CN113150150A

CN113150150A - TIM3 binding molecules and uses thereof

Info

Publication number: CN113150150A
Application number: CN202110200647.9A
Authority: CN
Inventors: 吴炯; 杨斌; 潘燕峰; 夏玉龙; 唐英杰; 金昕; 孙馨
Original assignee: Suzhou Hengkang Life Science Co ltd
Current assignee: Suzhou Hengkang Life Science Co ltd
Priority date: 2020-02-24
Filing date: 2021-02-23
Publication date: 2021-07-23
Anticipated expiration: 2041-02-23
Also published as: WO2021169948A1; CN113150150B

Abstract

The present invention provides a molecule that binds TIM3, which is capable of specifically recognizing endogenous and exogenous TIM3, and has activity to activate T cells, a useful therapeutic agent for the treatment of various cancers, especially hematologic tumors, like leukemia and lymphoma.

Description

TIM3 binding molecules and uses thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a molecule for binding TIM 3.

Background

T-cell immunoglobulins and mucin family-3 (T-cell immunoglobulin and mucin-domain binding-3, Tim3) proteins, which are members of the Tim family, are known as HAVCR2(Hepatitis A virus cellular receptor-2) proteins and are cell membrane receptors encoded by the HAVCR2 gene. It was found that Tim-3 is expressed in activated CD4⁺Th1、Th17、CD8⁺Tc1, and maintains immune tolerance by negatively regulating the function of the cells.

It is known in the art that T cell depletion is mediated by several immune checkpoint inhibitory receptors (e.g., PD1, TIM3, CTLA-1, LAG3, etc.), and that interaction of TIM3 and PD1 signaling pathways plays an important role in T cell depletion. Immune checkpoints play an important role in maintaining autoimmune tolerance and avoiding the immune system from attacking self organs, and many cancers achieve immune evasion by deregulating expression of immune checkpoint proteins. The removal of cancer cells in vivo by means of the immune function of the body through blocking immune checkpoints and restoring the body's own anti-tumor immune response has been one of the research directions of oncologists.

The inhibitory monoclonal antibody drugs aiming at immune check points CTLA-1 and PD-1 have achieved objective curative effects in clinical treatment of various tumors such as melanoma, renal cancer, lung cancer and the like. However, there are still many patients who cannot benefit from these two target therapies, and therefore it is desirable to find new drugs against immune checkpoints, such as Tim-3, to achieve treatment of diseases including tumors by inhibiting immune checkpoints.

Disclosure of Invention

The present invention relates to molecules, such as antibodies or antigen-binding fragments thereof, that bind to TIM3, which are rapidly and strongly internalized on cells expressing TIM3, such as tumor cells, and have significant therapeutic effects in the treatment of various diseases, such as tumors, particularly hematologic tumors, like leukemia and lymphoma. Moreover, they show strong immunostimulatory cytokine release in Mixed Lymphocyte Reaction (MLR), and thus have significant utility as immunostimulatory drugs.

In one aspect, the invention provides a TIM3 binding molecule comprising one or more amino acid sequences selected from the group consisting of: as shown in SEQ ID NO: 6, as shown in SEQ ID NO: 7, as shown in SEQ ID NO: 8, as shown in SEQ ID NO: 14, as shown in SEQ ID NO: 15 and the amino acid sequence as set forth in SEQ ID NO: 16. In one embodiment, a TIM3 binding molecule of the invention comprises a sequence as set forth in SEQ ID NO: 5 and/or the amino acid sequence as shown in SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof.

In one aspect, the invention relates to an antibody that binds TIM3, comprising: a light chain CDR1(LCDR1) comprising SEQ ID NO: 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, a light chain CDR2(LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, a light chain CDR3(LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, a heavy chain CDR1(HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, a heavy chain CDR2(HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to said sequence, and a heavy chain CDR3(HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to said sequence. In one embodiment, the present invention provides an antibody that binds TIM3, comprising an amino acid sequence set forth as SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to said sequence, and/or the amino acid sequence as set forth in SEQ ID NO: 13 or a heavy chain variable region amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto.

In one embodiment, the molecule that binds TIM3 is a TIM3 antagonist protein. In one embodiment, the molecule that binds TIM3 is a TIM3 antagonist antibody or antibody fragment. In one embodiment, the molecule that binds to TIM3 is a fusion protein that blocks the TIM3 signaling pathway.

In one embodiment, the above molecule that binds to TIM3 is an antibody or antigen-binding fragment thereof that binds to TIM3, e.g., a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody or antigen-binding fragment thereof. In a non-limiting embodiment, the antibody or antigen-binding fragment thereof that binds to TIM3 is a monoclonal antibody, scFv, Fab fragment, Fab 'fragment, f (ab)' fragment, bispecific antibody, immunoconjugate, or a combination thereof. In a non-limiting embodiment, the isolated antibody or fragment thereof that binds TIM3 specifically recognizes endogenous and exogenous TIM 3.

In a non-limiting embodiment, the molecule, antibody or antigen-binding fragment thereof that binds to TIM3 has the activity of promoting an immune response, activating an immune cell, e.g., activating a T cell.

In one embodiment, the molecule or antibody that binds TIM3 described above is a monospecific molecule or antibody fragment that binds TIM 3. In one embodiment, the molecule or antibody that binds TIM3 described above is a multispecific antibody or antibody fragment. In one embodiment, the multispecific antibody is a bispecific antibody. In one embodiment, the bispecific antibody comprises a second binding domain that binds a second biomolecule, wherein the second biomolecule is a cell surface antigen, such as a tumor antigen, for example selected from: tumor antigens of CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB 2.

In one aspect, the invention relates to an immunoconjugate comprising a therapeutic agent, such as a cytotoxic agent, linked to a molecule or antibody that binds to TIM3, as described above.

In one aspect, the invention relates to a pharmaceutical composition comprising a molecule, antibody, immunoconjugate described above that binds TIM3, and a pharmaceutically acceptable carrier.

In one aspect, the invention relates to an article of manufacture comprising a container holding a pharmaceutical composition as described above and a package insert, wherein the package insert is indicative of the use of the pharmaceutical composition.

In one embodiment, the above-described article of manufacture further comprises one or more containers containing one or more additional medicaments. In one embodiment, the other drug is an immunostimulatory antibody or a chemotherapeutic agent.

In one aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: 16.

in one aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: SEQ ID NO: 5 and/or SEQ ID NO: 13.

in one aspect, the invention also relates to the use of these isolated nucleic acid molecules in the preparation of immunotherapy-related drugs or cells (e.g., CAR-T cells, TCR-T cells).

The present invention relates to vectors, host cells comprising the above isolated nucleic acid molecules, and their use in the preparation of molecules and antibodies that bind to TIM 3.

In one aspect, the invention relates to a method of promoting an immune response in a subject, comprising contacting an immune cell in the subject with a molecule or antibody that binds to TIM3 as described above, thereby promoting an immune response in the subject. In one embodiment, the subject is a tumor-bearing subject or a virus-bearing subject.

In one aspect, the invention relates to a method of inhibiting tumor cell growth in a subject, comprising administering to the subject a molecule or antibody that binds to TIM3 as described above.

In one aspect, the invention relates to a method of treating a viral infection in a subject, comprising administering to the subject a molecule or antibody that binds to TIM3 as described above. In some embodiments, the above-described molecules or antibodies that bind TIM3 are used in combination with one or more other drugs, such as other antibodies (including anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, and other TIM3 antibodies), anti-cancer drugs, or anti-viral drugs.

In one aspect, the invention relates to the use of a molecule or antibody to which TIM3 described above binds for the preparation of a medicament.

Specifically, the present invention relates to:

1. a molecule that binds TIM3, comprising one or more amino acid sequences shown below:

as shown in SEQ ID NO: 6, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown as 6,

as shown in SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof, wherein,

as shown in SEQ ID NO: 8, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in figure 8,

as shown in SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, wherein,

as shown in SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown as 15,

as shown in SEQ ID NO: 16.

2. The molecule that binds TIM3 of claim 1, which is a TIM3 antagonist protein.

3. The molecule of

claim

1 or 2 that binds to TIM3, which is a TIM3 antagonistic antibody or antibody fragment.

4. The molecule of claims 1-3 that binds to TIM3, which is a fusion protein that blocks the TIM3 signaling pathway.

5. An antibody that binds TIM3, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises:

a light chain CDR1(LCDR1) comprising SEQ ID NO: 6 or an amino acid sequence having at least 90% homology to the sequence,

a light chain CDR2(LCDR2) comprising SEQ ID NO: 7 or an amino acid sequence having at least 90% homology to said sequence, and/or

A light chain CDR3(LCDR3) comprising SEQ ID NO: 8 or an amino acid sequence having at least 90% homology to said sequence;

the heavy chain variable region comprises:

heavy chain CDR1(HCDR1) comprising SEQ ID NO: 14 or an amino acid sequence having at least 90% homology to said sequence,

heavy chain CDR2(HCDR2) comprising SEQ ID NO: 15 or an amino acid sequence having at least 90% homology to the sequence, and/or

Heavy chain CDR3(HCDR3) comprising SEQ ID NO: 16 or an amino acid sequence which is at least 90% homologous to the sequence.

6. The antibody of claim 5, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 5 or a nucleotide sequence comprising an amino acid sequence substantially identical to SEQ id no: 5, or a variant thereof, and 5 is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence set forth in seq id no.

7. The antibody of claim 6, comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13 or a sequence comprising an amino acid sequence substantially identical to SEQ ID NO: 13, or a variant thereof, which has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence set forth in seq id No. 13.

8. The antibody of any one of claims 5-7, which is a chimeric, humanized, or human antibody.

9. The antibody of any one of claims 5-7, which is an antibody fragment that binds TIM 3.

10. The antibody of claim 9 which is Fab, Fab '-SH, Fv, scFv or (Fab')₂。

11. The antibody of any one of claims 5-7, which is a full length antibody.

12. The antibody of claim 11 which is an IgG antibody.

13. The antibody of any one of claims 5-12, which is a monospecific antibody or antibody fragment that binds TIM 3.

14. The antibody of any one of claims 5-12, which is a multispecific antibody or antibody fragment.

15. The antibody of claim 14, wherein the multispecific antibody is a bispecific antibody.

16. The antibody of claim 15, wherein the bispecific antibody comprises a second binding domain that binds a second biomolecule, wherein the second biomolecule is a cell surface antigen.

17. The antibody of claim 16, wherein the cell surface antigen is a tumor antigen.

18. The antibody of claim 17, wherein the tumor antigen is selected from the group consisting of: CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB 2.

19. An immunoconjugate comprising a therapeutic agent linked to the antibody of any one of claims 5-18.

20. The immunoconjugate of claim 19, wherein said therapeutic agent is a chemotherapeutic drug.

21. The immunoconjugate of claim 20, wherein said therapeutic agent is a cytotoxic agent.

22. An immunologically active polypeptide comprising the light chain variable region and/or the heavy chain variable region of the antibody of any one of claims 5-12.

23. A pharmaceutical composition comprising a molecule that binds to TIM3 of any one of claims 1-4, an antibody of any one of claims 5-18 or an immunoconjugate of any one of claims 19-21, or an immunologically active polypeptide of claim 22, and a pharmaceutically acceptable carrier.

24. An article of manufacture comprising a container holding the pharmaceutical composition of claim 23 and a package insert, wherein the package insert indicates the use of the pharmaceutical composition.

25. The article of manufacture of claim 24, further comprising one or more containers holding one or more additional medicaments.

26. The article of manufacture of claim 25, wherein the other drug is an antibody, a hormonal agent, or a chemotherapeutic agent.

27. An isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence selected from any one of seq id nos: SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 46. SEQ ID NO: 47 and SEQ ID NO: 48.

28. an isolated nucleic acid comprising a nucleotide sequence encoding the light chain variable region and/or the heavy chain variable region of the antibody of any one of claims 5-12.

29. A vector comprising the isolated nucleic acid of claim 27 or 28.

30. A host cell comprising the vector of claim 30.

31. The host cell of claim 30, wherein the host cell is a mammalian cell.

32. A method of making an antibody of any one of claims 5-12, comprising culturing the host cell of claim 30 and recovering the antibody that binds TIM 3.

33. A method of promoting an immune response in a subject, comprising administering to the subject a therapeutically effective amount of the molecule that binds TIM3 of any one of claims 1-4, the antibody of any one of claims 5-18 or the immunoconjugate of any one of claims 19-21, or the immunologically active polypeptide of claim 22.

34. The method of claim 33, wherein the subject is a tumor-bearing subject.

35. The method of claim 33, wherein the subject is a virus-bearing subject.

36. A method of inhibiting tumor cell growth in a subject, comprising administering to the subject a therapeutically effective amount of the molecule of any one of claims 1-4 that binds TIM3, the antibody of any one of claims 5-18 or the immunoconjugate of any one of claims 19-21, or the immunologically active polypeptide of claim 22.

37. A method of treating a viral infection in a subject, comprising administering to the subject a therapeutically effective amount of a molecule that binds TIM3 according to any one of claims 1 to 4, an antibody according to any one of claims 5 to 18 or an immunoconjugate according to any one of claims 19 to 21, or an immunologically active polypeptide according to claim 22.

38. The method of any one of claims 33-37, wherein the molecule that binds TIM3 of any one of claims 1-4, the antibody of any one of claims 5-18 or the immunoconjugate of any one of claims 19-21, or the immunologically active polypeptide of claim 22 is used in combination with one or more additional agents.

39. The method of claim 38, wherein the other drug is selected from one or more of the following: antibodies, anti-cancer drugs or anti-viral drugs.

40. The method of claim 39, wherein the antibody is selected from one or more of the following: anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies and other TIM3 antibodies.

Drawings

FIGS. 1A-1B show the results of Western blot analysis of anti-human TIM3 monoclonal antibody (clone 3G 11). Wherein, the left panel (A) is the incubation with TIM3 monoclonal antibody (1: 2000), the right panel (B) is the incubation with anti-human Fc monoclonal antibody, and anti-human GAPDH is used as the spotting control. In the figure, A is 293-6E cells transfected with cell lysate encoding TIM3(22-202aa) -Fc; B293-6E cells transfected with cell lysates encoding TIM1(21-290aa) -Fc; C293-6E cells transfected with cell lysates encoding TIM4(25-314aa) -Fc; D293-6E cells were transfected with cell lysates encoding the empty vector plasmids (control).

Fig. 2 is a result of identifying TIM3 expression in the following lysates by western blot examination of anti-human TIM3 mab (3G 11). Wherein A is activated human T cells (T cells activated by CD3 monoclonal antibody); b is Jurkat cells; c is 293A cell; d is 293A cells transfected with plasmid encoding TIM3(1-205aa) -GFP, the upper panel was incubated with TIM3 monoclonal antibody (1: 2000), the middle panel was incubated with GFP antibody, and the lower panel was incubated with GAPDH antibody as a spotting control.

FIG. 3 shows the results of an immunostaining assay for IF (immunofluorescence) to identify TIM3 monoclonal antibody specifically recognizing human exogenously transfected TIM 3. Wherein, the previous row of cells are dyed to enable HEK293 cells to only transfect a vector skeleton (an empty vector); the next row of cells was stained for HEK293 cells transfected with the eukaryotic expression vector encoding TIM 3.

FIG. 4 shows the flow assay of TIM3 expression using human peripheral blood to determine whether TIM3 recognizes endogenously expressed TIM 3. Wherein, the whole blood of the sample used for flow results is from the number of blood donors D2015, and D2015-Isotype is the antibody negative control of the same subtype; D2015-5B4, -6C7, -3G11A (3G11) is stained by TIM3 monoclonal antibody 5B4, 6C7 and 3G 11; D2015-BD was TIM3 monoclonal antibody positive control (BD product).

FIGS. 5A-5B are graphs showing the results of determining the affinity of TIM3 mab by ELISA. The upper panel (A) shows the result of TIM3 monoclonal antibody 3G11, and the lower panel (B) shows the result of TIM3 monoclonal antibody 5B 4.

FIG. 6 shows the results of determining the activation of T cells by TIM3 monoclonal antibodies 3G11 and 5B4 by detecting the real-time expression of IL-2 and IFNgamma using the real-time fluorescence quantitative nucleic acid amplification detection system (QPCR). Wherein Ctrl is mIgG (10 ug/ml); TIM3 mAb clones, each at 10 ug/ml; Anti-Tim3, positive control (Biolegend); gal-9, galectin-9,1 ug/ml; Ctrl-Gal control IgG isotype without Galectin-9.

FIG. 7 shows the result of using QPCR to detect IL-2 and IFNgamma real-time expression to determine the dose-dependent activation of T cells by TIM3 monoclonal antibody 3G 11. Wherein Ctrl-Gal is a control IgG isotype without Galectin-9.

FIG. 8 shows the result of using QPCR to detect IL-2 and IFNgamma real-time expression to determine that TIM3 monoclonal antibody 5B4 is dose-dependent activating T cells. Wherein Ctrl-Gal is a control IgG isotype without Galectin-9.

FIG. 9 shows the result of using TIM3 monoclonal antibodies 3G11 and 5B4 to enhance the cytotoxic effect of CIK (cytokine induced killer cells) on leukemia cells. Where E: T refers to the ratio of effector cells to target cells.

FIG. 10 shows the results of the inhibition of growth of CT26 colon cancer cells by the TIM3 monoclonal antibody alone or in combination with the PD1 monoclonal antibody.

Detailed Description

1. Definition of

The term "antibody" is used herein in a broad sense to encompass a variety of antibody structural molecules comprising one or more CDR domains disclosed herein that bind to TIM3, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fv, Fab '-SH, F (ab')₂) Linear antibodies, single chain antibody molecules (e.g., scFv), etc., so long as they exhibit the desired binding activity to TIM 3.

One skilled in the art may fuse one or more of the CDR domains disclosed herein with one or more other polypeptide sequences to make a functional fusion protein or polypeptide molecule that binds to the TIM3 molecule, such as a vaccine, a cell membrane receptor antagonist, a signaling pathway modulator, and a chimeric antigen receptor molecule, among others. For example, a TIM3 CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) molecule may be prepared using one or more CDR domains disclosed herein. These fusion protein molecules derived and prepared based on the sequence content disclosed in the present invention are also included in the protection scope of the present invention.

The modifier "monoclonal" in the term "monoclonal antibody" as used herein, means that the antibody is obtained from a substantially homogeneous population of antibodies, containing only minor amounts of naturally occurring mutations or mutations that occur during the course of monoclonal antibody preparation. Each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the antigen, as compared to a polyclonal antibody preparation that typically includes different antibodies directed against different epitopes. Monoclonal antibodies of the invention can be made by a variety of techniques, including but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus.

The terms "full-length antibody", "intact antibody" refer to an antibody having a structure substantially similar to a native antibody, and the terms are used interchangeably herein.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are 5 major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The constant domains of heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

"antibody fragment" refers to a portion of an intact antibody that comprises the antigen binding or variable region of the intact antibody. Antibody fragments such as Fab, Fab ', F (ab') and Fv fragments; diabodies (diabodies); single chain antibody molecules, such as single chain fv (scFv) molecules. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site and a residual "Fc" fragment. Pepsin treatment to yield F (ab')₂A fragment having two antigen binding sites and still being capable of cross-linking antigens.

The "Fab" fragment contains the constant region of the light chain and the first constant region of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one of the antibody hinge regionOne or more cysteines. Fab '-SH refers to Fab' with free thiol groups at the cysteines of the constant domains. F (ab')₂Antibody fragments were originally generated as pairs of Fab 'fragments with a hinge cysteine between the Fab' fragments.

An antibody fragment in which the "single chain Fv" or "scFv" antibody fragment exists as a single polypeptide chain, comprising the VH and VL regions of an antibody. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL regions.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. In a two-chain Fv, this region consists of a dimer of non-covalently tightly linked light and heavy chain variable regions. In a single chain Fv, the heavy and light chain variable regions may be covalently linked by a flexible peptide linker, such that the light and heavy chains can be connected in a "dimeric" structure analogous to that in a two chain Fv in which the 3 CDRs of each variable region interact to form an antigen binding site on the surface of a VH-VL dimer. The 6 CDRs collectively confer antigen binding specificity of the antibody.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 complementarity determining regions (CDR regions). (see, e.g., Kindt et al, Kuby Immunology, 6 th edition. A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "hypervariable region" or "HVR" as used herein refers to regions of an antibody variable domain which have regions of sequence hypervariability (also referred to as "complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen contacts"). Generally, an antibody comprises 6 HVRs (CDR regions): 3 are in VH (H1, H2, H3) and 3 are in VL (L1, L2, L3). Exemplary HVRs (CDR regions) herein include:

(a) hypervariable loops which occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) HVRs (CDR regions) occurring at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2) and 95-102(H3) (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts occurring at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2) and 93-101(H3) (MacCallum et al, J.mol.biol.262:732-745 (1996)); and

(d) combinations of (a), (b) and/or (c) comprising HVR (CDR region) amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3) and 94-102 (H3).

Unless otherwise indicated, HVR (CDR region) residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al (supra).

A "chimeric antibody" is an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody can comprise a murine variable region and a human constant region.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have undergone humanization.

A "human co-framework" is a framework representing the amino acid residues most commonly found in the selection of human immunoglobulin VL or VH framework sequences. Generally, human immunoglobulin VL or VH sequences are selected from a subset of variable domain sequences. In general, the sequence subgroups are subgroups as in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as described by Kabat et al (supra). In one embodiment, for the VH, the subgroup is subgroup III as described by Kabat et al (supra).

A "human antibody," which may also be referred to as a "human antibody," a "fully human antibody," or a "fully human antibody," is an antibody whose amino acid sequence corresponds to that produced by a human or produced by a human cell. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be prepared using a variety of techniques known in the art, including phage display library techniques and the like.

A "bispecific antibody" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be prepared by a variety of methods, including hybridoma fusion or ligation of Fab' fragments.

"homology" or "identity" between two amino acid sequences or nucleotide sequences refers to the percentage of amino acid residues or nucleotide residues that are identical between the two sequences. If the two sequences to be compared to each other are of different lengths, sequence "homology" or "identity" preferably refers to the percentage of nucleotide residues in the shorter sequence that are identical to the amino acid residues or nucleotide residues of the longer sequence. Sequence identity can be routinely determined using sequence analysis software commonly used in the art, such as the Wisconsin sequence analysis package.

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between binding partner members (e.g., antibody and antigen), unless otherwise indicated. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

"tumor antigen" refers to antigens that are newly present during the canceration of cells and to antigenic substances that are overexpressed. For example, the tumor antigen can be a newly produced protein during canceration, a specific degradation product of the protein, a structurally altered protein, a cryptic epitope exposed protein, an embryo antigen or a differentiation antigen with abnormal aggregation or abnormal high expression of various membrane protein molecules. Wherein the antigen specific to the tumor cell is referred to as "Tumor Specific Antigen (TSA)"; but not specific to a tumor, antigenic molecules also present on other tumor cells or normal cells are commonly referred to as "Tumor Associated Antigens (TAAs)", such as embryonic proteins, glycoprotein antigens, squamous cell antigens.

"antagonism" refers to the phenomenon in which one substance is repressed and restrained by another substance. For example, a molecule or antibody that binds to TIM3 inhibits or restricts, e.g., inhibits, the growth of a tumor cell, a cell (e.g., a tumor cell) that expresses TIM 3.

"fusion protein" refers to a protein molecule formed by connecting different proteins or polypeptides. Different proteins or polypeptides can be connected by chemical methods, and can also be realized by recombinant expression of DNA sequences with different sources through gene recombination technology. "fusion proteins" prepared by recombinant expression of DNA sequences derived from different sources by gene recombination techniques are sometimes referred to as "chimeric proteins".

"immunologically active peptide (polypeptide)" refers to a polypeptide or peptide having an activity of stimulating an immune response of a body, for example, a polypeptide or peptide having an activity of stimulating proliferation of lymphocytes, secretion of cytokines, or enhancing killing or phagocytosis of antigens of a body.

An "immune response" refers to the process by which an immune cell, upon exposure to an antigen (e.g., autoantigen, antigenic foreign body, mutant cell, or tumor cell), recognizes the antigen, activates proliferation, differentiates, forms effector cells or effector molecules to respond to the antigen or eliminates the antigen.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes; chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; various antineoplastic or anticancer agents are known in the art.

An "immunoconjugate" is a conjugate of an antibody and one or more heterologous molecules, including but not limited to cytotoxic agents.

"tumor" refers to the general term for various malignant or benign tumors. "malignancy" and "cancer" are used interchangeably herein.

A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domesticated animals, primates, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

The phrase "therapeutically effective amount" refers to an amount of a drug (e.g., a TIM3 binding molecule or antibody of the present invention) that, when administered to a subject, is sufficient to produce a therapeutic effect in the subject. For example, administration of a TIM3 binding molecule or antibody of the present invention to a subject to treat a tumor (cancer), administration of a "therapeutically effective amount" of a TIM3 binding molecule or antibody to a subject can reduce tumor cells (cancer cells); shrinking tumor cells (cancer cells); and/or to some extent inhibit tumor growth. In the case of tumor (cancer) treatment, the effect can be determined by measuring the size of the tumor. The therapeutically effective amount can be readily determined by one skilled in the art according to routine methods.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings concerning the use of such therapeutic products.

2. Antibodies, methods of manufacture, compositions, and articles of manufacture

1) Antibodies

The present invention relates to anti-TIM3 antibodies. In certain embodiments, the present invention provides an anti-TIM3 antibody comprising a binding domain comprising at least 1, 2,3, 4, 5, or 6 hypervariable regions (HVRs), or referred to as Complementarity Determining Regions (CDRs), selected from: (a) HVR-L1 (also referred to as light chain CDR1) comprising the amino acid sequence shown in SASSSVSSSHLY (SEQ ID NO: 6) or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; (b) HVR-L2 (also referred to as light chain CDR2) comprising the amino acid sequence shown by GTSNLAS (SEQ ID NO: 7) or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; (c) HVR-L3 (also referred to as light chain CDR3) comprising the amino acid sequence shown in HQWSSFPLT (SEQ ID NO: 8) or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; (d) HVR-H1 (also referred to as heavy chain CDR1) comprising the amino acid sequence shown by GFTFTDY (SEQ ID NO: 14) or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; (e) HVR-H2 (also referred to as heavy chain CDR2) comprising the amino acid sequence shown in RNKANGYT (SEQ ID NO: 15) or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% homology to this sequence; and (f) HVR-H3 (also referred to as heavy chain CDR3) comprising the amino acid sequence shown by DLDY (SEQ ID NO: 16) or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence. In some cases, the light chain Variable (VL) domain of an anti-TIM3 antibody may include a peptide

VDIVLTQTPAIMSASPGEKVTLTCSASSSVSSSHLYWYQQKPGSSPKLWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSFPLTFGAGPSHL (SEQ ID NO: 5), and/or a heavy chain Variable (VH) domain thereof which comprises an amino acid sequence having at least 80% sequence homology (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology) to the amino acid sequence set forth in SEQ ID NO: 5, and/or a VH domain thereof

An amino acid sequence having at least 80% sequence homology (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) to the amino acid sequence set forth in LIGAC-AWGFSETLLCTSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTKYSASVKGRFTISRDYSQSILYLQMNTLTAEDSATYFCARDLDYWGQGTFLTVSSKK (SEQ ID NO: 13).

In some embodiments, an anti-TIM3 antibody comprises the amino acid sequences set forth in SEQ ID NOs: 5 and SEQ ID NO: 13, and a light chain variable region and a heavy chain variable region amino acid sequence set forth in seq id No. 13.

2) Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab '-SH, (Fab')₂Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al, nat. Med.9: 129-. For scFv fragments see, for example, WO 93/16185.

Bifunctional antibodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161. Tri-and tetra-functional antibodies can be found, for example, in Hudson et al, nat. Med.9: 129-.

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (see, e.g., U.S. patent 6,248,516B 1).

Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E.coli or phage).

3) Chimeric and humanized antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Chimeric antibodies can be prepared, for example, in U.S. Pat. No. 4,816,567.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which all of the hvr (cdr) regions, or portions thereof, are derived from a non-human antibody and FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be substituted with corresponding residues from a non-human antibody to repair or improve the affinity of the antibody.

For humanized antibodies and methods for producing the same, reference is made to U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409.

4) Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art.

Human antibodies can be made by administering an immunogen to a modified transgenic animal, followed by challenge with an antigen to make whole human antibodies or whole antibodies with human variable regions. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal chromosome. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For methods of obtaining human antibodies from transgenic animals, see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 (describing the XENOMOUSETM technique); U.S. patent No. 5,770,429; U.S. Pat. No. 7,041,870 (describing the K-M technique); and U.S. application publication No. US 2007/0061900. The human variable regions derived from intact antibodies produced by such animals may be further modified, for example by combining them with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybridoma cell lines for the production of human monoclonal antibodies have been described, see, e.g., Boerner et al, J.Immunol.,147:86 (1991). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al, proc.natl.acad.sci.usa,103:3557-3562 (2006). Other methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiaondai Mianyixue,26(4):265-268(2006) (describing human-human hybridomas).

Human antibodies can also be prepared by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences can then be combined with the desired human constant domains.

In particular, antibodies of the invention with high affinity can be isolated by screening combinatorial libraries for antibodies with activity for binding TIM 3. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding characteristics. Such methods can be found, for example, in Lee et al, J.Immunol.methods 284(1-2):119-132 (2004).

In certain phage display methods, VH and VL gene lineages are individually cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, followed by screening for antigen-binding phage. Phage typically present antibody fragments as single chain fv (scfv) fragments or Fab fragments. Patents describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373 and U.S. Pat. No. 2005/0079574.

Antibodies or antibody fragments isolated from a human antibody library are considered herein as human antibodies or human antibody fragments.

5) Multispecific antibodies

In any of the above aspects, the anti-TIM3 antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies having binding specificities for at least two different sites. In certain embodiments, one binding specificity is for TIM3 and the other binding specificity is for any other antigen (e.g., a second biomolecule, e.g., a cell surface antigen, e.g., a tumor antigen). Accordingly, bispecific anti-TIM3 antibodies may have binding specificity for TIM3 and tumor antigens, such as CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, stepp 1, or TenB 2. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairings with different specificities, see WO 93/08829, WO2009/08025, and WO 2009/089004a1, among others.

6) Antibody variants

The antibodies of the invention encompass amino acid sequence variants of the anti-TIM3 antibodies of the invention. For example, antibody variants prepared to further improve the binding affinity and/or other biological properties of an antibody may be desirable. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as binding properties to the TIM3 antigen.

In certain embodiments, antibody variants are provided having one or more amino acid substitutions. Substitution mutants (including conservative substitution mutants or non-conservative substitution mutants) may be obtained by substituting at one or more sites in the hvr (cdr) region and/or FR region.

Amino acids can be grouped according to common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

(3) acidity: asp and Glu;

(4) alkalinity: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Conservative substitutions are defined as substitutions between amino acids of the same group, and non-conservative substitutions are defined as substitutions of an amino acid from one of the different classes by an amino acid from the other class. Amino acid substitutions may be introduced into an antibody of the invention and the product screened for a desired activity (e.g., retention/improvement of antigen binding or improvement of ADCC or CDC) to obtain an antibody variant of the invention.

The present invention encompasses antibody variants containing non-conservative mutations and/or conservative mutations obtained from the antibodies disclosed herein, so long as the variants still have the desired TIM3 binding activity.

One type of substitutional variant involves an antibody variant that replaces one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). In general, the resulting variants selected for further study will be modified (e.g., improved) with respect to the parent antibody in certain biological properties (e.g., increased affinity) and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more hvr (cdr) residues are mutated and the mutated antibody is displayed on a phage, and the mutated antibody is screened for a particular biological activity (e.g., binding affinity).

In certain embodiments, substitutions, insertions, or deletions may occur within one or more hvrs (cdrs) as long as such changes do not substantially impair the ability of the antibody to bind TIM 3. For example, conservative changes that do not substantially reduce binding affinity may be made in hvrs (cdrs). For example, such changes may occur outside of antigen-contacting residues in HVRs, e.g., conservative or non-conservative amino acid substitutions may occur at1, 2,3, 4, 5 amino acid residues in the FR region.

7) Recombination method

anti-TIM3 antibodies of the invention can be prepared using recombinant methods, for example, as described in U.S. patent No. 4,816,567. In one embodiment, isolated nucleic acids encoding anti-TIM3 antibodies described herein are provided. Such nucleic acids may encode the VL amino acid sequence and/or the VH amino acid sequence of an antibody. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., is transformed to have): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-TIM3 antibody is provided, wherein the method comprises culturing a host cell as provided above comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-TIM3 antibodies, nucleic acids encoding the antibodies are isolated (e.g., as described above) and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237. After expression, the antibody in the soluble fraction can be isolated from the bacterial cytoplasm and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Li et al, nat. Biotech.24:210-215 (2006).

Host cells suitable for expression of glycosylated antibodies may also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used to bind insect cells, particularly to transfect Spodoptera frugiperda cells.

Plant cell cultures may also be used as hosts. For example, U.S. patent No. 6,417,429 describes the technology of plantibodies for the production of antibodies in transgenic plants.

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be suitable. Other examples of suitable mammalian host cell lines are monkey kidney CV1 cell line transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 cells); baby hamster kidney cells (BHK); mouse sertoli cells (e.g., TM4 cells); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NS0 and Sp 2/0.

8) Immunoconjugates

The present invention also provides immunoconjugates comprising an anti-TIM3 antibody herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or chemotherapeutic drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody binds to one or more drugs, including but not limited to maytansine, orlistatin, dolastatin, methotrexate, vindesine, a taxane, a trichothecene (trichothecene), and CC 1065.

In another embodiment, the immunoconjugate comprises a conjugate of an anti-TIM3 antibody as described herein and an enzymatically active toxin, or fragment thereof, including but not limited to diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain, trichothecene toxin, and the like.

In another embodiment, the immunoconjugate comprises a radioactive conjugate formed by an anti-TIM3 antibody as described herein bound to a radioactive atom. A variety of radioisotopes are available for producing radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidoylmethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene).

9) Pharmaceutical preparation

Pharmaceutical formulations of anti-TIM3 antibodies of the present invention are prepared by mixing the antibody of the desired purity with one or more optional pharmaceutically acceptable carriers in lyophilized formulations or in aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; quaternary ammonium hexahydrochloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG).

An exemplary lyophilized antibody formulation is described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and/or anti-hormonal agents). Such active ingredients are suitably present in a combined form in an amount effective for the intended purpose.

10) Article of manufacture

In another aspect of the invention, an article of manufacture comprising an antibody or pharmaceutical composition of the invention is provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Such containers may be formed from a variety of materials, such as glass or plastic. The container holds the composition of the invention by itself or in combination with another composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for treating the selected tumor. In addition, the article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition therein, wherein the composition comprises another tumor therapy drug or another antibody. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that such compositions are useful for treating tumors. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The present invention will be described in detail below.

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced in view of the general description provided above.

Example 1: preparation of anti-TIM3 monoclonal antibody

(first) immunization of mice

1. Animals: balb/c mice were 3 mice per group, weighing 20 g/mouse. A total of 2 groups of mice were immunized.

2. Immunogen: the nucleotide sequence encoding the extracellular domain of TIM3 (amino acids 1-201 or amino acids 31-180) was digested by PCR and inserted into the vectors pcDNA3.1 and PET-32a (Biovector, cat # 3683689) to prepare recombinant proteins of the extracellular domains of plasmids pcDNA3.1-TIM3(1-201aa) and TIM3 (amino acids 31-180).

3. Hyperimmunity (SuperImmune)^TM): plasmid pcDNA3.1-TIM3(1-201aa) was used to prepare 0.6. mu.g/. mu.l PBS solution according to the super-immunization technique (super-immunization technique) described in the literature (immunization adherence and the processing of soluble compliance/DNA complexes in mice JC, L Tosic, RP Taylor-Clinical immunization and immunopathology,1989-Elsevier)^TM) Mice were immunized. The immunization was performed every two weeks, and 20 to 30. mu.l of blood was collected from the tail of the mouse 3 times after the immunization, and the serum was centrifuged to detect the antibody titer after the immunization by ELISA. If the antibody titer is higher than 1:10000,i.e., fusion after one booster immunization. If this titer is not achieved, immunization is continued until the titer is above 1: 10000. The immune process comprises the following steps: the first group was immunized 5 times with pcDNA3.1-TIM3(1-201aa), and the second group was immunized 3 times with pcDNA3.1-TIM3(1-201aa), followed by 1-2 booster immunizations with the above-mentioned TIM3 extracellular region recombinant protein.

(II) fusion screening

Spleen of the immunized mouse is prepared into single suspension cell for fusion with SP2/0 cell, and then clone screening is carried out.

Spleen and myeloma cell fusion:

(1) SP2/0 cells in logarithmic phase were discarded from the culture supernatant, D-Hank's solution was added to collect the cells, centrifuged at 1000rpm for 5min, and the supernatant was discarded and 45ml of D-Hank's solution was added to prepare a cell suspension.

(2) Spleen cells of the immunized mice were prepared into single suspension cells, and the spleen cells were added to the SP2/0 suspension (SP 2/0: spleen cells: 1: 5-10) and mixed well, followed by centrifugation at 1000rpm for 5 min.

(3) Discard the supernatant and flick the bottom of the tube with a hand to disperse the cells.

(4) Add 1ml of PEG4000 to the mixed cells of step "3", slowly add the drops to the tube and rotate the tube slowly, and then increase the drop rate slightly (PEG 4000 must be added within 1 minute). Standing for 1.5min, and immediately adding D-Hank's solution to 45 ml. The operation of step (4) was completed within 3 minutes.

(5) Centrifuging at 1000rpm for 5min, rapidly inverting the centrifuge tube, discarding supernatant, blowing off cell mass with a pipette, adding D-Hank's solution to 45ml, inverting, mixing, and centrifuging at 1000rpm for 5 min.

(6) The supernatant was discarded, the cells were blown off with a pipette, and HAT1640 medium containing 10% calf serum was added thereto and dispersed at 100. mu.l/well in a 96-well plate prepared in advance.

(7) HT1640 medium was changed 7-8 days after cell fusion.

Each fusion was performed in 8 96-well plates, and a total of 5 fusions were performed per group of immunized mice. After each fusion, the positive clones were subcloned 3 more times in succession.

Screening: clones were selected using CHO cells stably transfected to express TIM3 protein, using CHO cells as a negative control. Clones so screened ensure the identification of eukaryotically expressed TIM 3.

A total of 47 clones were obtained from the entire screen, and among the 47 clones, the 10 strains with the highest titers were selected for further biochemical and immunological identification.

Example 2: identification of anti-TIM3 monoclonal antibodies

1. The 10 clones with the highest titer were selected and subjected to a series of biochemical and immunological tests, the results of which are shown in Table 1(+ positive; -negative).

TABLE 1

Among the 10 selected clones described above, 3G11 and 5B4 clones were selected for further studies as follows.

2. Specificity for recognition by human-derived TIM3

Western blot detection procedure:

A. sample lysate preparation

(1) Collecting cells growing to about 80% and having good conditions, discarding the culture medium, adding a sample storage solution, and collecting the cells after fully and uniformly mixing;

(2) ultrasonically breaking the cells for 8 minutes until the sample is not sticky;

(3) and (3) carrying out dry heating on the sample EP tube at 100 ℃ for 10min, cooling, centrifuging at 12000rpm for 10min, sucking 60 mu L of supernatant, subpackaging and storing to-20 ℃, and avoiding repeated freeze thawing.

(4) Protein denaturation: and (3) putting the lysed cells into a drier, heating at 100 ℃, and preserving heat for 5-10 min.

(5) And (3) collecting a lysate: the cooked protein was centrifuged at 12000rpm for 10min at 4 ℃. The supernatant was aspirated and dispensed into small EP tubes, 60. mu.L per tube, and stored at-20 ℃ to avoid repeated freeze thawing.

B. Loading: thawing the treated sample, centrifuging for 2min, mixing, and collecting supernatant.

C. Electrophoresis: voltage 200V, electrophoresis for about 50 minutes.

D. Film transfer: the voltage is 60V, 1-2 h.

E. And (3) sealing: on a shaker, slowly shake at 70rpm for 1h at room temperature.

F. TIM3 antibodies to be detected: slowly shaking on a shaker at 70rpm for 1-2h at room temperature or overnight at 4 ℃; PBST rinsing 3 times.

G. Goat anti-mouse secondary antibody: slowly shaking by a shaking table at room temperature for 1 h; PBST rinsing 3 times.

H. And (6) developing.

FIG. 1 shows the result of Western blot analysis of anti-human TIM3 monoclonal antibody (clone 3G 11). Wherein, the left panel (A) is the incubation with TIM3 monoclonal antibody (1: 2000), the right panel (B) is the incubation with anti-human Fc monoclonal antibody, and anti-human GAPDH is used as the spotting control. In the figure, A is 293-6E cells transfected with cell lysate encoding TIM3(22-202aa) -Fc; B293-6E cells transfected with cell lysates encoding TIM1(21-290aa) -Fc; C293-6E cells transfected with cell lysates encoding TIM4(25-314aa) -Fc; D293-6E cells were transfected with cell lysates encoding the empty vector plasmids (control). As can be seen from the western blot analysis results of fig. 1, anti-human TIM3 mab (clone 3G11) specifically recognized only human TIM3, but not human TIM1 and human TIM4, which are members of the same family.

3. TIM3 monoclonal antibody recognizing human endogenous and exogenous TIM3

TIM3 was detected by western blot detection using anti-TIM3 mab (3G11) in the same procedure as above at a: activated T cells; b: jurkat cells; c: 293A cells; d: expression in cell lysates of 293A cells transfected with plasmids encoding TIM3(1-205aa) -GFP. As shown in fig. 2, the top panel was incubated with TIM3 mab (1: 2000), the middle panel with GFP antibody and the bottom panel with GAPDH antibody as spotting controls, indicating that TIM3 mab recognizes both human endogenous and exogenous TIM 3.

4. TIM3 monoclonal antibody specific recognition human exogenous transfection TIM3

An immunostaining detection step: A. fixing: and (3) lightly washing the cell slide with PBS, fixing the cell slide with 4% paraformaldehyde at room temperature for 15-20 min, and washing with PBS twice. B. And (3) sealing: mu.l blocking buffer/well, 45min at room temperature. Tim3 monoclonal antibody: standing at room temperature for 1h or overnight in a refrigerator at 4 ℃. D. Goat anti-mouse secondary antibody: incubate for 1h in the dark. E.4 ', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI) staining: discarding the secondary antibody, washing with washing buffer solution, sucking up residual liquid, adding DAPI working solution, and standing at room temperature in dark for 2-5 min. F. And (4) sealing sheet observation: discarding DAPI, washing with PBS and double distilled water for 5min, sucking off the residual liquid, reversely buckling the slide on the glass slide on which the anti-quenching blocking tablet is dropped, blocking, and observing.

The results are shown in FIG. 3. TIM3 mab (3G11) was purified with 1:1000 dilution, incubation at room temperature for 1 hour, incubation with Alexa 568-labeled secondary antibody for 1 hour, and photographing under Confocol microscope. In FIG. 3, the upper row of cells stained HEK293 cells transfected only the vector backbone (empty vector); the next row of cells was stained for HEK293 cells transfected with the eukaryotic expression vector encoding TIM 3. TIM3 mab (3G11) was purified with 1:1000 dilution, incubation at room temperature for 1 hour, incubation with Alexa 568-labeled secondary antibody for 1 hour, and photographing under Confocol microscope. DAPI staining is nuclear staining. The results indicate that TIM3 mab specifically recognizes human exogenously transfected TIM 3.

Example 3: monoclonal antibody recognition endogenous TIM3 detection

Flow assays of TIM3 expression using human peripheral blood were used to determine whether TIM3 mab recognizes endogenously expressed TIM 3. The experimental procedure was as follows: 1) PBMC were prepared from 5-10mL of whole blood cells. 2) If necessary, erythrocytes can be lysed by adding an erythrocyte lysis buffer. 3) PBMC cells were slowly transferred to a 15ml sterile tube (labeled). 4) 5ml of wash buffer (room temperature) was slowly added. 5) Centrifuge at 500g for 5 minutes. 6) The supernatant was discarded. 7) The cells were resuspended and then 6-10 ml of wash buffer was added. 8) Gently mix the cells, then pipette 2mL portions into another 3-5 15mL tubes (each tube containing at least 1X 10)⁶Individual cells) and then labeled and centrifuged at 500g for 5 minutes at room temperature. 9) The supernatant was decanted using a pipette to remove the remaining supernatant on the open end of the tube (100. mu.l remaining in the tube), and 11. mu.l of 1:10 diluted mouse anti-human TIM3 mAb, or 11. mu.l of 1:10 diluted mouse anti-GFP mAb was added to each tube as a control. Incubate at room temperature for 60 minutes. 10) 2-3mL of wash buffer was added to each tube and 500 deg.C at room temperatureg, centrifuge for 5 minutes. 11) The supernatant was decanted using a pipette to remove the remaining supernatant at the open end of the tube (100. mu.l remaining in the tube), and 11. mu.l of a 1:40 diluted goat anti-mouse IgG (H + L) secondary antibody Alexa Fluor 568(Life Technology batch No.: 1793903) was incubated at room temperature in the dark for 30 minutes. 12) 2mL of wash buffer was added to each tube and centrifuged at 500g for 5min at room temperature. 13) The supernatant was poured off and 200. mu.L of 1% paraformaldehyde in PBS was added. Vortex to resuspend the pellet and store in the dark at 4 ℃ prior to flow cytometry analysis. Analysis was performed within 24 hours.

The flow result shows that: various TIM3 monoclonal antibody clones (3G11, 5B4, and 6C7) recognized human monocyte endogenously expressed TIM3 with the results shown in fig. 4.

Example 4: affinity assay for TIM3 monoclonal antibody

The coating protein is 1.0ug/ml of the recombinant protein of the extracellular region of the TIM3 prepared in the above way. ELISA assays were performed with TIM3 mab in a gradient dilution. EC50 values were calculated. TIM3 mabs 3G11 and 5B4 have good affinity with measured EC50 values of 0.733nM and 0.82nM, respectively. The results are shown in FIG. 5.

Example 5: functional assay for TIM3 monoclonal antibody

1. Blocking TIM3 monoclonal antibody activating T cell activity

Normal human PBMCs were cultured for 3 days in RPMI supplemented with 10% fetal bovine serum, recombinant IL2(Cat #68-8779-82, ThermoFisher, USA)50U/ml and anti-CD 3 antibody (Cat #317301, OKT, BioLegend, San Diego, USA) to up-regulate the expression of Tim 3. anti-CD 3-stimulated PBMCs were incubated overnight at 37 degrees with 10. mu.g/ml anti-Tim3 monoclonal antibodies 3G11 and 5B4 or IgG isotype control or 10. mu.g/ml anti-Tim3 positive control (Cat #345001(F38-2E2), BioLegent, San Diego, USA) to block Tim3 interaction with Gal-9. The next day, Galetin-9 (Gal-9, which is Tim3 ligand) (Cat #754802, BioLegend, San Diego, USA) (1ug/ml) was added and incubated for 2 hours, and IL-2 and IFNgamma real-time expression was detected by QPCR assay of IL2 and IFNgamma mRNA. Ctrl as a control, no Galectin9 added; ctrl + Gal control Galectin9 was added.

In the experiment, the biological effect of the TIM3 monoclonal antibody is judged by Detecting the Real-time expression (QPCR, Real-time Quantitative PCR detection System) of IL-2 and IFNg. The experimental result shows that the TIM3 monoclonal antibody clones 3G11 and 5B4 have the functions of blocking the binding of a TIM3 receptor and Galectin9 and activating T cells. The results are shown in FIG. 6.

TIM3 MAb exhibiting dose-dependent T cell activating Effect

Three different doses of TIM3 mab (2, 10, 50ug/ml) were used in the experiments, which were performed as described above. The experimental results show that the TIM3 monoclonal antibodies 3G11 and 5B4 block the binding of TIM3 and ligand Galectin9 in a dose-dependent manner, and activate T cell functions. The results are shown in fig. 7 and 8, respectively.

3. The blocking TIM3 monoclonal antibody enhances the cytotoxicity effect of CIK on leukemia cells, CIK (cytokine-induced killer cells) is obtained by culturing human peripheral blood T cells in vitro, the CIK cells activated by the culture are used as Effector cells (Effect) and leukemia cells U937(Target) to be cultured in vitro in a mixed way, and the killing effect of the Effector cells on Target cells is detected under the condition of adding TIM 3. The percentage of specifically lysed target cells was judged by measuring the release of lactate dehydrogenase LDH. The results are shown in FIG. 9. T refers to the ratio of effector cells to target cells. The experimental result shows that TIM3 monoclonal antibodies 3G11 and 5B4 have the effect of enhancing the killing effect of CIK on tumor cells.

Example 6: humanized expression and detection of TIM3 monoclonal antibody

The TIM3 mab 3G11 clone was sequenced using Sanger dideoxy termination sequencing, the sequencing results are shown in the table below.

DESCRIPTION OF THE SEQUENCES	Serial number
		Nucleotide sequence for encoding variable region of light chain of 3G11 monoclonal antibody	SEQ ID NO：1
Nucleotide sequence of light chain CDR1 of monoclonal antibody encoding 3G11	SEQ ID NO：2
		Nucleotide sequence of light chain CDR2 of monoclonal antibody encoding 3G11	SEQ ID NO：3
Nucleotide sequence of light chain CDR3 of monoclonal antibody encoding 3G11	SEQ ID NO：4
		3G11 monoclonal antibody light chain variable region amino acid sequence	SEQ ID NO：5
3G11 monoclonal antibody light chain CDR1 amino acid sequence	SEQ ID NO：6
		3G11 monoclonal antibody light chain CDR2 amino acid sequence	SEQ ID NO：7
3G11 monoclonal antibody light chain CDR3 amino acid sequence	SEQ ID NO：8
		Nucleotide sequence for coding 3G11 monoclonal antibody heavy chain variable region	SEQ ID NO：9
Nucleotide sequence of heavy chain CDR1 of monoclonal antibody for encoding 3G11	SEQ ID NO：10
		Encoding 3G11 monoclonal antibody heavy chainCDR2 nucleotide sequence	SEQ ID NO：11
Nucleotide sequence of heavy chain CDR3 of monoclonal antibody for encoding 3G11	SEQ ID NO：12
		3G11 monoclonal antibody heavy chain variable region amino acid sequence	SEQ ID NO：13
3G11 monoclonal antibody heavy chain CDR1 amino acid sequence	SEQ ID NO：14
		3G11 monoclonal antibody heavy chain CDR2 amino acid sequence	SEQ ID NO：15
3G11 monoclonal antibody heavy chain CDR3 amino acid sequence	SEQ ID NO：16

Humanized TIM3 mab 3G11 was prepared by embedding six CDR regions of the heavy and light chains of the 3G11 monoclonal antibody into the constant framework of human IgG1 and LC kappa. Specifically, humanized chimeric antibody VL light chain plasmid pTT-LC (EcoR1-Leader-Sal1-VL-BsiW1-LC kappa) and VH heavy chain plasmid pTT-HC (EcoR1-Leader-Sal1-VH-Nhe1-CH1-CH2-CH3) were constructed, the plasmids were transfected in HEK293-6E in proportion, 100. mu.l of supernatant was taken after culture and tested, and the ELISA results were as follows:

the above results show that TIM3 mab (3G11) expressed after humanization (chimerism, or semi-humanization) specifically recognized the TIM3 recombinant protein as before humanization.

Example 7: in vivo experiment of TIM3 monoclonal antibody for inhibiting tumor growth

To test the ability of TIM3(3G11) monoclonal antibody to inhibit tumor growth in vivo, a tumor transplantation model using human CT26 colon cancer cells inoculated on NSG mice was used. 50 μ L of RPMI1640 medium (containing 2X 10)⁶Personal CT26 colon cancer cells) were mixed with 50 μ L basement membrane matrigel and each was injected subcutaneously into NSG mice on day 0. Mice were injected intraperitoneally with 100 μ g of different antibodies, TIM3 monoclonal antibody, PD1 monoclonal antibody (nivolumab (Opdivo, Bristol-Myers Squibb GmbH) on

days

6,9, 12, and 15, respectively&Co), TIM3 monoclonal antibody + PD1 monoclonal antibody (TIM3 monoclonal antibody and PD1 monoclonal antibody administered in combination), control antibody (mouse IgG, Sigma, # I5381). During the experiment, the size of the mouse tumor was determined, wherein the tumor volume was calculated using the following formula: tumor volume 1/2 × tumor length × tumor width²。

The inhibition of tumor growth in mice by the different antibodies is shown in figure 10, from which it can be seen that TIM3 antibody alone very significantly inhibited the growth of CT26 colon cancer compared to the control group (mouse IgG). It is worth emphasizing that the TIM3 antibody is more pronounced than the PD1 antibody alone against cancer. TIM3 in combination with PD1 antibody treatment acts synergistically to inhibit cancer further significantly inhibiting tumor growth and persistently reduce tumor burden compared to TIM3 or PD1 antibody alone.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments and application fields, and the above-described embodiments are merely illustrative and instructive examples and do not limit the scope of the claims of the present application. Many substitutions and modifications may be made by one of ordinary skill in the art in light of this disclosure without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Suzhou Hengkang Life sciences Co., Ltd

<120> TIM3 binding molecules and uses thereof

<130> PD00904R

<141> 2020-02-14

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ccaggatcct cccccaaact ctggatttat ggcacttcca acctggcttc tggagtccct 180

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gctgaagatg ctgcctctta tttctgccat cagtggagta gtttcccact cacgttcggt 300

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Met Tyr Pro Gly Asp Gly Ser Ile

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Claims

as shown in SEQ ID NO: 16.

2. The molecule that binds TIM3 of claim 1, which is a TIM3 antagonist protein.

3. The molecule of claim 1 or 2 that binds to TIM3, which is a TIM3 antagonistic antibody or antibody fragment.

the heavy chain variable region comprises:

10. The antibody of claim 9 which is Fab, Fab '-SH, Fv, scFv or (Fab')₂。

11. The antibody of any one of claims 5-7, which is a full length antibody.

12. The antibody of claim 11 which is an IgG antibody.

29. A vector comprising the isolated nucleic acid of claim 27 or 28.

30. A host cell comprising the vector of claim 30.

31. The host cell of claim 30, wherein the host cell is a mammalian cell.

34. The method of claim 33, wherein the subject is a tumor-bearing subject.

35. The method of claim 33, wherein the subject is a virus-bearing subject.