HK40040207A - Antibody binding tim-3 and use thereof - Google Patents

Description

TIM-3 binding antibodies and uses thereof

Technical Field

The present invention relates generally to an isolated monoclonal antibody, and in particular to a human monoclonal antibody that specifically binds to TIM-3 and has good therapeutic properties. Nucleic acid molecules encoding the antibodies, expression vectors, host cells, and methods for expressing the antibodies are also provided. The invention further provides immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies, and diagnostic and therapeutic methods using the anti-TIM-3 antibodies of the invention.

Background

Therapeutic antibodies are one of the fastest growing segments of the pharmaceutical industry, particularly monoclonal antibodies directed against cellular proteins associated with certain diseases.

One such target protein is T-cell immunoglobulin mucin-3, also known as TIM-3, a protein encoded by the HAVCR2 gene in humans. TIM-3 is an immune checkpoint and acts as a cell surface receptor on IFN γ -producing CD4+ T helper 1(Th1) and CD8⁺T cytotoxic 1(Tc1) cells, Th17 cells, regulatory T cells and innate immune cells (dendritic cells, NK cells and monocytes) (Monney L et al, (2002) Nature.415(6871): 536-41; Hastings WD et al, (2009) European Journal of immunology.39(9): 2492-501; Gao X et al, (2012) PLOS one.7(2): e 30676; Gleason MK et al, (2012) blood.119(13): 3064-72). Several ligands for TIM-3 were found, including galectin 9, PtdSer, HMGB1, and CEACAM 1. Of these, galectin 9 and Ptdser are ligands that primarily activate TIM-3, and ligand engagement limits CD4⁺Th1 and CD8⁺Duration and breadth of Tc1 cell response (Sabatos CA et al, (2003) Nat Immunol 4: 1102-10; Sabatos-Peyton CA et al, (2018) ONCOIMMUNOLOGY 7(2): e1385690)

Preclinical studies using antibody blocking TIM-3 for cancer treatment showed that activation of antigen-specific T cells at the tumor site was enhanced and tumor growth was disrupted. In addition, dual anti-TIM-3/anti-PD-1 antibody treatment cured the majority of mice with tumors that have been established to be extremely resistant to single antibody treatment (Ngiow SF et al, (2011) Cancer Res71: 3540-51).

Despite promising therapeutic effects, only a few anti-TIM-3 antibodies have been developed to date. One such antibody is MBG-453 to Novartis, a humanized antibody to ABTIM3 as described in US2015218274a1, which has been shown to block TIM-3-PtdSer interactions and is currently in phase I testing. Another anti-TIM-3 antibody is described in WO2017/079115, which inhibits the binding of TIM-3 to prolactin 9 (galectin-9).

Disclosure of Invention

The present invention provides an isolated monoclonal antibody, e.g., a human, mouse, chimeric or humanized monoclonal antibody, that binds to TIM-3 and has comparable or better pharmaceutical properties than existing anti-TIM-3 antibodies, e.g., ABTIM 3.

In one aspect, the invention relates to an isolated monoclonal antibody (e.g., a human antibody), or antigen-binding portion thereof, having a heavy chain variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 region, the CDR2 region, and the CDR3 region comprise amino acid sequences at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to: (1) 1, 5 and 9, respectively; (2) 2, 6 and 9, respectively; (3) 3, 7 and 10, respectively; or (4) SEQ ID NOS: 4, 8 and 11, respectively, wherein the antibody or antigen binding fragment thereof binds to TIM-3.

In one aspect, an isolated monoclonal antibody (e.g., a human antibody) or antigen-binding portion thereof of the invention comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NOs 24, 25, 26, or 27, wherein the antibody or antigen-binding fragment thereof binds to TIM-3. These amino acid sequences may be encoded by the nucleotide sequences set forth in SEQ ID NOS 37, 38, 39 and 40, respectively.

In one embodiment, the monoclonal antibody, or antigen binding portion thereof, of the invention comprises a light chain variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein said CDR1 region, said CDR2 region, and said CDR3 region comprise amino acid sequences at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to: (1) 12, 17 and 21, respectively; (2) 13, 17 and 21, respectively; (3) 14, 17 and 21, respectively; (4) 14, 18 and 21, respectively; (5) 15, 19 and 22, respectively; or (6) SEQ ID NOs 16, 20 and 23; wherein the antibody or antigen binding fragment thereof binds to TIM-3.

In one aspect, an isolated monoclonal antibody (e.g., a human antibody) or antigen-binding portion thereof of the invention comprises a monoclonal antibody comprising a heavy chain variable region having a heavy chain variable region as set forth in SEQ ID NOS: 28, 29 (X) ₁＝N、X₂(ii) S; or X₁＝Y、X₂＝S；X₁＝Y、X₂N), 30 or 31, wherein the antibody or antigen-binding fragment thereof binds to TIM-3, or a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical. These amino acid sequences may be encoded by the nucleotide sequences set forth in SEQ ID NOs 41, 42, 43, 44, 45, and 46, respectively.

In one aspect, an isolated monoclonal antibody or antigen-binding portion thereof of the invention comprises a heavy chain variable region and a light chain variable region each comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1, CDR2, and CDR3 regions of the heavy chain variable region and the CDR1, CDR2, and CDR3 regions of the light chain variable region comprise amino acid sequences at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to: (1) 1, 5, 9, 12, 17 and 21, respectively; (2) 2, 6, 9, 13, 17 and 21, respectively; (3) 2, 6, 9, 14, 17 and 21, respectively; (4) 2, 6, 9, 14, 18 and 21, respectively; (5) 3, 7, 10, 15, 19 and 22, respectively; or (6) SEQ ID NOs 4, 8, 11, 16, 20 and 23; wherein the antibody or antigen binding fragment thereof binds to TIM-3.

In one embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region and a light chain variable region comprising amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to: (1) is divided into24 and 28, respectively; (2) SEQ ID NOS: 25 and 29 (X), respectively₁＝N、X₂(ii) S; or X₁＝Y、X₂＝S；X₁＝Y、X₂N); (3) 26 and 30, respectively; or (4) SEQ ID NOS: 27 and 31, respectively, wherein the antibody or antigen binding fragment thereof binds to TIM-3.

In one embodiment, an isolated monoclonal antibody, or antigen-binding portion thereof, of the invention comprises a heavy chain comprising a heavy chain variable region and a heavy chain constant region, and a light chain comprising a light chain variable region and a light chain constant region, wherein the heavy chain constant region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:32 or 33, and the light chain constant region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:34, 35, or 36, and the heavy chain variable region and the light chain variable region comprise amino acid sequences as described above, wherein the antibody, or antigen-binding fragment thereof, binds to TIM-3. The amino acid sequences SEQ ID NO 32, 33, 34 and 36 may be encoded by the nucleotide sequences set forth in SEQ ID NO 47, 48, 49 and 50, respectively. The heavy chain constant region is specifically designed such that the anti-TIM 3 antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) on cells expressing TIM 3. For example, a human IgG1 heavy chain may comprise L234A, L235A, D265A, and/or P329A mutations for abrogating ADCC or CDC function.

The antibody of the invention may be a full length antibody, for example, of the IgG1, IgG2, or IgG4 isotype. In other embodiments, the antibodies of the invention may be single chain antibodies or consist of antibody fragments such as Fab or Fab' 2 fragments.

The antibodies of the invention or antigen binding portions thereof are present at about 2.05X 10^-9K of M or less_DBinding to human TIM-3, inhibiting the binding of TIM-3 to galectin 9, ptdser or other ligands, not cross-reacting with TIM-1 or TIM-4, inducing the internalization of TIM3 on the cell membrane, inducing the release of IL-2 and/or IFN γ by human T cells, not inducing AD on cells expressing TIM3CC or CDC, and/or enhances activation of antigen-specific CD4+ or CD8+ T cells. The antibodies of the invention, or antigen-binding portions thereof, have comparable, if not better, binding and/or blocking activity than prior art anti-TIM 3 antibodies, e.g., ABTIM 3.

The invention also provides an immunoconjugate comprising an antibody or antigen-binding portion thereof of the invention linked to a therapeutic agent, e.g., a cytotoxin. The invention also provides a bispecific molecule comprising an antibody or antigen-binding portion thereof of the invention linked to a second functional moiety (e.g., a second antibody) having a binding specificity different from that of the antibody or antigen-binding portion thereof. In another aspect, an antibody or antigen-binding portion thereof of the invention can be prepared as part of a Chimeric Antigen Receptor (CAR). The antibodies of the invention, or antigen binding portions thereof, may also be encoded by or used in conjunction with an oncolytic virus.

Also provided are compositions comprising an antibody or antigen-binding portion thereof or immunoconjugate, bispecific molecule or CAR of the invention and a pharmaceutically acceptable carrier.

The invention also encompasses nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of the invention, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors. Also provided is a method of making an anti-TIM 3 antibody using a host cell comprising an expression vector, the method comprising the steps of: (i) expressing the antibody in a host cell and (ii) isolating the antibody from the host cell or cell culture thereof.

In another embodiment, the invention provides a method of enhancing an immune response in a subject, the method comprising administering to the subject an antibody, or antigen-binding portion thereof, of the invention. In another embodiment, at least one additional immunostimulatory antibody may be administered with an antibody of the invention, or antigen-binding portion thereof, e.g., an anti-PD-1 antibody, an anti-LAG-3 antibody, and/or an anti-CTLA-4 antibody, such that an immune response (e.g., inhibition of tumor growth or stimulation of an anti-viral response) is enhanced in the subject. In one embodiment, the additional immunostimulatory antibody is an anti-PD-1 antibody. In another embodiment, the additional immunostimulatory agent is an anti-LAG-3 antibody. In yet another embodiment, the additional immunostimulant is an anti-CTLA-4 antibody. In yet another embodiment, the antibodies of the invention, or antigen binding portions thereof, are administered with a cytokine (e.g., IL-2 and/or IL-21) or a costimulatory antibody (e.g., anti-CD 137 and/or anti-GITR antibody). The antibody can be, for example, a mouse, human, chimeric, or humanized antibody.

In another embodiment, the invention provides a method of treating a tumor or cancer in a subject, the method comprising administering to the subject an antibody, or antigen-binding portion thereof, of the invention. The cancer may be a solid tumor or a non-solid tumor, including but not limited to B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal cancer. In some embodiments, the methods comprise administering a composition, bispecific molecule, immunoconjugate, CAR-T cell, or oncolytic virus encoding or carrying an antibody of the invention. In some embodiments, at least one additional anti-cancer antibody can be administered with an antibody or antigen-binding portion thereof of the invention, e.g., an anti-VISTA antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, and/or an anti-CTLA-4 antibody. In yet another embodiment, the antibodies of the invention, or antigen binding portions thereof, are administered with a cytokine (e.g., IL-2 and/or IL-21) or a costimulatory antibody (e.g., anti-CD 137 and/or anti-GITR antibody). In another embodiment, the antibody or antigen-binding portion thereof of the invention is administered with a chemotherapeutic agent, which may be a cytotoxic agent, such as epirubicin (epirubicin), oxaliplatin (oxaliplatin), and/or 5-fluorouracil (5-FU). The antibody of the invention may be, for example, a mouse, human, chimeric or humanized antibody.

In yet another embodiment, the invention provides a method of treating a viral infection in a subject, the method comprising administering to the subject an antibody, or antigen-binding portion thereof, of the invention.

In another aspect, the invention provides anti-TIM-3 antibodies and compositions of the invention for use in the aforementioned methods, or for use in the manufacture of a medicament for use in the aforementioned methods (e.g., for treatment).

Further features and advantages of the invention will become apparent from the following detailed description and examples which should not be construed as limiting. The contents of all references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated by reference herein.

Drawings

FIG. 1 shows the binding activity of anti-TIM-3 antibodies TIM3-6.12 (left panel), TIM3-6, TIM3-4G7 and TIM3-11 (right panel) to human TIM-3.

FIG. 2 shows the blocking activity of anti-TIM-3 antibodies of the present invention on human TIM-3-galectin 9 interaction.

FIG. 3 shows the binding activity of anti-TIM-3 antibodies of the present invention to TIM-3 expressed on CHO-K1-TIM3 cells.

Figure 4 shows IL-2 released from PBMCs treated with SEB, followed by treatment with anti-TIM-3 antibodies of the invention.

FIG. 5 shows the blocking activity of anti-TIM-3 antibodies of the present invention on the TIM-3-phosphatidylserine interaction.

FIG. 6 shows internalization of anti-TIM-3 antibodies of the invention by CHOK1-TIM3 cells.

Figure 7 shows that anti-TIM 3 antibodies of the invention do not bind to C1 q.

Figure 8 shows that anti-TIM 3 antibodies of the invention do not induce ADCC on CHOK1-TIM3 cells.

Figure 9 shows that anti-TIM 3 antibodies of the invention do not induce CDC on CHOK1-TIM3 cells.

Detailed Description

In order that the disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "TIM-3" refers to T cell immunoglobulin mucin-3. The term "TIM-3" encompasses variants, isoforms, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific for human TIM-3 proteins can cross-react with TIM-3 proteins from species other than humans. In other embodiments, an antibody specific for a human TIM-3 protein may be fully specific for the human TIM-3 protein and exhibit no cross-reactivity with other species or types, or it may cross-react with TIM-3 from some other species but not all others (e.g., cross-react with monkey TIM-3 but not mouse TIM-3).

The term "human TIM-3" refers to the human sequence of TIM-3, e.g., the complete amino acid sequence of human TIM-3 with Genbank accession number NP-116171.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complement) that cause selective damage, destruction, or elimination from the human body of invading pathogens, cells or tissues infected by pathogens, cancer cells, or in the case of autoimmune or pathological inflammation, normal human cells or tissues.

The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. Intact antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V)_H) And a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region comprises one domain, namely CL. The VH and VL regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), which alternate with more conserved regions, termed Framework Regions (FRs). 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, FR 4. Heavy load The variable regions of the chains and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a TIM-3 protein). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, i.e., consisting of V_L、V_H、C_LAnd C_H1Monovalent fragments consisting of domains; (ii) f (ab')₂Fragments, i.e. bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) from V_HDomains and C_H1Domain-forming Fd fragments; (iv) by a single-armed V_LDomains and V_H(iii) an Fv fragment consisting of a domain; (v) from V_HdAb fragments consisting of domains (Ward et al (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) nanobodies, i.e., heavy chain variable regions comprising a single variable domain and two constant domains. Furthermore, despite the two domains V of the Fv fragment _LAnd V_HAre encoded by separate genes, but they can be joined using recombinant methods by synthetic linkers that enable them to be made into a single protein chain, wherein the V_LRegion and V_HThe regions pair to form monovalent molecules (known as single chain fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. These single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a TIM-3 protein is substantially free of antibodies that specifically bind antigens other than the TIM-3 protein). However, isolated antibodies that specifically bind to human TIM-3 proteins can be cross-reactive to other antigens, such as TIM-3 proteins from other species. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the invention may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo). 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 have been grafted onto human framework sequences.

The term "mouse antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from mouse germline immunoglobulin sequences. The mouse antibodies of the invention can include amino acid residues that are not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "mouse antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.

The term "chimeric antibody" refers to an antibody prepared by combining genetic material from a non-human source with genetic material from a human. Or more generally, a chimeric antibody is an antibody having genetic material from one species with genetic material from another species.

The term "humanized antibody" as used herein refers to an antibody from a non-human species whose protein sequence has been modified to increase similarity to naturally occurring antibody variants of humans.

The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity with variable regions, wherein both framework and CDR regions are derived from human germline immunoglobulin sequences.

The term "isotype" refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

The phrases "an antibody that recognizes an antigen" and "an antibody having specificity for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen".

The term "antibody derivative" refers to any modified form of an antibody, e.g., a conjugate of an antibody with another agent or antibody.

As used herein, an antibody that "specifically binds to human TIM-3" is intended to refer to an antibody that binds to a human TIM-3 protein (and possibly a TIM-3 protein from one or more non-human species) but does not substantially bind to a non-TIM-3 protein. Preferably, the antibody is "high affinity", i.e., 5x10 ^-9K of M or less_DBinds to human TIM-3 protein.

The term "substantially not binding" a protein or cell as used herein means not binding to said protein or cell or not binding to said protein or cell with high affinity, i.e. 1 x 10^-6M or greater, more preferably 1X 10^-5M or greater, more preferably 1X 10^-4M or greater, more preferably 1X 10^-3M or greater, even more preferably 1X 10^-2K of M or greater_DBinding to said protein or cell.

The term "K" as used herein_{Association of}"or" K_a"intended to refer to the association rate of a particular antibody-antigen interaction, and the term" K "as used herein_Dissociation"or" K_d"intended to refer to specific antibody-antigen interactionsThe rate of dissociation. The term "K" as used herein_D"intended to mean the dissociation constant, which is defined by K_dAnd K_aRatio of (i.e. K)_d/K_a) Obtained and expressed as molar concentration (M). K of antibody_DValues may be determined using methods recognized in the art. K for determining antibodies_DBy using surface plasmon resonance, preferably using a biosensor system, such as Biacore^TMProvided is a system.

The term "high affinity" for an IgG antibody refers to 1X 10 for the target antigen ^-6M or less, more preferably 5X 10^-8M or less, even more preferably 1X 10^-8M or less, even more preferably 5X 10^-9M or less and even more preferably 1X 10^-9K of M or less_DThe antibody of (1). However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding of IgM isotype refers to having 10^-6M or less, more preferably 10^-7M or less, even more preferably 10^-8K of M or less_DThe antibody of (1).

The term "IC₅₀"also referred to as half maximal inhibitory concentration" refers to the concentration of antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of antibody.

The term "EC₅₀"also referred to as half maximal effective concentration, refers to the concentration of antibody that induces a response halfway between the baseline and maximum values after a specified exposure time.

The terms "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" as used herein refer to the mechanism of cell-mediated immune defense whereby effector cells of the immune system actively lyse target cells, e.g. tumor cells, whose membrane surface antigens have been bound by antibodies. The antibodies of the present invention do not induce ADCC on cells expressing TIM3 to protect immune cells.

The term "complement-dependent cytotoxin" or "CDC" generally refers to the effector function of IgG and IgM antibodies that, when bound to surface antigens, trigger the classical complement pathway, induce the formation of membrane attack complexes and target cell lysis. The antibodies of the present invention do not induce CDC on TIM 3-expressing cells and thus protect immune cells.

The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows, and horses.

Various aspects of the invention are described in more detail in the following subsections.

anti-TIM-3 antibodies with advantageous functional properties

The antibodies of the invention specifically bind to human TIM-3. The antibodies of the invention are preferably at 5x10^-9K of M or less_DMore preferably at 2.5x10^-9K of M or less_DBinds to human TIM-3 protein.

The antibodies of the invention inhibit the binding of TIM-3 to galectin 9, ptdser or other ligands. The antibodies of the present invention do not cross-react with TIM-1 or TIM-4. The antibodies of the invention induce the internalization of TIM3 on the cell membrane. The antibodies of the invention induce the release of IL-2 and/or IFN γ by human T cells and enhance the activation of antigen-specific CD4+ or CD8+ T cells. The antibodies of the present invention do not induce ADCC or CDC on cells expressing TIM3 to protect immune cells.

The binding activity of the antibodies of the invention is comparable to, if not better than, the prior art anti-TIM 3 antibodies, e.g., ABTIM 3. In one embodiment, the antibodies of the invention may inhibit the binding of TIM-3 to galectin 9 at much lower concentrations than ABTIM 3.

Preferred antibodies of the invention are fully human monoclonal antibodies.

Monoclonal anti-TIM-3 antibodies

Preferred antibodies of the invention are monoclonal antibodies having the structural and chemical characteristics described below and in the examples belowAnd (3) a body. V against TIM-3 antibody_HThe amino acid sequences are set forth in SEQ ID NO. 24, 25, 26 or 27. V against TIM-3 antibody_LThe amino acid sequence is shown in SEQ ID NO 28, 29, 30 or 31. The amino acid sequence ID numbers of the heavy/light chain variable regions of the antibodies are summarized in Table 1 below, with some clones sharing the same V_HOr a CDR sequence. The heavy chain constant region is specifically designed such that the anti-TIM 3 antibody does not induce ADCC or CDC on cells expressing TIM 3. The heavy chain constant region may have the amino acid sequence set forth in SEQ ID NO 32 or 33, and the light chain constant region may have the amino acid sequence set forth in SEQ ID NO 34, 35 or 36.

The CDR regions in Table 1 have been determined by the Kabat numbering system. However, it is well known in the art that CDR regions can also be determined by other systems such as Chothia, CCG, and IMGT systems/methods based on heavy/light chain variable region sequences.

TIM3-6.10, TIM3-6.11 and TIM3-6.12 differ in the light chain variable region by one or two amino acid residues, resulting in slightly different affinities to human TIM-3. The three antibodies also had Q at amino acid position 106 compared to K in TIM3-6. Such amino acid modifications make these antibodies more stable under stress.

TABLE 1 amino acid sequence of anti-TIM-3 antibodies

V of other anti-TIM-3 antibodies binding to human TIM-3_HAnd V_LThe sequences (or CDR sequences) may be related to the V of an anti-TIM-3 antibody of the present invention_HAnd V_LSequences (or CDR sequences) "mix and match". Preferably, when V_HAnd V_LChains (or CDRs within such chains) when mixed and matched, are derived from a particular V_H/V_LPaired V_HSequence is structurally similar V_HAnd (4) replacing the sequence. Also, preferably from a particular sourceV_H/V_LPaired V_LSequence is structurally similar V_LAnd (4) replacing the sequence.

Thus, in one embodiment, an antibody or antigen-binding portion thereof of the invention comprises:

(a) a heavy chain variable region comprising an amino acid sequence set forth in table 1 above; and

(b) variable region of light chain comprising amino acid sequence as set forth in Table 1 above, or V of another anti-TIM-3 antibody_LWherein the antibody specifically binds to human TIM-3.

In another embodiment, an antibody or antigen-binding portion thereof of the invention comprises:

(a) The CDR1, CDR2, and CDR3 regions of the heavy chain variable regions listed in table 1 above; and

(b) the CDR1, CDR2, and CDR3 regions of the light chain variable regions listed in table 1 above, or the CDRs of another anti-TIM-3 antibody, wherein the antibody specifically binds to human TIM-3.

In yet another embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable CDR2 region of an anti-TIM-3 antibody that combines the CDRs of other antibodies that bind to human TIM-3, e.g., CDR1 and/or CDR3 from the heavy chain variable region of a different anti-TIM-3 antibody, and/or CDR1, CDR2, and/or CDR3 from the light chain variable region thereof.

Furthermore, it is well known in the art that the CDR3 domain alone, independent of the CDR1 domain and/or the CDR2 domain, can determine the binding specificity of an antibody to a homologous antigen, and that multiple antibodies with the same binding specificity can be predictably produced based on a common CDR3 sequence. See, e.g., Klimka et al, British J.of Cancer 83(2):252-260 (2000); beiboer et al, J.mol.biol.296:833-849 (2000); rader et al, Proc. Natl. Acad. Sci. U.S.A.95:8910-8915 (1998); barbas et al, J.Am.chem.Soc.116:2161-2162 (1994); barbas et al, Proc.Natl.Acad.Sci.U.S.A.92:2529-2533 (1995); ditzel et al, J.Immunol.157:739-749 (1996); berezov et al, BIAjournal 8: Scientific Review 8 (2001); igarashi et al, J.biochem (Tokyo) 117:452-7 (1995); bourgeois et al, J.Virol 72:807-10 (1998); levi et al, Proc.Natl.Acad.Sci.U.S.A.90:4374-8 (1993); polymenis and Stoller, J.Immunol.152:5218-5329 (1994); and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. patent nos. 6,951,646; 6,914,128, respectively; 6,090,382; 6,818,216, respectively; 6,156,313, respectively; 6,827,925, respectively; 5,833,943, respectively; 5,762,905, and 5,760,185. Each of these references is incorporated herein by reference in its entirety.

Thus, in another embodiment, an antibody of the invention comprises CDR2 of the heavy chain variable region of an anti-TIM-3 antibody and at least CDR3 of the heavy chain variable region and/or the light chain variable region of an anti-TIM-3 antibody, or CDR3 of the heavy chain variable region and/or the light chain variable region of another anti-TIM-3 antibody, wherein said antibody is capable of specifically binding to human TIM-3. These antibodies preferably (a) compete for binding to TIM-3; (b) functional characteristics are reserved; (c) binding to the same epitope; and/or (d) has similar binding affinity as an anti-TIM-3 antibody of the present invention. In yet another embodiment, the antibody may further comprise CDR2 of the light chain variable region of an anti-TIM-3 antibody or CDR2 of the light chain variable region of another anti-TIM-3 antibody, wherein the antibody is capable of specifically binding to human TIM-3. In another embodiment, an antibody of the invention may comprise CDR1 of the heavy chain variable region and/or light chain variable region of an anti-TIM-3 antibody or CDR1 of the heavy chain variable region and/or light chain variable region of another anti-TIM-3 antibody, wherein said antibody is capable of specifically binding to human TIM-3.

Conservative modifications

In another embodiment, an antibody of the invention comprises a heavy chain variable region sequence and/or a light chain variable region sequence of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that differ from an anti-TIM-3 antibody of the invention by one or more conservative modifications. It will be appreciated in the art that certain conservative sequence modifications may be made that do not remove antigen binding. See, e.g., Brummell et al (1993) Biochem 32: 1180-8; de Wildt et al (1997) prot. Eng.10: 835-41; komissarov et al (1997) J.biol.chem.272: 26864-26870; hall et al (1992) J.Immunol.149: 1605-12; kelley and O' Connell (1993) biochem.32: 6862-35; Adib-Conquy et al (1998) int. Immunol.10: 341-6; and Beers et al (2000) Clin. Can. Res.6: 2835-43.

Thus, in one embodiment, the antibody comprises a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence and/or a light chain variable region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence, wherein:

(a) the heavy chain variable region CDR1 sequence comprises the sequence listed in table 1 above and/or conservative modifications thereof; and/or

(b) The heavy chain variable region CDR2 sequence comprises the sequence listed in table 1 above and/or conservative modifications thereof; and/or

(c) The heavy chain variable region CDR3 sequence comprises the sequence listed in table 1 above and/or conservative modifications thereof; and/or

(d) The light chain variable region CDR1 sequence and/or CDR2 sequence and/or CDR3 sequence comprises the sequences listed in table 1 above and/or conservative modifications thereof; and is

(e) The antibodies specifically bind to human TIM-3.

The term "conservative sequence modification" as used herein is intended to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CDR region of an antibody of the invention can be substituted with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the function described above) using the functional assays described herein.

Engineered and modified antibodies

V with anti-TIM-3 antibodies of the invention may be used_Hsequence/V_LAntibodies to one or more of the sequences as starting materials to engineer the modified antibodies to make the antibodies of the invention. Can be modified by modifying one or both variable regions (i.e., V)_HAnd/or V_L) The antibody is engineered with one or more residues, e.g., within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, the antibody may be engineered by modifying residues within one or more constant regions, for example to alter one or more effector functions of the antibody.

In certain embodiments, CDR grafting may be used to engineer the variable regions of antibodies. Antibodies interact with a target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Since the CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann et al (1998) Nature 332: 323-327; Jones et al (1986) Nature 321: 522-525; Queen et al (1989) Proc. Natl. Acad. See. U.S. A.86: 10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Thus, another embodiment of the invention relates to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising a CDR1 sequence comprising the sequence of the invention as described above,CDR2 and CDR3 sequences; and/or a light chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence comprising the sequences of the invention as described above. Although these antibodies comprise V of the monoclonal antibody of the invention_HAnd V_LBut they may comprise different framework sequences.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/VBase), and Kabat et al (1991) (cited above); tomlinson et al (1992), J.mol.biol.227: 776-798; and Cox et al (1994), Eur.J. Immunol.24:827-836, the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in gene bank databases. For example, the following heavy chain germline sequences found in HCo7 HuMAb mice may be attached to the genbank accession number: 1-69(NG __0010109, NT __024637 and BC070333), 3-33(NG __0010109 and NT __024637) and 3-7(NG __0010109 and NT __ 024637). As another example, the following heavy chain germline sequences found in HCo12 HuMAb mice may be attached to the genbank accession number: 1-69(NG __0010109, NT __024637 and BC070333), 5-51(NG __0010109 and NT __024637), 4-34(NG __0010109 and NT __024637), 3-30.3(CAJ556644) and 3-23(AJ 406678).

Antibody protein sequences were compared to compiled protein sequence databases using one of the sequence similarity search methods known to those skilled in the art as Gapped BLAST (Altschul et al (1997) (supra)).

Preferred framework sequences for use in the antibodies of the invention are those that are structurally similar to the framework sequences used in the antibodies of the invention. V_HThe CDR1 sequence, CDR2 sequence, and CDR3 sequence may be grafted onto a framework region having a sequence identical to a sequence present in a germline immunoglobulin gene from which the framework sequence is derived, orCDR sequences can be grafted onto framework regions containing one or more mutations compared to the germline sequence. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

Another type of variable region modification is to modify V_HAnd/or V_LAmino acid residues within the CDR1 region, CDR2 region, and/or CDR3 region are mutated to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations and the effect on antibody binding or other functional property of interest can be assessed in an in vitro or in vivo assay as described herein and provided in the examples. Preferably, conservative modifications (as known in the art) are introduced. The mutation may be an amino acid substitution, addition or deletion, but is preferably a substitution. In addition, typically no more than 1, 2, 3, 4 or 5 residues within a CDR region are altered.

Thus, in another embodiment, the invention provides an isolated anti-LAG-3 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising: (a) v comprising an amino acid sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the invention_HA CDR1 region; (b) v comprising an amino acid sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the invention_HA CDR2 region; (c) v comprising an amino acid sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the invention_HA CDR3 region; (d) v comprising an amino acid sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the invention_LA CDR1 region; (e) v comprising an amino acid sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the invention_LA CDR2 region; and (f) contains or has 1 of the present inventionV of an amino acid sequence of 2, 3, 4 or 5 amino acid substitutions, deletions or additions _LA CDR3 region.

Engineered antibodies of the invention include those in which V has been paired_HAnd/or V_LFramework residues within antibodies are modified, for example, to improve the properties of those antibodies. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to germline sequences that are the source of the antibody.

Another type of framework modification involves mutating one or more residues within the framework regions or even within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization" and is described in more detail in U.S. patent publication No. 20030153043.

In addition to or as an alternative to modifications within the framework or CDR regions, the antibodies of the invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. In addition, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody), or it may be modified to alter its glycosylation, thereby again altering one or more functional properties of the antibody.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. Such a process is further described in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the antibody has impaired staphylococcal protein a (SpA) binding relative to native Fc hinge domain SpA binding. This method is described in more detail in U.S. Pat. No. 6,165,745.

In yet another embodiment, the glycosylation of the antibody is modified. For example, antibodies can be made that are aglycosylated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen. See, for example, U.S. Pat. nos. 5,714,350 and 6,350,861.

Another modification contemplated by the present disclosure to the antibodies herein is pegylation. The antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibodies. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy polyethylene glycol or aryloxy polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EPO 154316 and EP 0401384.

Physical Properties of antibodies

The antibodies of the present invention may be characterized by their various physical properties to detect and/or distinguish their different classes.

For example, an antibody may contain one or more glycosylation sites in the light chain variable region or the heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or altered pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem41: 673-. Glycosylation is known to occur at motifs containing N-X-S/T sequences. In some cases, it is preferred to have an anti-TIM-3 antibody that does not contain variable region glycosylation. This can be achieved by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.

In a preferred embodiment, the antibody does not contain asparagine isomerization sites. Deamidation of asparagine may occur on the N-G or D-G sequence and result in the production of isoaspartic acid residues, which introduces kinks into the polypeptide chain and reduces its stability (isoaspartic acid action).

Each antibody will have a unique isoelectric point (pI) which typically falls within a pH range of 6 to 9.5. The pI values of IgG1 antibodies typically fall within a pH range of 7-9.5 and IgG4 antibodies typically fall within a pH range of 6-8. It is speculated that antibodies with pI values outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, an anti-TIM-3 antibody having a pI value falling within the normal range is preferred. This can be achieved by selecting antibodies with pI values in the normal range or by mutating charged surface residues.

Nucleic acid molecules encoding the antibodies of the invention

In another aspect, the invention provides nucleic acid molecules encoding the heavy and/or light chain variable regions or CDRs of the antibodies of the invention. The nucleic acid may be present in whole cells, cell lysates, or partially purified or substantially pure form. Nucleic acids are "isolated" or "become substantially pure" when purified by standard techniques to remove other cellular components or other contaminants, such as other cellular nucleic acids or proteins. The nucleic acids of the invention may be, for example, DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by a hybridoma (e.g., a hybridoma prepared from a transgenic mouse carrying human immunoglobulin genes, as described further below), cdnas encoding the light and heavy chains of the antibody prepared by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene libraries.

Preferred nucleic acid molecules of the invention include V encoding a TIM-3 monoclonal antibody_HAnd V_LOr a CDR sequence. Once the code V is obtained_HSection and V_LThe DNA fragments of the segments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, V will be encoded_LOr V_HIs operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments are maintained in frame.

Can be obtained by encoding V_HDNA of (3) and a DNA encoding a heavy chain constant region (C)_H1、C_H2And C_H3) Is operably linked to encode V_HIsolated DNA of the region is converted to the full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art and DNA fragments encompassing these regions may beObtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the gene encoding V can be _HIs operably linked to another DNA molecule encoding only the constant region of heavy chain CH 1.

Can be obtained by encoding V_LIs operably linked to another DNA molecule encoding a light chain constant region CL to encode V_LThe isolated DNA of the region was converted to the full-length light chain gene (as well as the Fab light chain gene). The sequence of the human light chain constant region gene is known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region.

To generate the scFv gene, V will be encoded_HAnd V_LWith a DNA fragment encoding a flexible linker, e.g. encoding an amino acid sequence (Gly)₄-Ser)₃Is operably linked such that V_HSequence and V_LThe sequence may be expressed as a continuous single chain protein, wherein V_LRegion and V_HThe regions are joined by flexible linkers (see, e.g., Bird et al (1988) Science 242: 423-.

Generation of monoclonal antibodies of the invention

Monoclonal antibodies (mAbs) of the invention can be produced using well-known somatic hybridization (hybridoma) techniques of Kohler and Milstein (1975) Nature 256: 495. Other embodiments for generating monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See, e.g., U.S. Pat. nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are specifically incorporated herein by reference in their entirety.

In a preferred embodiment, the antibody of the invention is a human monoclonal antibody. Such human monoclonal antibodies to human TIM-3 may be used to carry aTransgenic or transchromosomal mice that are part of the human immune system and do not carry the mouse system are produced. These transgenic and transchromosomal mice include what are referred to herein as HuMAb Mouse, respectively^TMAnd KM Mouse^TMAnd are collectively referred to herein as "human Ig mice".

HuMAb Mouse^TM(Medarex^TMCompany) contains a human immunoglobulin gene minilocus (minioci) encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences and targeted mutations that inactivate endogenous mu and kappa chain loci (see, e.g., Lonberg et al (1994) Nature 368(6474): 856-859). Thus, the mice display reduced mouse IgM or kappa expression and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG kappa monoclonal antibodies (Lonberg et al (1994) (supra); reviewed in Lonberg (1994) Handbook of Experimental Pharmacology (A Handbook of Experimental Pharmacology), 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern.Rev.Immunol.13: 65-93; and Harding and Lonberg (1995) Ann.N.Y.Acad.Sci.764: 536: 546). HuMAb Mouse ^TMThe preparation and use of (A) and the genomic modifications carried by such mice are further described in Taylor et al (1992) Nucleic Acids Research 20: 6287-6295; chen et al (1993) International Immunology 5: 647-656; tuaillon et al (1993) Proc. Natl. Acad. Sci. USA 90: 3720-3724; choi et al (1993) Nature Genetics4: 117-123; chen et al (1993) EMBO J.12: 821-830; tuaillon et al (1994) J.Immunol.152: 2912-2920; taylor et al (1994) International Immunology 6: 579-; and Fishwild et al (1996) Nature Biotechnology 14: 845-. See also U.S. Pat. nos. 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,789,650, respectively; 5,877,397, respectively; 5,661,016, respectively; 5,814, 318; 5,874,299, respectively; 5,770,429, respectively; and 5,545,807; PCT publication nos. WO 92/03918; WO 93/12227; WO 94/25585; WO 97/13852; WO 98/24884; WO 99/45962 and WO 01/14424, the contents of which are incorporated herein by reference in their entirety.

In another embodiment, the human of the inventionAntibodies can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice are referred to herein as "KM Mouse ^TM", and is described in detail in PCT publication WO 02/43478. Modified forms of such mice that also comprise homozygous disruption of the endogenous Fc γ RIIB receptor gene are also described in PCT publication WO 02/43478 and are referred to herein as "KM/FCGR 2D mice". In addition, mice carrying HCo7 or HCo12 heavy chain transgenes, or both, may be used.

Additional transgenic animal embodiments include Xenomouse (Abgenix corporation, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963). Additional embodiments include "TC mice" (Tomizuka et al (2000) Proc. Natl. Acad. Sci. USA 97: 722-. The contents of these patents and publications are specifically incorporated by reference herein in their entirety.

In one embodiment, the human monoclonal antibodies of the invention are prepared using phage display methods to screen libraries of human immunoglobulin genes. See, e.g., U.S. Pat. nos. 5,223,409; 5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108, respectively; 6,172,197, respectively; 5,885,793, respectively; 6,521,404; 6,544,731, respectively; 6,555,313, respectively; 6,582,915, respectively; and 6,593,081, the contents of which are incorporated by reference herein in their entirety.

Human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted so that a human antibody response can be generated following immunization. See, for example, U.S. patent nos. 5,476,996 and 5,698,767, the contents of which are incorporated by reference herein in their entirety.

In another embodiment, human anti-TIM-3 antibodies are prepared using phage display, wherein the phage comprise nucleic acids encoding antibodies produced in transgenic animals previously immunized with TIM-3. In a preferred embodiment, the transgenic animal is a HuMab, KM or Kirin mouse. See, for example, U.S. patent No. 6,794,132, the contents of which are incorporated by reference herein in their entirety.

Immunization of human Ig mice

In one embodiment of the invention, human Ig mice are immunized with purified or enriched preparations of TIM-3 antigen, recombinant TIM-3 protein, or cells expressing TIM-3 protein. See, e.g., Lonberg et al (1994) (supra); fishwild et al (1996) (supra); PCT publication WO 98/24884 or WO 01/14424, the contents of which are incorporated herein by reference in their entirety. In a preferred embodiment, 6-16 week old mice are immunized with 5 μ g to 50 μ g of LAG-3 protein. Optionally, a portion of LAG-3 fused to a non-LAG-3 polypeptide is used.

In one embodiment, TIM-3 antigen used in complete Freund's adjuvant is used for Intraperitoneal (IP) or Intravenous (IV) immunization of transgenic mice, followed by subsequent IP or IV immunization with antigen used in incomplete Freund's adjuvant. In other embodiments, adjuvants other than freund's adjuvant or whole cells in the absence of adjuvant are used. Plasma may be screened by ELISA and fusion may be performed using cells from mice with sufficient titers of anti-TIM-3 human immunoglobulin.

Production of hybridomas producing human monoclonal antibodies of the invention

To generate hybridomas that produce the human monoclonal antibodies of the invention, spleen and/or lymph node cells from immunized mice can be isolated and fused with an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual (Antibodies: A Laboratory Manual), Cold Spring Harbor Press, New York, Cold Spring Harbor Publications, N.Y..

Generation of transfectomas producing monoclonal antibodies of the invention

Antibodies of the invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody genes are linked into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody genes.

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)) in Academic Press, San Diego, Calif. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from Cytomegalovirus (CMV), simian virus 40(SV40), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the β -globin promoter. Furthermore, the regulatory elements are composed of sequences from different sources, such as the SR α promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of the human T cell leukemia virus type 1 (Takebe et al (1988) mol.cell.biol.8: 466-472). The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or separate expression vectors. In a preferred embodiment, the variable regions are used to render the V by inserting them into expression vectors that already encode the heavy and light chain constant regions of the desired isotype_HSegment and theOne or more C's in a carrier_HThe segments are operably connected and V_LSegment and C within the carrier_LThe segments are operably linked to produce a full-length antibody gene of any antibody isotype. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may also carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically, a selectable marker gene confers resistance to a drug, such as G418, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR-host cells in the case of methotrexate selection/amplification) and the neo gene (for G418 selection).

To express the light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" of various forms is intended to cover the usually used to introduce exogenous DNA into prokaryotic or eukaryotic host cells in a variety of techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably mammalian host cells, is most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a correctly folded and immunologically active antibody.

Preferred mammalian host cells for expression of recombinant antibodies of the invention include chinese hamster eggsNest (CHO cells) (including dhfr)^-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-. In particular, another preferred expression system for use with NSO myeloma cells is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or, more preferably, to secrete the antibody into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Immunoconjugates

The antibodies of the invention can be conjugated to therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide or hydrazone linker. More preferably, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Cit, Ser or Glu. ADCs can be prepared as described in the following documents: U.S. patent nos. 7,087,600; 6,989,452, respectively; and 7,129,261; PCT publications WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; U.S. patent publication 20060024317; 20060004081, respectively; and 20060247295, the disclosures of which are incorporated herein by reference.

Bispecific molecules

In another aspect, the disclosure features bispecific molecules comprising one or more antibodies of the invention linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand of a receptor), to produce bispecific molecules that bind to at least two different binding sites or target molecules. Thus, a "bispecific molecule" as used herein includes molecules having three or more specificities.

In one embodiment, the bispecific molecule has a third specificity in addition to the anti-Fc binding specificity and the anti-TIM-3 binding specificity. The third specificity may be for an anti-Enhancer Factor (EF), such as a molecule that binds to a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell. For example, the anti-enhancer factor may bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) or other immune cells, thereby causing an increase in the immune response against the target cell.

Bispecific molecules can have many different forms and sizes. At one end of the size spectrum, the bispecific molecule retains the traditional antibody format except that it does not have two binding arms with the same specificity, but two binding arms each with a different specificity. At the other extreme, is a bispecific molecule consisting of two single-chain antibody fragments (scFv's) connected by a peptide chain, the so-called Bs (scFv) ₂Constructs. A bispecific molecule of intermediate size comprises two different f (ab) fragments linked by a peptidyl linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, e.g., Kufer et al (cited above); cao and Suresh, Bioconjugate Chemistry,9(6),635-644 (1998); and van Spriel et al, Immunology Today,21(8),391-397(2000) and references cited therein.

Pharmaceutical composition

In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more antibodies of the invention formulated with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present invention may also be administered in combination therapy with, for example, another immunostimulant, an anti-cancer agent, an anti-viral agent, or a vaccine, such that the anti-TIM-3 antibodies enhance the immune response against the vaccine.

The pharmaceutical composition may comprise any number of excipients. Excipients that may be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending aids, solubilizers, colorants, flavorants, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents and combinations thereof. The selection and use of suitable excipients is taught in Gennaro's eds, Remington: The Science and Practice of Pharmacy (Remington: Science and Practice of Pharmacy), 20 th edition (Lippincott Williams & Wilkins 2003), The disclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect it against the action of acids and other natural conditions that may inactivate it. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the antibodies of the invention may be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

The pharmaceutical compositions may be in the form of a sterile aqueous solution or dispersion. They may also be formulated as microemulsions, liposomes or other ordered structures suitable for high drug concentrations.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration, and will generally be that amount of the composition which produces a therapeutic effect. Generally, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, in one hundred percent, in combination with a pharmaceutically acceptable carrier.

The dosing regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and to maintain uniform dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit doses for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained release formulation, in which case less frequent administration is required.

For administration of the antibody, the dose is in the range of about 0.0001 to 100mg per kg of body weight of the host, and more typically 0.01 to 5mg per kg of body weight of the host. For example, the dose may be 0.3mg per kg body weight, 1mg per kg body weight, 3mg per kg body weight, 5mg per kg body weight or 10mg per kg body weight or in the range of 1mg/kg-10 mg/kg. Exemplary treatment regimens entail administering once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every 3 months, or once every 3 months to 6 months. A preferred dosing regimen for an anti-TIM-3 antibody of the present invention comprises 1mg per kg body weight or 3mg per kg body weight, via intravenous administration, wherein the antibody is administered using one of the following dosing schedules: (i) once every four weeks for six doses and then once every three months; (ii) once every three weeks; (iii) administered at 3mg per kg body weight followed by 1mg per kg body weight once every three weeks. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1 μ g/mL to 1000 μ g/mL, and in some methods, to achieve a plasma antibody concentration of about 25 μ g/mL to 300 μ g/mL.

A "therapeutically effective dose" of an anti-TIM-3 antibody of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disorders or disability due to the affliction with the disease. For example, for treating a subject with a tumor, a "therapeutically effective dose" preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%, relative to an untreated subject. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.

The pharmaceutical compositions may be controlled release formulations including implants, transdermal patches and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

The therapeutic composition can be administered via a medical device, such as (1) a needleless hypodermic injection device (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 and 4,596,556); (2) micro infusion pumps (U.S. patent No. 4,487,603); (3) transdermal devices (U.S. patent No. 4,486,194); (4) infusion devices (U.S. Pat. nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. nos. 4,439,196 and 4,475,196); the disclosures of these documents are incorporated herein by reference.

In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the present invention cross the blood-brain barrier, they may be formulated in liposomes that may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g., U.S. Pat. nos. 4,522,811; 5,374,548, respectively; 5,416,016; and 5,399,331; v. ranade (1989) j.clin.pharmacol.29: 685; umezawa et al, (1988) biochem. Biophys. Res. Commun.153: 1038; blueman et al (1995) FEBS Lett.357: 140; m. Owais et al (1995) Antimicrob. Agents Chemother.39: 180; briscoe et al (1995) am.J.Physiol.1233: 134; schreier et al (1994) J.biol.chem.269: 9090; keinanen and Laukkanen (1994) FEBS Lett.346: 123; and Killion and Fidler (1994) Immunomethods 4: 273.

Uses and methods of the invention

The antibodies (compositions, bispecific molecules, and immunoconjugates) of the invention have a number of in vitro and in vivo utilities, including, for example, enhancing immune responses by blocking TIM-3. Such antibodies can be administered to cells in culture in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of circumstances. Accordingly, in one aspect, the invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject an antibody, or antigen-binding portion thereof, of the invention, such that the immune response in the subject is modulated. Preferably, the response is enhanced, stimulated or upregulated.

Preferred subjects include human patients in need of an enhanced immune response. The methods are particularly useful for treating human patients suffering from conditions that can be treated by enhancing an immune response, such as a T cell-mediated immune response. In a particular embodiment, the method is particularly useful for treating cancer in vivo. To achieve antigen-specific immunity enhancement, an anti-TIM-3 antibody may be administered with the antigen of interest or the antigen may already be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject). When an antibody to TIM-3 is administered with another agent, the two may be administered in either order or simultaneously.

In view of the ability of the anti-TIM-3 antibodies of the present invention to inhibit the binding of TIM-3 to galectin 9 or Ptdser molecules and to activate antigen-specific CD4+ or CD8+ T cells, the present invention also provides in vitro and in vivo methods of using the antibodies to enhance or up-regulate antigen-specific T cell responses. For example, the invention provides a method of enhancing an antigen-specific T cell response, the method comprising contacting the T cell with an antibody of the invention such that the antigen-specific T cell response is enhanced or up-regulated.

The invention also provides a method for stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, the method comprising administering to the subject an antibody of the invention, thereby stimulating an immune response (e.g., an antigen-specific T cell response) in the subject. In a preferred embodiment, the subject is a tumor-bearing subject and an immune response against the tumor is stimulated. In another preferred embodiment, the subject is a virus-bearing subject and an immune response against the virus is stimulated.

In another embodiment, the invention provides a method for inhibiting the growth of a tumor cell in a subject, the method comprising administering to the subject an antibody of the invention, thereby inhibiting the growth of the tumor in the subject. In yet another embodiment, the invention provides a method for treating a viral infection in a subject, the method comprising administering to the subject an antibody of the invention, thereby treating the viral infection in the subject.

These and other methods of the present invention are discussed in more detail below.

Cancer treatment

Blockade of TIM-3 by antibodies may enhance the immune response of patients to cancer cells. In one aspect, the invention relates to the use of an anti-TIM-3 antibody to treat a subject in vivo, thereby inhibiting the growth of a cancerous tumor. anti-TIM-3 antibodies may be used alone to inhibit the growth of cancerous tumors. Alternatively, anti-TIM-3 antibodies may be used in combination with other immunogenic agents, standard cancer treatments, or other antibodies, as described below.

Accordingly, in one embodiment, the present invention provides a method of inhibiting tumor cell growth in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TIM-3 antibody or antigen-binding portion thereof. Preferably, the antibody is a chimeric, human or humanized anti-TIM-3 antibody.

Preferred cancers for which growth can be inhibited using the antibodies of the invention include cancers that are generally responsive to immunotherapy.

Combination therapy

In another aspect, the invention provides combination therapy methods in which an anti-TIM-3 antibody (or antigen-binding portion thereof) of the invention is co-administered with one or more additional antibodies effective to stimulate an immune response, thereby further enhancing, stimulating, or up-regulating the immune response in a subject. In one embodiment, the present invention provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an anti-TIM-3 antibody and one or more additional immunostimulatory antibodies, such as an anti-LAG-3 antibody, an anti-PD-1 antibody, and/or an anti-CTLA-4 antibody, thereby stimulating an immune response in the subject, e.g., to inhibit tumor growth or stimulate an anti-viral response.

In another embodiment, the invention provides a method for treating a hyperproliferative disease (e.g., cancer), comprising administering to a subject an anti-TIM-3 antibody and another antibody, e.g., an anti-LAG-3 antibody, an anti-PD-1 antibody, and/or a CTLA-4 antibody.

In certain embodiments, the combination of therapeutic antibodies discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions comprising each antibody in a pharmaceutically acceptable carrier.

Optionally, the combination of anti-TIM-3 and one or more additional antibodies (e.g., anti-CTLA-4 antibodies and/or anti-LAG-3 antibodies and/or anti-PD-1 antibodies) can be further combined with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) j. immunol.173: 4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1, and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. The combined TIM-3 and CTLA-4 and/or LAG-3 and/or PD-1 blockade may be further combined with a vaccination regimen, such as any of the vaccination regimens discussed in detail above with respect to monotherapy using anti-TIM-3 antibodies.

Combined TIM-3 and CTLA-4 and/or LAG-3 and/or PD-1 blockade may also be further combined with standard cancer treatments. For example, TIM-3 and CTLA-4 and/or LAG-3 and/or PD-1 blockade in combination may be effectively combined with a chemotherapeutic regimen. In these cases, it is possible to reduce the dose of other chemotherapeutic agents administered with the combinations of the present disclosure (Mokyr et al (1998) Cancer Research 58: 5301-5304). An example of such a combination is the combination of anti-TIM-3 and anti-CTLA-4 antibodies and/or anti-LAG-3 antibodies and/or anti-PD-1 antibodies further in combination with amifostine for the treatment of melanoma. Another example is the combination of anti-TIM-3 and anti-CTLA-4 antibodies and/or anti-LAG-3 antibodies and/or anti-PD-1 antibodies further in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific basis for the combined use of TIM-3 and CTLA-4 and/or LAG-3 and/or PD-1 blockade with chemotherapy is that cell death as a result of the cytotoxic effects of most chemotherapeutic compounds should lead to elevated levels of tumor antigens in the antigen presenting pathway. Other combination therapies that may produce a synergistic effect by cell death with combined TIM-3 and CTLA-4 and/or LAG-3 and/or PD-1 blockade include radiation therapy, surgery or hormone deprivation. Each of these protocols produces a source of tumor antigens in a host. The angiogenesis inhibitor may also be combined with a combined TIM-3 and CTLA-4 and/or LAG-3 and/or PD-1 blockade. Inhibition of angiogenesis causes tumor cell death, which may be the source of tumor antigens supplied to the host antigen presentation pathway.

The disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, gene bank (Genbank) sequences, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

Examples

Example 1 phage panning, screening and affinity maturation

Phage libraries

Antibody single chain phage display libraries are generated by cloning a repertoire of light chain variable regions (VL) and heavy chain variable regions (VH). Heavy and light chain repertoires were generated by PCR amplification of human lymphocytes collected mainly from the periphery. The VL repertoire was mixed with the VH repertoire and PCR was performed with overlapping primers. The final form of the antibody is a single chain Fv (scFv) with a VH fragment and a VL fragment connected by a flexible linker peptide (GGGGSGGGGSGGGGS (SEQ ID NO: 51)).

Phage library panning against human TIM3

Selection of phage particles displaying specific scFv fragments was performed in Immuno 96MicroWell^TMPerformed on plates (Nunc, Denmark). First, 50. mu.g/ml TIM3 recombinant protein (Acrobiosystems, Cat # TM3-H5229) in Phosphate Buffered Saline (PBS) was coated onto plates overnight at 4 ℃. After blocking with 2% (w/v) milk powder in PBS (2% MPBS), a solution containing about 10% was added ¹¹Library of individual phage particles and plates were incubated at room temperature (RT; 25-28 ℃) for 2 hours. Unbound phage were eliminated by washing 10-20 times with PBS containing 0.1% Tween 20 (PBS-T) followed by 10-20 washes with PBS. Bound phage were eluted by incubation with 50. mu.l of 1. mu.g/. mu.l trypsin for 10 min followed by 50. mu.l of 50mM glycine hydrochloride (pH 2.0) neutralized immediately after 10 min with 50. mu.l of 200mM Na2HPO4(pH 7.5). Four rounds of panning were performed.

Phage screening

From the third and fourth rounds of panning output, phages were selected and tested for binding to human TIM3, human TIM3(AcroBiosystems, catalog # TM3-H5229) was coated on 96-well plates at 0.1 μ g/mL, single clonal phages were added to the plates, unbound phages were washed off, and binding was detected by anti-M13 secondary antibody (Abcam, catalog # ab 50370).

ELISA positive clones were sequenced and 10 unique sequences were identified, including clones TIM3-6, TIM3-4G7 and TIM 3-11. The amino acid sequence ID numbers of the heavy/light chain variable regions of anti-TIM 3 antibodies TIM3-6, TIM3-4G7 and TIM3-11 are summarized in Table 1.

Affinity maturation

To increase the binding affinity of TIM3-6, two phage libraries of VH and VL were constructed for panning. After 4 rounds of panning, the test variants were screened by ELISA for positive binding to human TIM3(AcroBiosystems, catalog # TM 3-H5229). The off-rate rating of the positive variants was determined by Octet Red 96 (Fortebio). Clones with increased off-rates were selected and converted to full-length IgG for analysis. The amino acid sequence ID numbers of anti-TIM 3 antibodies TIM3-6.10, TIM3-6.11 and TIM3-6.12 are summarized in Table 1.

The nucleotide sequences encoding the heavy and light chains of the anti-TIM 3 antibody were inserted into the expression vector pcdna3.1 (Invitrogen). ExpicHO was used according to the manufacturer's instructions^TMExpression System (ThermoFisher) vectors were co-transfected into CHO-S cells. Transfected cells in ExpicHO^TMThe expression medium was cultured for 12 days, and then the culture supernatant was harvested and sent for purification by protein a affinity chromatography (GE Healthcare).

Example 2 physicochemical analysis

Antibody TIM3-6 was tested in size exclusion chromatography. In particular, 100mM sodium phosphate +100mM Na was used₂SO₄20 μ G of sample was injected on a TSK G3000SWXL column as running buffer, pH 7.0. The run time was 30 minutes. All measurements were performed on an Agilent1220 HPLC. Data were analyzed using OpenLAB software.

The major peak in SEC for antibody TIM3-6 was above 95%, indicating high purity and integrity of the purified antibody.

Example 3 specific binding of anti-TIM-3 antibodies to human TIM-3

ELISA assay was used to determine the relative binding activity of the antibody to recombinant human TIM-3.

Carbonate buffer (pH 9.6, 1.59g sodium carbonate and 2.93g sodium bicarbonate in 1L water) was dissolved by incubation at 4 ℃ overnightThe human TIM-3 protein (Acrobiosystems, Cat # TM3-H5229) was immobilized on 96-well plates. The plates were then blocked with 1% BSA in PBS for one hour at 37 ℃. After blocking, plates were washed three times with PBST (PBS containing 0.05% Tween 20). Serially diluted anti-TIM-3 antibodies TIM3-6.12, TIM3-6, TIM3-11 and TIM3-4G7, human IgG control (prepared according to US20190016800a1, having the amino acid sequences listed in SEQ ID NO: 52) and ABTIM3 analogue (used as reference antibody, prepared according to US 2015/0218274a1, having the amino acid sequences of the heavy and light chains listed in SEQ ID NOs: 53 and 54) in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) were incubated with the immobilized proteins, respectively, for one hour at 37 ℃. After binding, the plates were washed 3 times with PBST, incubated with peroxidase-labeled donkey anti-human IgG (Jackson Immuno Research) diluted 1/15,000 in binding buffer for one hour at 37 ℃, washed again, developed with TMB and developed with 1M H ₂SO₄And (6) terminating.

The absorbance at 450nm to 620nm is measured. Cloned EC binding to human TIM-3₅₀And a representative binding curve is shown in figure 1.

The results indicate that the anti-TIM 3 antibody specifically binds to human TIM-3, with the binding activity of antibody TIM3-6.12 being comparable to that of the ABTIM3 analog.

Example 4 affinity of anti-TIM-3 antibodies

By usingSurface plasmon resonance of the T200 system (Biacore, GE Healthcare) was used to measure the kinetic binding activity of anti-TIM-3 antibodies to human TIM-3(Acrobiosystems, Cat # TM 3-H5229).

Approximately 6800RU of anti-human IgG (Fc) antibody (GE catalog # BR-1008-39) was immobilized on a CM5 sensor chip by amine coupling chemistry. Antibodies (TIM3-6.10, TIM3-6.11, TIM3-6.12) were injected onto the surface of the immobilized goat anti-human IgG antibody. HBS-EP + buffer was used as the running buffer. Human TIM-3 protein was injected onto antibody surfaces at various concentrations ranging from 1.56nM to 50 nM.After each injection cycle, the CM5 chip surface was regenerated using 3M magnesium chloride solution injections. Background subtraction binding sensorgrams were used to analyze association Ka and dissociation Kd, and equilibrium dissociation constant K_D. The resulting data set was fitted to a 1:1 Langmuir binding model using Biacore T200 evaluation software.

Table 2 below summarizes the affinity of anti-TIM-3 antibodies for recombinant human TIM-3.

TABLE 2 affinity of anti-TIM-3 antibodies for recombinant human TIM-3

Antibody #	Ka(M-1S-1)	Kd(S-1)	KD(M)
				TIM3-6.10	2.02E+05	4.15E-04	2.05E-09
TIM3-6.11	2.30E+05	3.44E-04	1.50E-09
				TIM3-6.12	2.23E+05	3.70E-04	1.66E-09

The results show that the three antibodies have similar affinities for recombinant human TIM-3, with antibody TIM3-6.11 having the highest affinity.

Example 5 anti-TIM-3 antibodies do not cross-react with human TIM-1

ELISA assays were used to determine the relative binding activity of antibodies to human TIM-1.

Human TIM-1(Sino biological, Cat #11051-HNCH) was immobilized on 96-well plates by incubation at 4 ℃ overnight. Non-specific binding sites were blocked with 1% BSA in PBS for one hour at 37 ℃. After blocking, plates were washed three times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of anti-TIM-3 antibody TIM3-6, ABTIM3 analogs and human IgG controls were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with the immobilized proteins for one hour at 37 ℃. After binding, the plates were washed 3 times with PBST, incubated with peroxidase-labeled donkey anti-human IgG (Jackson Immuno Research) diluted 1/15,000 in binding buffer for one hour at 37 ℃, washed again, developed with TMB and developed with 1M H ₂SO₄And (6) terminating.

The absorbance at 450nm to 620nm is measured. The results indicate that TIM3-6 does not cross-react with human TIM-1.

Example 6 anti-TIM-3 antibodies do not cross-react with human TIM-4

ELISA assays were used to determine the relative binding activity of anti-TIM-3 antibodies to human TIM-4.

Human TIM-4(Sino biological, Cat #12161-H08H) was immobilized on 96-well plates by incubation at 4 ℃ overnight. Non-specific binding sites were blocked with 1% BSA in PBS for one hour at 37 ℃. After blocking, plates were washed three times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of anti-TIM-3 antibody TIM3-6, ABTIM3 analogs and human IgG controls were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with the immobilized proteins for one hour at 37 ℃. After binding, plates were washed with PBST 3Next, a 1/15,000 dilution of peroxidase-labeled donkey anti-human IgG (Jackson Immuno Research) in binding buffer was incubated at 37 ℃ for one hour, washed again, developed with TMB and developed with 1M H₂SO₄And (6) terminating.

The absorbance at 450nm to 620nm is measured. The results indicate that TIM3-6 does not cross-react with human TIM-4.

Example 7 anti-TIM-3 antibodies block the interaction of galectin 9 with TIM-3

To evaluate the inhibitory effect of anti-TIM-3 antibodies on the human TIM-3/galectin 9 interaction, HTRF blocking assays were performed using a commercially available kit (Cisbio, catalog #63ADK000CTLPEB) in which Eu3+ cryptate labeled TIM3 reacted with Tag-Gal 9.

To test antibodies in this assay, serial dilutions of anti-TIM-3 antibodies TIM3-6, TIM3-11, TIM3-4G7, F38-2E2(ebioscience, catalog #16-3109-85), ABTIM3 analogs, and human IgG controls were added to TIM3-EuK protein/Tag-Gal 9 protein mixtures, respectively. The resulting mixture was incubated at room temperature for 1 hour and then the fluorescence emission was read. IC blocking interaction of galectin 9 and TIM-3₅₀Values and representative curves are shown in fig. 2.

The results showed that anti-TIM 3 antibodies TIM3-6, TIM3-11 and TIM3-4G7 had lower IC than ABTIM3 analogues and F38-2E2₅₀Values blocked the interaction between galectin 9 and TIM-3, indicating a better blocking activity. Antibody TIM3-6 has optimal blocking activity.

Example 8 binding of anti-TIM-3 antibodies to CHO-K1-TIM3 expressed cell-surface TIM-3

anti-TIM-3 antibodies were tested for their ability to bind to human TIM-3 stably expressed on CHO-K1 cells. Chinese hamster ovary epithelial cell CHO-K1 cell line (ATCC, catalog # CCL-61) maintained at 37 ℃ with 5% CO ²In F-12K medium containing 10% FBS in a humidified incubator. Polyethyleneimine (MW25K, 23966-2, Polyscience) was diluted to 1mg/mL and added to pcDNA3.1 vector containing TIM3 cDNA (NP-116171.3) and incubated with it at 37 ℃ for 10 minutes. Will be mixed withThe compounds were added to cell cultures and incubated with them for 3 hours for DNA transfection.

anti-TIM 3 antibody was serially diluted in PBS buffer containing 0.5% BSA. The antibody was added to CHO-K1-TIM3 cells and incubated with them for 30 minutes at 4 ℃. Cells were pelleted (3 min, 600 Xg), washed once with PBS buffer containing 0.5% BSA and pelleted again. Then, the cells were incubated with PE conjugated AffiniPure goat anti-human IgG (Fc γ fragment specific) (Jackson ImmunoResearch catalog #109-116-098) diluted 1:100 at 4 ℃ for 30 minutes. Cells were washed twice as described above and resuspended in PBS buffer. The cells were then sent to a BD Accuri C5 flow cytometer (BD Bioscience) for binding activity analysis. Calculation of EC₅₀The value is obtained. Representative curves of antibody binding to TIM3 are shown in figure 3.

The results indicate that TIM3-6.12 specifically binds to human TIM-3 stably expressed on CHO-K1 cells and that its binding activity is comparable to that of ABTIM3 analogues.

Example 9 anti-TIM-3 antibodies induce IL-2 release from human T cells

Functional activity of anti-TIM 3 antibodies on primary T cells was assessed using human PBMC cultures stimulated with superantigen SEB.

Human PBMCs from healthy donors were stimulated with SEB (Toxin Technology, catalog # BT202) for 48 hours. Serial dilutions of antibodies TIM3-6.12, F38-2E2(ebioscience, catalog #16-3109-85) and human IgG controls were added to and incubated with PBMC cultures for 3 days, respectively. IL-2 levels in the supernatants were then measured using the human IL-2DuoSet ELISA kit (R & D, catalog # DY 202).

As shown in FIG. 4, antibody TIM3-6.12 induced IL-2 release by T cells at a concentration of 30. mu.g/mL.

Example 10 anti-TIM 3 antibody blocks the interaction of TIM-3 with phosphatidylserineBlockade of phosphatidylserine-TIM-3 interaction was determined as follows.

Briefly, Jurkat T cells (CBTCCCAS, Clone E6-1) were incubated with 1. mu.g/mL of anti-human CD95(Fas) antibody (Clone EOS9.1, Biogems, catalog #08011-25-500) for 16 hours. When Jurkat T cells are induced to undergo apoptosis, phosphatidylserine is turned to the extracellular surface of the cell to which TIM-3 may bind.

Mu.l (40. mu.g/mL) of human TIM3-mFc protein (amino acid sequence listed in SEQ ID NO: 55) was mixed with 25. mu.l of serially diluted antibody (starting at 1. mu.g/mL) in annexin V binding buffer (Biolegend catalog 422201) and incubated with it for 20 min at Room Temperature (RT). The mixture was then added to 2X 10 in 50. mu.l of binding buffer (PBS containing 0.5% BSA) ⁵Jurkat T cells. After incubation at 4 ℃ for 40 min, the cells were pelleted (3 min, 600 Xg), washed once with binding buffer containing 0.5% BSA and pelleted again. PE conjugated AffiniPure goat anti-mouse IgG (subclass 1+2a +2b +3) (Fc γ fragment specific) (Jackson ImmunoResearch, catalog #115-115-164) diluted 1:100 was then added to the cells and the cells were analyzed with a BD Accuri C5 flow cytometer.

As shown in FIG. 5, anti-TIM 3 antibody TIM3-6.12 blocked the interaction of TIM 3-phosphatidylserine, IC thereof₅₀Values were similar to ABTIM3 analogs.

Example 11 internalization of CHOK1-TIM3 cells against TIM3 antibody

The anti-TIM 3 antibody was first labeled with the pHAb amine reactive dye (Promega, G9845), a pH sensitive dye that fluoresces at pH values less than 7.0, according to the manufacturer's instructions.

CHOK1-TIM3 produced in example 8 was cultured in DMEM/F12 medium (Gibco) containing 10% fetal bovine serum. Cells in log phase were collected, 50. mu.l of 20. mu.g/ml dye-labeled antibody was added, and incubated at 37 ℃ for 2 hours, 6 hours, or 24 hours. The cells were then sent for analysis using a BD Accuri C5 flow cytometer.

As shown in FIG. 6, a fluorescent signal was detected, indicating that the anti-TIM-3 antibody TIM3-6.12 may have been internalized into endosomes (pH 6.0-6.5) and lysosomes (pH 4.5-5.5) within the cells.

Example 12 anti-TIM-3 antibodies do not bind to C1q

C1q binding is the first step in Complement Dependent Cytotoxicity (CDC). To test the binding activity of the antibodies to C1q, an ELISA binding assay was performed.

Briefly, 96-well polystyrene ELISA plates were coated with antibody TIM3-6.12, rituximab analogue (used as positive control, prepared according to US5736137, with SEQ ID NO:56 and 57 as heavy and light chain amino acid sequences), human IgG control at a concentration ranging from 0.94-60. mu.g/mL in PBS. After overnight incubation at 4 ℃, plates were washed three times with PBST and then blocked with 200 μ l PBS containing 1% BSA for one hour at 37 ℃. Plates were washed three times with PBST and then 0.05. mu.g/well of C1q (Calbiochem, Cat #204876) diluted in PBS containing 0.05% Tween20 and 0.5% BSA was added. After one hour incubation at 37 ℃, plates were washed three times with PBST and 50 μ Ι _ of anti-C1 q antibody-HRP (1:400, abcam, catalog # ab46191) was added to each well. Plates were incubated for one hour at room temperature and then washed three times with PBST. Then, 100. mu.L of a substrate of HRP, 3',5,5' -tetramethylbenzidine (TMB: Thermo catalog #34028) was added to each well, and the plate was incubated at room temperature for 20 minutes. The reaction is applied to 1M H ₂SO₄The reaction was terminated and absorbance was measured at 450nM using a microplate reader.

As shown in FIG. 7, antibody TIM3-6.12 did not bind to C1 q.

Example 12 anti-TIM 3 antibodies do not induce antibody-dependent cell-mediated cytotoxicity (ADCC)

In CHO-K1-TIM3Antibody-dependent cell-mediated cytotoxicity (ADCC) assays were performed on the cells. CHO-K1-produced in example 8TIM3Cells were seeded at a density of 10,000 cells per well and preincubated for 30 minutes with 100nM or 10nM anti-TIM 3 antibody in assay buffer (phenol red-free MEM medium + 1% FBS). PBMC effector cells from healthy donors were added to elicit ADCC effects with E/T ratios of 10:1, 25:1 or 50: 1. The ADCC effect of rituximab analogs on Raji (CBTCCCAS, catalog # TCHu 44) was used as an internal control to ensure assay quality. At 37 deg.C, 5% CO₂After incubation in the incubator for 24 hours, cell supernatants were collectedReleased LDH was measured using a cytotoxic LDH assay kit (Dojindo, catalog # CK 12). OD read on F50(Tecan)_490nmAbsorbance of (d) in (d). The percentage of cell lysis was calculated according to the following formula,

cell lysis% -100 × (OD)_{Sample (I)}-OD_{Target cells plus effector cells})/(OD_{Maximum release}-OD_{Minimum release}). Data were analyzed by Graphpad Prism.

As shown in FIG. 8, the anti-TIM 3 antibody TIM3-6.12 did not have ADCC activity on CHO-K1-Tim3 cells.

Example 13 anti-TIM 3 antibodies do not induce Complement Dependent Cytotoxicity (CDC)

In CHO-K1-TIMComplement Dependent Cytotoxicity (CDC) assays were performed on 3 cells. CHO-K1-produced in example 8TIM3 cells were seeded at a density of 5,000 cells per well and preincubated for 30 minutes with 100nM or 10nM antibody in assay buffer (phenol red-free MEM medium + 1% FBS). Plasma from healthy donors at concentrations of 10 vol%, 20 vol% and 50 vol% were then added to the plates to induce CDC effects. At 37 deg.C, 5% CO₂After 4 hours incubation in the incubator, Cell-Titer Glo reagent (Promega, catalog # G7572) was added to the cells and RLU data were read on F200 (Tecan). The percentage of cell lysis was calculated according to the following formula,

cell lysis% (% 100 × (1- (RLU)_{Sample (I)})/(RLU_{Cell + NHP}) NHP) wherein NHP represents normal human plasma.

Data analyzed by Graphpad Prism showed that anti-TIM 3 antibody TIM3-6.12 had no CDC activity on CHO-K1-TIM3 cells.

Example 14 pharmacokinetics of anti-TIM 3 antibodies in rats

The pharmacokinetic profile of TIM3-6.12 was evaluated in rats. For the study, TIM3-6.12 was injected intravenously into rats at a dose of 10 mg/kg. Blood samples were taken at various time points between 0 and 360 hours (0-15 days). All samples were processed as plasma and stored frozen at-70 ℃ to-86 ℃ until analysis. The concentration of TIM3-6.12 present in the serum was determined by ELISA.

Table 3 shows the pharmacokinetic properties determined as above.

TABLE 3 summary of the pharmacokinetic Properties of TIM3-6.12

AUC_last(area under the plasma level time curve from 0 to the final measurable plasma drug concentration at time t), AUC_{INF_obs}(area under the concentration-time curve 0- ∞), Vz __obs(volume of distribution), Cl __obs(clearance rate).

The sequences in this application are summarized below.

Claims (12)

1. An isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region comprise the amino acid sequences of:

(1) 2, 6 and 9, respectively;

(2) 1, 5 and 9, respectively;

(3) 3, 7 and 10, respectively; or

(4) 4, 8 and 11, respectively;

wherein the antibody or antigen binding fragment thereof binds to TIM-3.

2. The antibody, or antigen-binding portion thereof, of claim 1, comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO 25, 24, 26, or 27.

3. The antibody, or antigen binding portion thereof, of claim 1, further comprising a light chain variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 region, the CDR2 region, and the CDR3 region comprise the amino acid sequences of:

(1) 14, 18 and 21, respectively;

(2) 12, 17 and 21, respectively;

(3) 13, 17 and 21, respectively;

(4) 14, 17 and 21, respectively;

(5) 15, 19 and 22, respectively; or

(6) 16, 20 and 23, respectively.

4. The antibody or antigen binding portion thereof of claim 1, further comprising a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID No. 29, 28, 30, or 31, wherein X is in SEQ ID No. 29₁Y and X₂N; or X₁Is N and X₂(ii) S; or X₁Y and X₂＝S。

5. Separated one from the otherA monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to: (1) are SEQ ID NO 25 and 29, respectively, wherein X in SEQ ID NO 29₁Y and X₂N; or X₁Is N and X₂(ii) S; or X₁Y and X₂(ii) S; (2) 24 and 28, respectively; (3) 26 and 30, respectively; or (4) SEQ ID NOS: 27 and 31, respectively.

6. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, comprising a heavy chain constant region having an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID No. 33 or 32, and/or a light chain constant region having an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID No. 35, 34, or 36.

7. The antibody or antigen-binding portion thereof of claim 1, which (a) binds to human TIM 3; (b) does not bind to TIM-1; (c) does not bind to TIM-4; (d) inhibiting the binding of TIM-3 to galectin-9; (e) inhibiting the binding of TIM-3 to phosphatidylserine; (f) inducing the T cells to release IL-2; (g) does not induce ADCC on cells expressing TIM 3; and/or (h) does not induce CDC on cells expressing TIM 3.

8. The antibody or antigen binding portion thereof of claim 1, which is a human, mouse, chimeric or humanized antibody.

9. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of claim 1, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, further comprising an antineoplastic agent.

11. A method of inhibiting tumor growth in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 9.

12. A method of enhancing an immune response in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 9.