-binding molecule of the present disclosure (for purposes of the present disclosure, such administration regimens are considered the administration of an antigen-binding molecule "in combination with" an additional therapeutically active component).

[00141] The present disclosure includes pharmaceutical compositions in which an antigen-binding protein of the present disclosure is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.

Administration Regimens

[00142] According to certain embodiments of the present disclosure, multiple doses of an antigen-binding protein (e.g., a bispecific antigen-binding protein that specifically binds TAA and CD3) and/or population of T cells comprising yd T cells may be administered to a subject over a defined time course. The methods according to this aspect of the disclosure comprise sequentially administering to a subject in need thereof multiple doses of an antigen-binding molecule and/or populations of T cells comprising yd T cells. As used herein, "sequentially administering" means that each dose is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present disclosure includes methods which comprise sequentially administering to the patient a single initial dose, followed by one or more secondary doses, and optionally followed by one or more tertiary doses.

[00143] The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the antigenbinding molecule and/or population of T cells comprising yd T cells, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of an antigen-binding molecule and/or population of T cells comprising yd T cells contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

[00144] In some embodiments, each secondary and/or tertiary dose of antigen-binding protein is administered 1 to 26 (e.g., 1 , 1/2, 2, 2/2, 3, 3/2, 4, 4/2, 5, 5/2, 6, 6/2, 7, 7/2, 8, 8/2, 9, 9¹/₂, 10, 10/₂, 11 , 11 /₂, 12, 12¹/₂, 13, 13¹/₂, 14, 14¹/₂, 15, 15¹/₂, 16, 16¹/₂, 17, 17¹/₂, 18, 18¹/₂, 19, 19/2, 20, 20¹/₂, 21 , 211/2, 22, 22¹/₂, 23, 23¹/₂, 24, 24¹/₂, 25, 25¹/₂, 26, 26¹/₂, or more) weeks after the immediately preceding dose. In some embodiments, each secondary and/or tertiary dose of population of T cells comprising yd T cells is administered 1 to 26 (e.g., 1 , 1¹/₂, 2, 2¹/₂, 3, 3¹/₂, 4, 4¹/₂, 5, 5¹/₂, 6, 6/2, 7, 7/2, 8, 8/2, 9, 9/2, 10, I O/2, 11 , H /2, 12, 12¹/₂, 13, 13/2, 14, 14/2, 15, 15/2, 16, I 6/2, 17, 17/2, 18, I 8/2, 19, 19/2, 20, 20¹/₂, 21 , 211/a, 22, 22¹/₂, 23, 23/2, 24, 24/2, 25, 25/2, 26, 26/2, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[00145] The methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of an antigen-binding molecule (e.g., an anti-TAA antibody or a bispecific antigen-binding molecule that specifically binds TAA and CD3) and/or population of T cells comprising yd T cells. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[00146] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination. [00147] In some embodiments, the antigen-binding molecule (e.g., a bispecific antigenbinding molecule that specifically binds a TAA and CD3) is administered to a subject as a weight-based dose. A "weight-based dose" (e.g., a dose in mg/kg) is a dose of the antibody or the antigen-binding fragment thereof or the bispecific antigen-binding molecule that will change depending on the subject's weight.

[00148] In some embodiments, an antigen-binding protein (e.g., an antibody or the antigenbinding fragment thereof or a bispecific antigen-binding molecule) is administered to a subject as a fixed dose. A "fixed dose" (e.g., a dose in mg) means that one dose of the antibody or the antigen-binding fragment thereof or the bispecific antigen-binding molecule is used for all subjects regardless of any specific subject-related factors, such as weight. In one particular embodiment, a fixed dose of an antibody or the antigen-binding fragment thereof or a bispecific antigen-binding molecule of the disclosure is based on a predetermined weight or age.

[00149] In general, a suitable dose of an antigen binding protein described herein can be in the range of about 0.001 to about 200.0 milligram per kilogram body weight of the recipient, generally in the range of about 1 to 50 mg per kilogram body weight. For example, the antibody or the antigen-binding fragment thereof or the bispecific antigen-binding molecule can be administered at about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1 .5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg per single dose. Values and ranges intermediate to the recited values are also intended to be part of this disclosure.

[00150] In some embodiments, the antigen binding molecule of the disclosure is administered as a fixed dose of between about 1 mg to about 2500 mg. In some embodiments, the antigen binding molecule of the disclosure is administered as a fixed dose of about 1 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, about 30 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about

525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about

675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about

825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1500 mg, about 2000 mg, or about 2500 mg. Values and ranges intermediate to the recited values are also intended to be part of this disclosure.

EXAMPLES

[00151] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[00152] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

EXAMPLE 1. Generation of Anti-CD3 Antibodies and Characterization Thereof

[00153] Anti-CD3 antibodies are obtained by immunizing a VELOCIMMUNE® mouse (/.e., an engineered mouse comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions) with cells expressing CD3 or with DNA encoding CD3. The antibody immune response is monitored by a CD3-specific immunoassay. When a desired immune response is achieved splenocytes are harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce CD3-specific antibodies. Using this technique several anti-CD3 chimeric antibodies (/.e., antibodies possessing human variable domains and mouse constant domains) are obtained. In addition, several fully human anti-CD3 antibodies are isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in US 2007/0280945A1 .

[00154] Anti-CD3 antibodies are tested for their ability to bind to human T-cells and induce their proliferation. Binding is assessed using Jurkat cells (a CD3+ human T-cell line), while proliferation of Peripheral Blood Mononuclear Cells (PBMC) is assessed using ATP catalyzed quantification (CellTiter Gio®). Anti-CD3 antibody OKT3 acts as a positive control and irrelevant isotype matched antibodies serve as negative controls.

[00155] FACS data is acquired using the following protocol: Cells at 2x10⁵ per well are incubated with serially diluted antibodies for 30 min on ice. Post incubation, cells are washed and secondary antibody is added and incubated for an additional 30 minutes. After incubation, cells are washed, re-suspended in cold PBS containing 1% BSA and analyzed by flow cytometry with viable Jurkat cells gated by side and forward scatters. The EC50S for cell binding titration are determined using Prism software with values calculated using a 4- parameter non-linear regression analysis.

[00156] Proliferation data is acquired using the following protocol: Human PBMC (5x10⁴/ well) are incubated with a 3-fold serial dilution of anti-CD3 and a fixed concentration of a commercial anti-CD28 antibody (200ng/ml) in 96 well plates for 72 h at 37°C. Following incubation, CellTiter Gio® is added and luminescence is measured using a VICTOR X5 multi-label plate reader (PerkinElmer). The EC₅o of cell viability (ATP catalyzed quantification) is calculated using a 4-parameter non-linear regression analysis in GraphPad Prism.

Example 2. Generation of Bispecific Antibodies that Bind CD3 and a TAA

[00157] Bispecific antibodies comprising an anti-CD3-specific binding domain and an anti-TAA-specific binding domain are constructed using standard methodologies wherein a heavy chain and a light chain from an anti-CD3 antibody are combined with a heavy chain from an anti-TAA antibody, e.g., an anti-CD20 antibody. The anti-CD3 antibodies used to construct the bispecific antibodies are obtained by immunizing a Veloclmmune® mouse with cells expressing CD3 or with DNA encoding CD3. The anti-CD20 antibodies used to construct the bispecific antibodies are as set forth in US 7,879,984, the entire contents of which are expressly incorporated herein by reference.

[00158] CD20 x CD3 bispecific antibodies and control constructs are tested via FACS for their ability to bind to Jurkat (CD3+, CD20 - human T-cell line), Raji (CD3-, CD20+ Human B-cell line), or cynomolgus PBMCs ("mkT cells").

[00159] FACS data is acquired using the following protocol: Cells at 2x10⁵ per well are incubated with serially diluted antibodies for 30 min on ice. Post incubation, cells are washed and appropriate secondary (Jurkat, RAJI cells) or cocktail of secondary antibodies (for cyno PBMC) is added and incubated for an additional 30 minutes. After incubation, cells are washed, re-suspended in cold PBS containing 1% BSA and analyzed by flow cytometry on a BD FACS Canto II. Jurkat and Raji cells are gated by side and forward scatters, while cynomolgus T cells are also gated in a CD2+CD4+ population. The EC₅oS for cell binding titration are determined using Prism software with values calculated using a 4-parameter non-linear regression analysis.

Example 3. Generation of Bispecific Antibodies that Bind CD3 and a TAA

[00160] Bispecific antibodies comprising i) an anti-CD3-specific binding domain and ii) an anti-CD20-specific binding domain or an anti-PRLR-specific binding domain were generated and utilized in a cytotoxicity assay in combination with ex vivo expanded Vd2 yd T cells. In short, effector cells were prepared by expanding yd T cells with zoledronate and IL- 2, and independently expanding op T cells using CD3/CD28 beads and IL-2. In this 24 hour assay, for the yd T cells, the effector cell:T cell ratio was 1 :1 and for the op T cells, the effector cell:T cell ratio was 5:1 . Primary B cells, Daudi cells, Raji cells, and SKBR cells were tested (FIG. 1). In this assay, ex vivo expanded Vd2 yd T cells induced strong target cell killing in combination with the CD3 x TAA bispecific antibody. Moreover, these Vd2 yd T cells showed similar activity as op T cells in CD3 bispecific-mediated target cell killing.

Example 4. Cytotoxic effects of anti-TAA x CD3 bispecific antibodies

Expansion and preparation of Effector T cells for killing assays.

[00161] In brief, human ap⁺T cells were generated by incubating healthy donor peripheral blood mononuclear cells (PBMCs) with O-CD3/CD28 Dynabeads (1 :1) in media containing recombinant human IL-2 (30 lU/mL). Dynabeads were removed at day 7 of culture and ap⁺T cells were subsequently harvested at day 9 of expansion for use in killing assays. Human Vd1 ⁺ and Vd2⁺ yd T cells were generated by incubating healthy donor peripheral blood mononuclear cells (PBMCs) with plate-bound agonistic a-Vd1 or a-Vd2 antibodies in media containing recombinant human IL-2 (100 lU/mL). At day 7 of cultures, cell cultures were transferred to new plates not containing agonistic a-Vd antibodies and continued to culture in media with IL-2. Alternatively, Vd2 were generated by incubating healthy donor PBMCs in media containing Zoledronate (5 pM) with recombinant human IL-2 (100 lU/mL). Between day 11 and day 14 of culture, residual ap ⁺ T cells were depleted by negative magnetic bead selection and Vd1 ⁺ or Vd2⁺yd T cells were harvested for use in killing assays. Throughout expansion, cell cultures were maintained between 0.5-1 x 10⁶ live cells/mL and final T cell purity was assessed by flow cytometry using viability staining and detection antibodies for hCD45, hCD3, hTCR op , hTCR yd, hVd1 , and hVd2.

Assessment of tumor cell killing by yd T cells when combined with CD3 x TAA Bispecific Antibodies

[00162] T umor cell killing activity was assessed by combining target tumor cell lines with effector T cells at various effector:target ratios and CD3 x tumor associated antigen (TAA) Bispecific Antibodies (BsAb). Target cell viability was assessed by target viability staining with Near-IR dye via flow cytometry (FIG. 2 and FIG. 3), live-cell imaging of target cell lines constitutively expressing green fluorescent protein (GFP) using an Incucyte live cell imager (FIG. 4), or target cell counts in target cell lines constitutively expressing luciferase and readout of luminescence after substrate addition using an Envision plate reader (FIG. 5). Specifics for the data in FIG. 2, FIG. 3, FIG. 4, and FIG. 5 are as follows: • FIG. 2: Effector T cells (ap+, Vd1 +, or V62+ T cells) were incubated at a 3:1 ratio with target cells (Raji) with a serial dilution of anti-CD20 x CD3 or non-targeting isotype control Bispecific antibodies (BsAb). After 24 hours, target cell viability was determined by flow cytometry.

• FIG. 3: Effector T cells (ap+, Vd1 +, or Vd2+ T cells) were incubated at a 3:1 ratio with target cells (22RV1 ) with a serial dilution of anti-STEAP2 x CD3 or non-targeting isotype control Bispecific antibodies (BsAb). After 48 hours, target cell viability was determined by flow cytometry.

• FIG. 4: Effector T cells (ap+, Vd1+, or Vd2+ T cells) were incubated at a 5:1 ratio with target cells (C4-2 - GFP+) with anti-PSMA x CD3 or non-targeting isotype control Bispecific antibodies (BsAb) at 1 pg/mL. Target cell counts were determined using live cell imaging (Incucyte) of GFP+ target cells over time.

• FIG. 5: Effector T cells (ap+ or V52+ T cells) were incubated at various ratios with target cells (OVCAR3-Luc) with a serial dilution of anti-MUC16 x CD3 or nontargeting isotype control Bispecific antibodies (BsAb). After 24 hours, target cell counts were measured by relative light units (RLU) after addition of luciferase substrate.

[00163] Both Vd1 and Vd2 yd T cells mediated effective Raji cell killing when combined with an anti-CD20 x CD3 bispecific antibody (BsAb) (FIG. 2), effective 22RV1 cell killing when combined with an anti-STEAP2 x CD3 BsAb (FIG. 3), and robust C4-2 cell killing when combined with an anti-PSMA x CD3 BsAb (FIG. 4). In addition, Vd2 yd T cells also mediated effective killing of OVCAR3 cells when combined with an anti-MUC16 x CD3 BsAb (FIG. 5). Overall, both Vd1 and Vd2 yd T cells exhibited robust and comparable target cell killing as ap⁺T cells when combined with anti-CD3 x TAA bispecific antibodies.

Claims

Claims What is claimed is:

1 . A method for treating a cancer in a subject, said method comprising administering to the subject: a) an antigen-binding protein, wherein said antigen-binding protein binds to CD3 and to a tumor-associated antigen; and b) a population of T cells comprising yd T cells.

2. The method of claim 1 , wherein said population of yd T cells is an ex vivo expanded population of yd T-cells.

3. The method of claim 2, wherein said population of yd T cells is expanded with an aminobisphosphonate.

4. The method of claim 3, wherein said aminobisphosphonate is zoledronate.

5. The method of any one of claims 3 or 4, wherein said aminobisphosphonate is combined with IL-2 to induce expansion.

6. The method of claim 2, wherein said population of yd T cells is expanded with one or more antibodies.

7. The method of any one of claims 1 -6, wherein said antigen-binding protein is an antibody or antigen-binding fragment thereof.

8. The method of claim 7, wherein said antibody is a multispecific antibody.

9. The method of claim 8, wherein said antibody is a bispecific antibody.

10. The method of claim 9, wherein said tumor-associated antigen is CD20.

1 1 . The method of any one of claims 1 -10, wherein said population of T cells comprises >80%,

>85%, >90%, >95%, >96%, >97%, >98%, >99%, or 100% yd T cells.

12. The method of any one of claims 1 -11 , wherein said population of T cells is autologous to said subject.

13. The method of any one of claims 1 -11 , wherein said population of T cells is allogeneic to said subject.

14. The method of any one of claims 1 -13, wherein said population of yd T cells has been modified to express a chimeric antigen receptor.

15. The method of claim 14, wherein said chimeric antigen receptor binds a tumor-associated antigen.

16. A composition comprising: a) an antigen-binding protein, wherein said antigen-binding protein binds to CD3 and to a tumor-associated antigen; and b) a population of T cells comprising yd T cells.

17. The composition of claim 16, wherein said population of yd T cells is an ex vivo expanded population of yd T-cells.

18. The composition of claim 17, wherein said population of yd T cells is expanded with an aminobisphosphonate.

19. The composition of claim 18, wherein said aminobisphosphonate is zoledronate.

20. The composition of any one of claims 18 or 19, wherein said aminobisphosphonate is combined with IL-2 to induce expansion.

21 . The composition of claim 17, wherein said population of yd T cells is expanded with one or more antibodies.

22. The composition of any one of claims 16-21 , wherein said antigen-binding protein is an antibody or antigen-binding fragment thereof.

23. The composition of claim 22, wherein said antibody is a multispecific antibody.

24. The composition of claim 23, wherein said antibody is a bispecific antibody.

25. The composition of claim 24, wherein said tumor-associated antigen is CD20.

26. The composition of any one of claims 16-25, wherein said population of T cells comprises

>80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%, or 100% yd T cells.

27. The composition of any one of claims 16-26, wherein said population of T cells is autologous to said subject.

28. The composition of any one of claims 16-26, wherein said population of T cells is allogeneic to said subject.

29. The composition of any one of claims 16-28, wherein said population of yd T cells has been modified to express a chimeric antigen receptor.

30. The composition of claim 29, wherein said chimeric antigen receptor binds a tumor- associated antigen.