WO2021175191A1

WO2021175191A1 - Anti-tim-3 antibody and use thereof

Info

Publication number: WO2021175191A1
Application number: PCT/CN2021/078473
Authority: WO
Inventors: 匡智慧; 荆华; 陈炳良; 刘军建
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2020-03-02
Filing date: 2021-03-01
Publication date: 2021-09-10

Abstract

Provided are an antibody or an antigen-binding fragment thereof that specifically bind to TIM-3, and a composition containing the antibody or antigen-binding fragment thereof. Also provided are a corresponding encoding nucleic acid, a host cell, and use of the antibody or antigen binding fragment thereof in therapy and diagnosis.

Description

Anti-TIM-3 antibody and its use

This application is based on the application with the CN application number 202010134434.6 and the filing date of March 2, 2020, and claims its priority. The disclosure of the CN application is incorporated into this application as a whole this time.

The present invention relates to novel antibodies and antibody fragments that specifically bind to TIM-3, and compositions containing the antibodies or antibody fragments. In addition, the present invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, host cells containing them, and related uses. In addition, the present invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments (especially in combination with PD-1 pathway antibodies).

Background technique

T cell immunoglobulin and mucin domain-3 (TIM3), also known as hepatitis A virus cell receptor 2 (HAVCR2), is a type I transmembrane protein that serves as a target for second-generation immune checkpoint drugs One is expressed on the surface of a variety of immune cells, such as T cells, NK cells, and macrophages. Like many inhibitory receptors (eg, PD-1 and CTLA-4), TIM3 expression is associated with many types of chronic diseases, including cancer.

Several potential ligands for TIM3 have been identified: Galectin-9, HMGB1, Semaphorin-4A, CEACAM-1, ILT-4 and Phosphatidylserine (PtdSer or PS). PtdSer is an important cell membrane component and is usually located in the inner lobules of the cell membrane. But when the cell undergoes apoptosis, PtdSer is redistributed and exposed to the outer membrane. This redistribution is also observed in many tumor cell lines. The binding of TIM3 to PtdSer may be essential for phagocytosis and cross-presentation.

Studies have shown that TIM3 has a close relationship with the inhibitory receptor PD-1. For example, many tumor-specific T cells express PD-1 and TIM3, and these T cells have been shown to be more dysfunctional compared to T cells expressing only PD-1 or TIM3. See Fourcade J et al., J Exp Med. 207: 2175-2186 (2010).

Studies have shown that the Tim-3 signaling pathway plays a negative regulatory role in immune regulation and inhibits the anti-tumor T cell response. Therefore, blocking the Tim-3 signaling pathway can significantly improve the anti-tumor effect of T cells. Specifically, T cells expressing Tim-3 may exhibit a depletion phenotype characterized by impaired cytotoxic function, effector cytokine production, and proliferation. In this regard, it has been shown that anti-Tim-3 antibodies can restore anti-tumor immunity in some murine cancer models.

Antibodies against human Tim-3 are known. WO2016161270 describes a humanized antibody against human Tim-3. At present, a number of anti-tumor experiments against human Tim-3 antibodies have been carried out clinically. However, to date, no antibodies targeting Tim-3 have been approved for therapeutic use in humans.

Therefore, there is still a need to provide antibodies that bind to human Tim-3 with strong affinity, enhance the therapeutic effect when combined with PD-1 pathway antibodies, and are also expected to overcome the drug resistance caused by PD-1 pathway antibody therapy.

Summary of the invention

The present invention provides a novel Tim-3 antibody that binds Tim-3 with higher affinity, especially human and cynomolgus Tim-3, which can enhance the ability of PD-1 pathway antibodies to activate T cells and enhance PD-1 Anti-tumor activity of pathway antibodies.

In mouse tumor models, anti-Tim-3 antibody combined with anti-PD-1 antibody can achieve better anti-tumor effects than PD-1 antibody alone. Therefore, the combination therapy of anti-Tim-3 antibody and anti-PD-1 antibody may not only improve the curative effect of anti-PD-1/anti-PD-L1 antibody, but also hope to overcome the resistance caused by anti-PD-1/anti-PD-L1 antibody treatment. It has been proved that the anti-Tim-3 monoclonal antibody combined with the anti-PD-1 monoclonal antibody of the present invention has better efficacy than the PD-1 single drug in vivo and in vitro.

In some aspects, the present invention relates to the following embodiments:

1. An antibody or antigen-binding fragment thereof that binds to TIM-3, which comprises

The three complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO: 8, 15 or 26, and the 3 complementarity determining regions of the light chain variable region shown in SEQ ID NO: 9, 16 or 27 Area LCDR.

2. The antibody or antigen-binding fragment thereof of embodiment 1, which comprises

(i) The 3 complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO: 8 and the 3 complementarity determining regions LCDR of the light chain variable region shown in SEQ ID NO: 9;

(ii) The 3 complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO: 15 and the 3 complementarity determining regions LCDR of the light chain variable region shown in SEQ ID NO: 16; or

(iii) The three complementarity determining regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 26, and the three complementarity determining regions LCDR of the light chain variable region as shown in SEQ ID NO: 27.

3. The antibody or antigen-binding fragment thereof of embodiment 1, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR1 includes or consists of the amino acid sequence of SEQ ID NO:1;

Under the Kabat rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 2 or 12;

Under the Kabat rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3; and

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 5 or 14;

Under the Kabat rule, LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 6;

Under the Kabat rule, LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 7;

or

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 19;

Under the Kabat rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 20;

Under the Kabat rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21; and

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 23;

Under the Kabat rule, LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 24;

Under the Kabat rule, LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25;

or

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 4 or 13;

Under the Abm rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3;

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

or

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 22;

Under the Abm rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21;

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25.

4. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, which comprises a light chain variable region and/or a heavy chain variable region, wherein

(a) Heavy chain variable region

(i) The amino acid sequence comprising SEQ ID NO: 8, 15 or 26 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% The identical amino acid sequence or consists of said amino acid sequence; or

(ii) It comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, 15 or 26; or

(iii) Comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared with the amino acid sequence shown in SEQ ID NO: 8, 15 or 26; and/or

(b) Light chain variable region

(i) The amino acid sequence comprising SEQ ID NO: 9, 16 or 27 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% The identical amino acid sequence or consists of said amino acid sequence; or

(ii) It comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, 16 or 27; or

(iii) An amino acid sequence comprising or consisting of 1-10 amino acid substitutions, insertions or deletions compared with the amino acid sequence shown in SEQ ID NO: 9, 16 or 27.

5. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 3, which comprises

(i) Contains amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 8 Sequence or heavy chain variable region consisting of the amino acid sequence, and/or comprising the amino acid sequence shown in SEQ ID NO: 9 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or light chain variable regions consisting of said amino acid sequences;

(ii) Contains amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 15 Sequence or heavy chain variable region consisting of the amino acid sequence, and/or comprising the amino acid sequence shown in SEQ ID NO: 16 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or light chain variable region consisting of said amino acid sequence; or

(iii) Contains amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 26 Sequence or heavy chain variable region consisting of the amino acid sequence, and/or comprising the amino acid sequence shown in SEQ ID NO: 27 with at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence with 96%, 97%, 98% or 99% identity or a light chain variable region composed of said amino acid sequence.

6. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 3, which comprises

(i) Containing the amino acid sequence shown in SEQ ID NO: 8 or the heavy chain variable region composed of the amino acid sequence, and/or containing the amino acid sequence shown in SEQ ID NO: 9 or consisting of the amino acid sequence Light chain variable region;

(ii) Containing the amino acid sequence shown in SEQ ID NO: 15 or the heavy chain variable region composed of the amino acid sequence, and/or containing the amino acid sequence shown in SEQ ID NO: 16 or consisting of the amino acid sequence Light chain variable region; or

(iii) Containing the amino acid sequence shown in SEQ ID NO: 26 or the heavy chain variable region composed of the amino acid sequence, and/or containing the amino acid sequence shown in SEQ ID NO: 27 or consisting of the amino acid sequence Light chain variable region.

7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, which comprises

(a) Heavy chain

(i) The amino acid sequence comprising SEQ ID NO: 10, 17 or 28 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity or consisting of said amino acid sequence; or

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 10, 17 or 28; or

(iii) Comprising or consisting of an amino acid sequence having 1-20 amino acid changes compared with the amino acid sequence shown in SEQ ID NO: 10, 17 or 28;

and / or

(b) Light chain

(i) The amino acid sequence comprising SEQ ID NO: 11, 18 or 29 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity or consisting of said amino acid sequence; or

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 11, 18 or 29; or

(iii) An amino acid sequence having 1-20 amino acid changes compared with the amino acid sequence shown in SEQ ID NO: 11, 18, or 29, or consisting of the amino acid sequence.

8. The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, which comprises a heavy chain and/or a light chain, wherein

(i) The heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 10 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 11 , 95%, 96%, 97%, 98%, or 99% identical amino acid sequence or consist of said amino acid sequence;

(ii) The heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 17 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 18 , 95%, 96%, 97%, 98%, or 99% identical amino acid sequence or consist of said amino acid sequence;

(iii) The heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 28 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 29 , 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consist of said amino acid sequence.

9. The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, which comprises a heavy chain and/or a light chain, wherein

(i) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 11;

(ii) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 18;

(iii) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29.

10. The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1 or IgG2 or IgG3 or IgG4.

11. The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 10, wherein the antibody is a monoclonal antibody.

12. The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody.

13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 12, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g. scFv) or (Fab') ₂ , single domain antibody, double antibody (dAb) or linear antibody.

14. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13, wherein the antibody is a bispecific or multispecific antibody molecule, preferably, the bispecific antibody molecule is also compatible with PD-1, PD- L1 or PD-L2 binding.

15. An isolated nucleic acid that encodes the light chain variable region or the heavy chain variable region, or the light chain or the heavy chain in the TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14.

16. A vector comprising the nucleic acid of embodiment 15, preferably the vector is an expression vector.

17. A host cell containing the nucleic acid of embodiment 15 or the vector of embodiment 16, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO Cells, such as 293F cells or CHO-S cells or ExpiCHO cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

18. A method for preparing an antibody or antigen-binding fragment thereof that binds to TIM-3, the method comprising: The host cell of embodiment 17 is cultured under conditions, and the antibody or antigen-binding fragment thereof is optionally isolated. Optionally, the method further comprises recovering the antibody or antigen-binding fragment thereof that binds to TIM-3 from the host cell .

19. An immunoconjugate comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 and other substances, such as a cytotoxic agent.

20. A pharmaceutical composition comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or the immunoconjugate of embodiment 19, and optionally one or more other treatments Agents, such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

21. A pharmaceutical combination comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or the immunoconjugate of embodiment 19, and a PD-1 pathway antibody such as an anti-PD-1 antibody Or anti-PD-L1 antibody or anti-PD-L2 antibody, and optionally one or more other therapeutic agents, such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

22. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14, or The immunoconjugate of embodiment 19, or the pharmaceutical composition of embodiment 20.

23. The method of embodiment 22, which further comprises co-administering a PD-1 pathway antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, to the subject.

24. A method of preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical combination of embodiment 21.

25. The method of any one of embodiments 22-24, wherein the tumor is a cancer, and more preferably, the cancer has elevated levels (e.g., nucleic acid or protein levels) of TIM-3 and/or elevated levels (E.g. at the nucleic acid or protein level) PD-L1 or PD-1 or PD-L2.

26. The method of any one of embodiments 22-25, wherein the tumor is a tumor that is resistant to treatment with PD-1 pathway antibodies, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies .

27. The method of any one of embodiments 22-26, wherein the method further comprises administering to the patient one or more therapies, such as treatment modalities and/or other therapeutic agents, preferably, the treatment modalities include surgical treatment and/ Or radiotherapy, other therapeutic agents are selected from chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

28. A method for detecting TIM-3 in a sample, the method comprising

(a) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14; and

(b) Detect the formation of a complex between the antibody or antigen-binding fragment thereof and TIM-3; optionally, the antibody is detectably labeled.

The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered as limiting the scope of the present invention, and changes easily conceived by those skilled in the art will be included in the spirit of the present invention and the protection scope of the appended claims.

Description of the drawings

Figure 1 shows the determination of the binding ability of the chimeric antibody of the present invention to the human Tim-3CHO-S stably transfected cell line by flow cytometry.

Figure 2 shows the determination of the binding ability of the humanized antibody of the present invention to the human Tim-3CHO-S stably transfected cell line by flow cytometry.

Figure 3 shows the determination of the binding ability of the humanized antibody of the present invention to CD4+T by flow cytometry.

Figure 4 shows the determination of the ability of the humanized antibody of the present invention to block Tim-3 from binding to PtdSer by the MOA method.

Figure 5 shows the ability of the humanized antibody of the present invention to activate CD4+ T cells to release IL-2.

Figure 6 shows the results of the NK cell activation experiment, where A shows the percentage of cells expressing NKG2D on the cell surface, and B shows the percentage of CD107a expressing on the cell surface

Figure 7 shows the results of the in vivo drug efficacy test of the humanized antibody molecule of the present invention.

Figure 8 shows the effect of the antibody of the present invention on the body weight of mice.

Figure 9 shows the detection of the Tm of the antibody Hz4-3.6 of the present invention by the DSF method.

Figure 10 shows the accelerated stability of the antibody Hz4-3.6 of the present invention.

Detailed description of the invention

I. Definition

Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

To interpret this specification, the following definitions will be used, and as long as appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

The term "about" when used in conjunction with a numerical value means to encompass a numerical value within a range having a lower limit that is 5% smaller than the specified numerical value and an upper limit that is 5% larger than the specified numerical value.

As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.

As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.

The term "T cell immunoglobulin and mucin domain-3" or "TIM-3" as used herein refers to a receptor that is a member of the T cell immunoglobulin and mucin domain (TIM) family of proteins. The main ligand of TIM3 includes phosphatidylserine (PtdSer). TIM3 is also known as hepatitis A virus cell receptor 2 (HAVCR2), T cell immunoglobulin mucin receptor 3, TIM3, TIMD3, TIMD-3, kidney injury molecule-3, KIM-3, and CD366. The term "TIM-3" includes any variant or isoform of TIM3 that is naturally expressed by the cell. Therefore, the antibodies described herein can cross-react with TIM-3 from a species other than human (e.g., cynomolgus monkey TIM3). TIM-3 or any variants and isoforms thereof can be isolated from the cells or tissues in which they are naturally expressed, or produced recombinantly using techniques well known in the art and/or those described herein. In one embodiment, the amino acid sequence of human TIM-3 is shown in SEQ ID NO:34.

The terms "anti-TIM-3 antibody", "anti-TIM-3", "TIM-3 antibody" or "TIM-3 binding antibody" as used herein refer to antibodies that can bind with sufficient affinity ( Human or cynomolgus) TIM-3 or variants and isotypes thereof so that the antibody can be used as a diagnostic and/or therapeutic agent that targets (human or cynomolgus) TIM-3. In one embodiment, the degree of binding of the anti-TIM-3 antibody to non-(human or cynomolgus) TIM-3 protein is less than about 10% of the binding of the antibody to (human or cynomolgus) TIM-3. 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more, such as, for example, by radioimmunoassay (RIA) or biofilm thin-layer interferometry Or MSD measurement or surface plasmon resonance (SPR) measurement.

"Antibody fragment" refers to a molecule that is different from an intact antibody, which contains a part of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibodies (such as scFv); single-domain antibodies; Specific antibodies or fragments thereof; camelid antibodies; and bispecific antibodies or multispecific antibodies formed from antibody fragments.

As used herein, the term "epitope" refers to a portion of an antigen (e.g., TIM-3) that specifically interacts with an antibody molecule.

An "antibody that binds to the same or overlapping epitope" as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody in a competition assay. The binding of the antigen, on the contrary, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen in the competition assay.

An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Determination (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253).

An antibody that inhibits (for example, competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen . Conversely, the reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g. equilibrium dissociation constant). Methods of determining affinity are known in the art.

An antibody that shows the same or similar binding affinity and/or specificity as the reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody Affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.

A "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ( "Antigen contact point") area. CDR is mainly responsible for binding to antigen epitopes. The CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (on the World Wide Web imgt.cines.fr/on), and based on affinity propagation clustering using a large number of crystal structures North CDR definition.

For example, according to different CDR determination schemes, the residues of each CDR are as follows.

The CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).

Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

Unless otherwise specified, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), it refers to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

In one embodiment, the CDR of the heavy chain variable region of the antibody of the present invention is determined according to the following rules

(1) VH CDR1 is determined in accordance with Abm rules, and VHCDR2 and VHCDR3 are provided in accordance with Kabat rules; or

(2) VH CDR1, 2, and 3 are determined in accordance with Abm's rules.

In one embodiment, the CDR of the light chain variable region of the antibody of the invention is determined according to the Kabat rule.

It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As those skilled in the art know, the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined by Kabat or Chothia can be replaced by conserved amino acid residues.

"Antibody in the form of IgG" refers to the form of IgG to which the constant region of the heavy chain of the antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different. For example, an antibody in the form of IgG4 means that its heavy chain constant region is derived from IgG4.

A "humanized" antibody refers to an antibody comprising amino acid residues derived from non-human CDR and amino acid residues derived from human FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of the non-human antibody, and all Or substantially all FRs correspond to those of human antibodies. The humanized antibody optionally may comprise at least a portion of the constant region of an antibody derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source, using a human antibody library or other human Antibody coding sequence. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as, for example, by radioimmunoassay (RIA) or biofilm thin-layer interferometry or MSD. Measurement method or surface plasmon resonance (SPR) measurement.

An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.

The term "PD-1 pathway antibody" as used herein includes antibodies that specifically bind to PD-1 or its binding partner, such as PD-L1 or PD-L2. In one aspect, the PD-1 pathway antibody reduces, blocks, inhibits, eliminates, or interferes with signal transduction derived from the interaction of PD-1 with one or more of its ligands (such as PD-L1, PD-L2) . In some embodiments, the PD-1 pathway antibody is a PD-1 antibody, a PD-L1 antibody, or a PD-L2 antibody.

As used herein, the terms "anti-PD-1/PD-L1/PD-L2 antibody", "anti-PD-1/PD-L1/PD-L2", "PD-1/PD-L1/PD-L2 antibody" or "An antibody that binds to PD-1/PD-L1/PD-L2" refers to an antibody that can bind to the PD-1/PD-L1/PD-L2 protein or a fragment thereof with sufficient affinity. In one embodiment, the anti-PD-1/PD-L1/PD-L2 antibody binds to a non-PD-1/PD-L1/PD-L2 protein to a lesser degree than the antibody binds to PD-1/PD-L1/ About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of PD-L2 binding, as, for example, by radioimmunoassay ( RIA) or biofilm thin-layer interferometry or MSD measurement or surface plasmon resonance (SPR) measurement.

The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressive agents). ).

The term "cytotoxic agent" used in the present invention refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.

"Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer or diseases of the immune system.

The term "small molecule drugs" refers to low molecular weight organic compounds capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As a therapeutic agent, small molecules can penetrate cells better than large molecules, are less susceptible to degradation, and are less likely to trigger an immune response.

The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressive agents.

As used herein, "immunosuppressive agents", "immunosuppressive drugs" or "immunosuppressants" are therapeutic agents used to suppress or prevent the activity of the immune system in immunosuppressive therapy.

The "functional Fc region" possesses the "effector function" of the native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., antibody variable domain), and can be assessed using a variety of assays, such as those disclosed herein.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

The term "effective amount" refers to the amount or dose of the antibody or fragment or conjugate or composition or combination of the present invention, which, after being administered to a patient in single or multiple doses, produces the desired effect in patients in need of treatment or prevention .

"Therapeutically effective amount" refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effect of the antibody or antibody fragment or its conjugate or composition or combination is less than the therapeutically beneficial effect. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (eg tumor volume) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% And still more preferably at least about 80% or 90%.

"Prophylactically effective amount" refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations. Included herein is the mutant progeny with the same function or biological activity that is screened or selected in the initially transformed cell.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of enzymatic labeling, can catalyze a chemical change of a detectable substrate compound or composition. The term is intended to cover the direct labeling of the probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody and the indirect labeling of the probe or antibody by reaction with another reagent that is directly labelled.

"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., , Mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to more than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) confirmed. For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

"Isolated nucleic acid encoding anti-TIM-3 antibody or fragments thereof" refers to one or more nucleic acid molecules that encode antibody heavy or light chains (or fragments thereof), including such in a single vector or separate vectors Nucleic acid molecules, and such nucleic acid molecules that are present at one or more locations in the host cell.

The calculation of sequence identity between sequences is performed as follows.

In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, the first and second amino acid sequences or nucleic acid sequences may be used for optimal alignment. Gaps can be introduced in one or both or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.

Mathematical algorithms can be used to achieve sequence comparison between two sequences and calculation of percent identity. In a preferred embodiment, the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used. Available), use Blossum 62 matrix or PAM250 matrix and

gap weight

16, 14, 12, 10, 8, 6 or 4 and

length weight

1, 2, 3, 4, 5 or 6, to determine the difference between two amino acid sequences Percent identity. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 are used.

Length weights

1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossom 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

You can also use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), using E. Meyers and W. Miller algorithms that have been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determine the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.

As used herein, the term "hybridizes under stringent conditions (e.g., under low stringency, medium stringency, high stringency, or very high stringency conditions)" describes hybridization and washing conditions. Instructions for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in the references and either method can be used. The specific hybridization conditions mentioned in this article are as follows: 1) Low stringency hybridization conditions are in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by at least 50°C (for low stringency conditions, you can Increase the washing temperature to 55°C) Wash twice in 0.2X SSC, 0.1% SDS; 2) Medium stringency hybridization conditions are about 45°C in 6X SSC, and then at 60°C in 0.2X SSC, 0.1% Wash one or more times in SDS; 3) High-stringency hybridization conditions are in 6X SSC at about 45°C, and then wash one or more times in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) Very high stringency hybridization conditions are washing one or more times in 0.5M sodium phosphate, 7% SDS at 65°C, and then in 0.2X SSC, 0.1% SDS at 65°C. The very high stringency condition (4) is the preferred condition and one that should be used unless otherwise specified.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by various means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is usually characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include colon cancer, rectal cancer, colorectal cancer, including metastatic forms of those cancers.

The term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.

The term "pharmaceutical adjuvant" refers to diluents, adjuvants (for example Freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. administered together with the active substance.

The term "pharmaceutical composition" refers to a composition that is present in a form that allows the biological activity of the active ingredients contained therein to be effective, and does not contain other substances that have unacceptable toxicity to the subject to which the composition is administered. Ingredients.

The term "pharmaceutical combination" refers to non-fixed combination products or fixed combination products, including but not limited to kits and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (for example, (i) anti-TIM-3 antibody or antigen-binding fragment thereof, and (ii) PD-1 pathway antibody or antigen-binding fragment thereof) are simultaneously, without Specific time limits or sequential administration to the patient at the same or different time intervals, wherein such administration provides a preventive or therapeutically effective level of two or more active agents in the patient. In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof and the PD-1 pathway antibody or antigen-binding fragment thereof used in the pharmaceutical combination are administered at a level not exceeding the level when they are used alone. The term "fixed combination" means that two or more active agents are simultaneously administered to a patient in the form of a single entity. Preferably, the dosage and/or time interval of two or more active agents are selected so that the combined use of each part can produce an effect greater than that achieved by using any one component alone in the treatment of diseases or conditions. Each component may be in the form of a separate preparation, and the preparation form may be the same or different.

The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g. radiation therapy or surgery) to treat the diseases described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the respective active ingredients in multiple or separate containers (e.g., tablets, capsules, powders and liquids). The powder and/or liquid can be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in the treatment of the conditions or conditions described herein.

As used herein, "treatment" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of drugs before the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.

The term "vector" when used herein refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

"Subject/patient/individual sample" refers to a collection of cells or fluid obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from the subject's pregnancy or development at any time. Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and so on.

II. Antibodies

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention binds to TIM-3 (e.g., human TIM-3 or cynomolgus TIM-3) with high affinity. In some embodiments, TIM-3 It is human TIM-3. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention has a higher binding affinity to human or cyno TIM-3 than known TIM-3 antibodies, such as the TIM-3 antibody of WO2016161270, for example, referred to herein as It is an anti-TIM-3 antibody of TSR-022. In some embodiments, the affinity of antibodies is determined by biofilm thin-layer interferometry or surface plasmon resonance methods, such as Biacore.

In some embodiments, the anti-TIM-3 antibody of the present invention, in the following equilibrium dissociation constant (K _D) human TIM-3 binding, the K _D of less than about 10 nM, preferably less than or equal to about 6nM, 5.5 nM, 5nM, 4.5nM, 4nM, 3.5nM, 3nM, 2.5nM, and in some embodiments, the K _{D is} between the aforementioned values (inclusive).

In some embodiments, the anti-TIM-3 antibody of the present invention, in the following equilibrium dissociation constant (K _D) in combination with cynomolgus TIM-3, the K _D of less than about 11 nM, preferably less than or equal to about 5nM , 4.5nM, 4nM, 3.5nM, 3nM, 2.5nM, 2nM, 1.5nM, in some embodiments, the K _{D is} between the above-mentioned values (inclusive).

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to Tim-3 on the cell surface. In some embodiments, Tim-3 is overexpressed on the cell surface. In some embodiments, the cell is a CHO cell, such as CHO-S. In some embodiments, the cells are NK cells. In some embodiments, the binding is detected using flow cytometry. In some embodiments, the antibody of the present invention binds to TIM overexpressed on CHO cells with an EC50 less than or equal to about 1.5nM, 1.4nM, 1.3nM, 1.2nM, 1.1nM, 1nM, 0.9nM, 0.8nM, 0.7nM -3.

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof bind to CD4+ T cells.

In some embodiments, the antibody or antigen-binding fragment thereof of the invention blocks the binding of TIM-3 to its ligand. In one embodiment, the ligand is PtdSer, such as PtdSer on the surface of apoptotic cells. In one embodiment, the cells are L363 cells.

In some embodiments, the combination of the antibody or its antigen-binding fragment of the present invention and the PD-1 pathway antibody or its antigen-binding fragment can effectively activate CD4+ T cells, preferably better than the PD-1 pathway antibody alone, more preferably It is better than PD-1 pathway antibody combined with other anti-TIM-3 antibodies (such as TSR-022) to activate. In some embodiments, the PD-1 pathway antibody is an anti-PD-1 antibody, such as IBI308.

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention can effectively activate NK cells. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention can enhance the expression of NKG2D and/or CD107a on the surface of NK cells.

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention can be used in the treatment of cancer in combination with the PD-1 pathway antibody or antigen-binding fragment thereof. Preferably, its efficacy is better than that of the PD-1 pathway antibody alone. In some embodiments, the antibodies or antigen-binding fragments of the present invention and the PD-1 pathway antibody or antigen-binding fragments can effectively inhibit tumor growth, and the tumor inhibition rate is greater than or equal to about 60%, 65%, or 70% .

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention has good thermal stability. In some embodiments, the thermal stability of the antibody or antigen-binding fragment thereof of the present invention is detected by differential scanning fluorescence method. Preferably, the T _{m is} greater than or equal to 65°C, 66°C, 67°C, 68°C, or 69°C.

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention has good long-term thermal stability and accelerated stability. In one embodiment, the antibody or fragment thereof of the present invention maintains a purity of at least 95%, 96%, 97%, 97.5% for at least 7, 8, 9, 10, 11, 12, 13, 14 days, and its binding There was no significant change in the ability of cells expressing human Tim-3.

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the VH comprises

(i) The three complementarity determining regions (CDRs) contained in the VH as shown in SEQ ID NO: 8, 15 or 26, or

(ii) Relative to the sequence of (i), the three CDR regions contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequences in the three CDR regions.

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the VL comprises:

(i) The three complementarity determining regions (CDRs) contained in the VL shown in SEQ ID NO: 9, 16 or 27; or

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein

(a) The VH includes

(ii) Relative to the sequence of (i), a sequence containing at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions; and / or

(b) The VL includes:

In a preferred embodiment, the VH comprises an amino acid sequence selected from SEQ ID NO: 8, 15 or 26, or consists of the amino acid sequence.

In a preferred embodiment, VL comprises an amino acid sequence selected from SEQ ID NO: 9, 16 or 27, or consists of said amino acid sequence.

In a preferred embodiment, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises the three complementarity determining regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 8, 15 or 26, and the HCDR as shown in SEQ ID NO: 8, 15 or 26. The three complementarity determining regions LCDR of the light chain variable region shown by NO: 9, 16 or 27.

In a preferred embodiment, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises the three complementarity determining regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 8 or 15, and the HCDR as shown in SEQ ID NO: The three complementarity determining regions of the light chain variable region shown by 9 or 16 are LCDR.

In a more preferred embodiment, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein

(i) The VH includes complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence of SEQ ID NO:1 or 19, or consists of the amino acid sequence, or HCDR1 includes the amino acid sequence of SEQ ID NO:1 The amino acid sequence of or 19 has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequence; HCDR2 contains an amino acid selected from SEQ ID NO: 2 or 4 or 12 or 13 or 20 or 22 The sequence, or consists of the amino acid sequence, or HCDR2 contains one, two or three changes (preferably amino acid substitutions, Preferably conservative substitution) amino acid sequence; HCDR3 includes the amino acid sequence of SEQ ID NO: 3 or 21 or consists of the amino acid sequence, or HCDR3 includes the amino acid sequence of SEQ ID NO: 3 or 21 that has one, two, or The amino acid sequence of three changes (preferably amino acid substitutions, preferably conservative substitutions);

and / or

(ii) Wherein said VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, where LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or 14 or 23, or LCDR1 comprises the same sequence as SEQ ID NO: : Compared with the amino acid sequence of 5 or 14 or 23, the amino acid sequence has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions); LCDR2 includes the amino acid sequence of SEQ ID NO: 6 or 24 or is composed of the amino acid sequence Sequence composition, or LCDR2 includes an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 6 or 24; LCDR3 includes an amino acid sequence selected from SEQ ID NO: 7 Or the amino acid sequence of 25 or consists of the amino acid sequence, or LCDR3 contains an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 7 or 25 .

In a preferred embodiment, the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Kabat rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3;

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR3 includes the amino acid sequence of SEQ ID NO: 7 or consists of the amino acid sequence.

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Kabat rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21;

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

(a) The VH includes

(i) The combination of HCDR1, HCDR2 and HCDR3 shown in Table A; or

(ii) A variant of the HCDR combination of (i), the variant comprising a total of at least one and no more than 5, 4, 3, 2 or 1 amino acid changes in the three CDR regions (preferably amino acid substitutions, preferably Conservative substitution);

and / or

(b) The VL includes

(i) The combination of LCDR1, LCDR2 and LCDR3 shown in Table A; or

(ii) A variant of the LCDR combination of (i), the variant comprising a total of at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably amino acid substitutions) in the three CDR regions Conservative substitution).

In a preferred embodiment, the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a complementarity determining region ( CDR) HCDR1, HCDR2 and HCDR3 and said VL comprises (CDR) LCDR1, LCDR2 and LCDR3, wherein the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in said antibody or antigen-binding fragment thereof is as follows (table A) shows:

Table A: Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in the antibody of the present invention or its antigen-binding fragment

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,

(a) Heavy chain variable region VH

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 8, 15 or 26; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 8, 15 or 26 The amino acid sequence of an amino acid change (preferably an amino acid substitution, more preferably an amino acid conservative substitution) is composed of the amino acid sequence, and preferably, the amino acid change does not occur in the CDR region;

and / or

(b) Light chain variable region VL

(i) The amino acid sequence comprising SEQ ID NO: 9, 16 or 27 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% An identical amino acid sequence or consist of said amino acid sequence;

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 9, 16 or 27; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 9, 16 or 27 The amino acid sequence of an amino acid change (preferably an amino acid substitution, more preferably an amino acid conservative substitution) is composed of the amino acid sequence, and preferably, the amino acid change does not occur in the CDR region.

(a) Heavy chain variable region VH

(i) It contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from SEQ ID NO: 8 or 15 The amino acid sequence of or consists of the amino acid sequence; or

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 8 or 15; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO: 8 or 15 The amino acid sequence (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region;

and / or

(b) Light chain variable region VL

(i) It contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from SEQ ID NO: 9 or 16. The amino acid sequence of or consists of the amino acid sequence;

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 9 or 16; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO: 9 or 16 The amino acid sequence (preferably amino acid substitution, more preferably amino acid conservative substitution) is composed of the amino acid sequence, and preferably, the amino acid change does not occur in the CDR region.

In a preferred embodiment, the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof The combination of the heavy chain variable region VH and the light chain variable region VL included is shown in the following table (Table B):

Table B: Exemplary combinations of heavy chain variable region VH and light chain variable region VL in the antibody or antigen-binding fragment of the present invention

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and/or light chain, wherein

(a) Heavy chain

(i) The amino acid sequence comprising SEQ ID NO: 10, 17 or 28 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or a 99% identical amino acid sequence or consist of said amino acid sequence;

(iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 10, 17 or 28 The amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the Amino acid changes do not occur in the variable region of the heavy chain;

and / or

(b) Light chain

(i) The amino acid sequence comprising SEQ ID NO: 11, 18 or 29 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or a 99% identical amino acid sequence or consist of said amino acid sequence;

(iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 11, 18 or 29 The amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the Amino acid changes do not occur in the variable region of the light chain.

(a) Heavy chain

(i) The amino acid sequence comprising SEQ ID NO: 10 or 17 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % Identity amino acid sequence or consists of said amino acid sequence;

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 17; or

(iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 10 or 17 The amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or composed of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the amino acid change Does not occur in the variable region of the heavy chain;

and / or

(b) Light chain

(i) The amino acid sequence comprising SEQ ID NO: 11 or 18 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % Identity amino acid sequence or consists of said amino acid sequence;

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 11 or 18; or

(iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 11 or 18 The amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change Does not occur in the variable region of the light chain.

In a preferred embodiment, the present invention provides an anti-TIM-3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain and a light chain, wherein the combination of the heavy chain and the light chain contained in the antibody or the antigen-binding fragment thereof is shown in the following table (Table C) shows:

Table C: Exemplary combinations of heavy and light chains in the antibodies of the present invention or antigen-binding fragments thereof

In some embodiments, the heavy chain and/or light chain of the anti-TIM-3 antibody or fragment thereof of the present invention further comprises a signal peptide sequence, for example, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 35).

In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDR (for example, in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain.

In some embodiments, the substitutions are conservative substitutions. Conservative substitution refers to the replacement of an amino acid by another amino acid in the same category, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid. Replacement. Exemplary permutations are shown in Table D below:

Table D

原始残基Original residue	示例性置换Exemplary permutation	优选的保守氨基酸置换Preferred conservative amino acid substitutions
Ala(A)Ala(A)	Val、Leu、IleVal, Leu, Ile	ValVal
Arg(R)Arg(R)	Lys、Gln、AsnLys, Gln, Asn	LysLys
Asn(N)Asn(N)	Gln、His、Asp、Lys、ArgGln, His, Asp, Lys, Arg	GlnGln
Asp(D)Asp(D)	Glu、AsnGlu, Asn	GluGlu
Cys(C)Cys(C)	Ser、AlaSer, Ala	SerSer
Gln(Q)Gln(Q)	Asn、GluAsn, Glu	AsnAsn
Glu(E)Glu(E)	Asp、GlnAsp, Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn、Gln、Lys、ArgAsn, Gln, Lys, Arg	ArgArg
Ile(I)Ile(I)	Leu、Val、Met、Ala、Phe、正亮氨酸Leu, Val, Met, Ala, Phe, Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸、Ile、Val、Met、Ala、PheNorleucine, Ile, Val, Met, Ala, Phe	IleIle
Lys(K)Lys(K)	Arg、Gln、AsnArg, Gln, Asn	ArgArg
Met(M)Met(M)	Leu、Phe、IleLeu, Phe, Ile	LeuLeu
Phe(F)Phe(F)	Trp、Leu、Val、Ile、Ala、TyrTrp, Leu, Val, Ile, Ala, Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	Val、SerVal, Ser	SerSer
Trp(W)Trp(W)	Tyr、PheTyr, Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp、Phe、Thr、SerTrp, Phe, Thr, Ser	PhePhe
Val(V)Val(V)	Ile、Leu、Met、Phe、Ala、正亮氨酸Ile, Leu, Met, Phe, Ala, Norleucine	LeuLeu

In certain embodiments, the substitution occurs in the CDR region of the antibody. Generally, the obtained variant has a modification (e.g., improvement) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties that are substantially retained of the parent antibody. An exemplary substitution variant is an affinity matured antibody.

In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence so as to create or remove one or more glycosylation sites. When the antibody contains an Fc region, the carbohydrate attached to it can be changed. In some applications, modifications to remove unwanted glycosylation sites can be useful, such as removing fucose moieties to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733 ). In other applications, galactosylation can be modified to modify complement dependent cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants to change one or more functional properties of the antibody, such as serum half-life, Complement binding, Fc receptor binding, and/or antigen-dependent cytotoxicity. An Fc region variant may include a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In one embodiment of the present invention, the antibody described herein introduces substitution mutations in the Fc region to improve the binding ability to human FcRn, in order to extend its half-life in vivo.

In certain embodiments, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAbs", in which one or more residues of the antibody are replaced with cysteine residues. Cysteine engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. The part suitable for antibody derivatization includes, but is not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerin), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention has one or more of the following characteristics:

(i) Showing the same or similar binding affinity and/or specificity to TIM-3 as the antibody of the present invention (such as CH4-3, Hz4-3.6 or CH5-17);

(ii) Inhibit (e.g., competitively inhibit) the binding of the antibody (e.g. CH4-3, Hz4-3.6 or CH5-17) of the present invention to TIM-3;

(iii) It binds to the same or overlapping epitope with the antibody of the present invention (e.g. CH4-3, Hz4-3.6 or CH5-17);

(iv) Compete with the antibody of the present invention (such as CH4-3, Hz4-3.6 or CH5-17) for binding to TIM-3;

(v) Having one or more biological properties of the antibody of the present invention (for example, CH4-3, Hz4-3.6 or CH5-17).

In some embodiments, the anti-TIM-3 antibody of the present invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4.

In some embodiments, the anti-TIM-3 antibody is a monoclonal antibody.

In some embodiments, the anti-TIM-3 antibody is humanized.

In some embodiments, the anti-TIM-3 antibody is a human antibody.

In some embodiments, the anti-TIM-3 antibody is a chimeric antibody.

In some embodiments, at least part of the framework sequence of the anti-TIM-3 antibody is a human consensus framework sequence.

In one embodiment, the anti-TIM-3 antibody of the present invention also encompasses its antibody fragments, preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibodies (such as scFv) or ( Fab') ₂ , single domain antibody, double antibody (dAb) or linear antibody.

In certain embodiments, the anti-TIM-3 antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity for TIM-3 and a second binding specificity for PD-1 or its ligand (eg, PD-L1 or PD-L2). In one embodiment, the bispecific antibody molecule binds to TIM-3 and PD-1, PD-L1 or PD-L2. Multispecific antibody molecules can have any combination of binding specificities for the aforementioned molecules.

III. The nucleic acid of the present invention and the host cell containing it

In one aspect, the invention provides a nucleic acid encoding any of the above anti-TIM-3 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as pcDNA3.1. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (such as CHO cells (such as CHO-S or ExpiCHO) or 293 cells (such as 293F)) or other cells suitable for preparing antibodies or antigen-binding fragments thereof . In another embodiment, the host cell is prokaryotic.

In one aspect, the invention provides a nucleic acid encoding any anti-TIM-3 antibody or fragment thereof described herein. The nucleic acid may include a nucleic acid encoding the amino acid sequence of the light chain variable region and/or the heavy chain variable region of an antibody, or a nucleic acid encoding the amino acid sequence of the light chain and/or heavy chain of the antibody.

For example, the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 8-11, 15-18, 26-29, or a nucleic acid that encodes an amino acid sequence selected from SEQ ID NO: 8-11, 15. The amino acid sequence shown in any one of -18, 26-29 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% The identity of the amino acid sequence of the nucleic acid.

The present invention also covers nucleic acids that hybridize with the following nucleic acids under stringent conditions or nucleic acids that have one or more substitutions (such as conservative substitutions), deletions or insertions with the following nucleic acids: comprising a code selected from SEQ ID NO: 8- 11. A nucleic acid containing a nucleic acid sequence of the amino acid sequence shown in any one of 11, 15-18, and 26-29; or a nucleic acid containing a nucleic acid sequence selected from SEQ ID NO: 8-11, 15-18, and 26-29 The amino acid sequence of the nucleic acid has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of the nucleic acid sequence.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include but are not limited to viruses, plasmids, cosmids, lambda phage or yeast artificial chromosomes (YAC). In one embodiment, the vector is pTT5 vector or pcDNA3.1.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, and see also Charlton, Methods in Molecular Biology, Volume 248 (BKCLo, editor, Humana Press, Totowa, NJ, 2003) , Pages 245-254, which describe the expression of antibody fragments in E. coli). After expression, the antibody can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains where the glycosylation pathway has been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, a mammalian cell line modified to be suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293HEK or 293F or 293 cells, such as, for example, Graham et al., J. Gen Virol. 36:59 (1977)) and so on. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:216 (1980), CHO-S cells, ExpiCHO, etc.; And myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (BKCLo, Humana Press, Totowa , NJ), pages 255-268 (2003).

IV. Production and purification of antibody molecules of the present invention

In one embodiment, the present invention provides a method for preparing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment), wherein the method includes a method suitable for expressing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment). ) The host cell is cultured under the condition of nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated. In a certain embodiment, the method further comprises recovering the antibody molecule of the invention or a fragment thereof (preferably an antigen-binding fragment) from the host cell.

In one embodiment, a method for preparing an antibody molecule of the present invention is provided, wherein the method comprises, under conditions suitable for expression of the antibody, culturing the antibody (for example, any one polypeptide chain and/or multiple polypeptide chains) The nucleic acid or the host cell containing the expression vector of the nucleic acid, as provided above, and the antibody is optionally recovered from the host cell (or host cell culture medium).

In order to recombinantly produce the antibody molecule of the present invention, the nucleic acid encoding the antibody (such as the antibody described above, such as any one polypeptide chain and/or multiple polypeptide chains) is isolated and inserted into one or more vectors for use in the host Further cloning and/or expression in the cell. Such nucleic acids are easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains).

The antibody molecules prepared as described herein can be purified by known existing techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be obvious to those skilled in the art. The purity of the antibody molecule of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, and the like.

V. Assay

The anti-TIM-3 antibodies provided herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities by a variety of assays known in the art. On the one hand, the antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot, and the like. The binding to TIM-3 can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, it is measured using radioimmunoassay (RIA) or biofilm thin-layer interferometry or MSD assay or surface plasmon resonance (SPR).

On the other hand, a competition assay can be used to identify antibodies that compete with any of the anti-TIM-3 antibodies disclosed herein for binding to TIM-3. In certain embodiments, such competitive antibodies bind to the same or overlapping epitopes (e.g., linear or conformational epitopes) as bound by any of the anti-TIM-3 antibodies disclosed herein. The epitopes bound by antibodies are known, for example, see Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) for an exemplary method.

The present invention also provides an assay method for identifying anti-TIM-3 antibodies with biological activity. Biological activities can include, for example, binding to TIM-3 (for example, binding to human and/or cynomolgus TIM-3), binding to cell surface TIM-3, binding to T cells, and blocking TIM-3 and its ligands Function, activation of T cells and activation of NK cells. An antibody having such biological activity in vivo and/or in vitro is also provided.

In certain embodiments, the antibodies of the invention are tested for such biological activity.

The present invention also provides methods for identifying properties of antibodies, such as properties related to druggability. The properties related to druggability include, for example, thermal stability, such as long-term thermal stability and accelerated stability.

Cells for use in any of the above in vitro assays include cell lines that naturally express TIM-3 or are engineered to express TIM-3. Such cells also include cell lines transfected with TIM-3 expressing TIM-3 and TIM-3 encoding DNA that does not normally express TIM-3.

It is understood that the immunoconjugate of the present invention can be used to replace or supplement the anti-TIM-3 antibody to perform any of the aforementioned assays.

It is understood that a combination of an anti-TIM-3 antibody and another active agent can be used to perform any of the aforementioned assays.

VI. Immunoconjugates

In some embodiments, the present invention provides immunoconjugates comprising any of the anti-TIM-3 antibodies provided herein and other substances, such as therapeutic agents, including chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small Molecular drugs or immunomodulators (e.g. anti-inflammatory or immunosuppressive agents). In one embodiment, the other substance is, for example, a cytotoxic agent, which includes any agent that is harmful to cells.

In some embodiments, the immunoconjugate is used to prevent or treat cancer.

VII. Pharmaceutical compositions and pharmaceutical preparations

In some embodiments, the present invention provides a composition comprising any anti-TIM-3 antibody or fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises pharmaceutical excipients. In one embodiment, the composition, for example, a pharmaceutical composition, comprises the anti-TIM-3 antibody of the present invention or a fragment or immunoconjugate thereof, and a combination of one or more other therapeutic agents.

The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-TIM-3 antibodies or immunoconjugates thereof, or compositions comprising polynucleotides encoding anti-TIM-3 antibodies (including pharmaceutical compositions or Pharmaceutical preparations). In certain embodiments, the composition comprises one or more TIM-3 binding antibodies or fragments thereof, or one or more polynucleotides encoding one or more anti-TIM-3 antibodies or fragments thereof . These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, etc. that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. It is also possible to use saline solutions and aqueous dextrose and glycerol solutions as liquid carriers, especially for injectable solutions.

Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skimmed milk, glycerin , Propylene, glycol, water, ethanol, etc. For the use and use of excipients, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

If desired, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, and saccharin.

The composition of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

It can be prepared by mixing the antibody of the present invention with the required purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). The pharmaceutical preparation of the antibody is preferably in the form of a lyophilized preparation or an aqueous solution.

The pharmaceutical composition or preparation of the present invention may also contain more than one active ingredient that is required for the specific indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as chemotherapeutics, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulators. The active ingredients are suitably present in combination in an amount effective for the intended use. In one embodiment, the other antibody is a PD-1 pathway antibody.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules.

VIII. Drug combinations and kits

In some embodiments, the present invention also provides a pharmaceutical combination comprising the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention, or an immunoconjugate thereof, and a PD-1 pathway antibody, and optionally one Or a variety of other therapeutic agents (such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, etc.).

Another object of the present invention is to provide a kit of medicines, which contains the drug combination of the present invention, preferably the kit is in the form of a drug dosage unit. Thereby, dosage units can be provided according to the dosing schedule or drug administration interval.

In one embodiment, the kit of the present invention contains in the same package:

-A first container containing a pharmaceutical composition comprising an anti-TIM-3 antibody or an antigen-binding fragment thereof;

-A second container containing a pharmaceutical composition containing PD-1 pathway antibodies.

In some embodiments, the PD-1 pathway antibodies include anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies.

In a specific embodiment, the PD-1 antibody includes the anti-PD-1 antibody described in WO2017025016, CN108473977B and WO2017133540 and other patent applications/patents of the same family, and the entire content of the patent or patent application (including definitions of terms) Was introduced into this article. Wherein, preferably, the anti-PD-1 antibody is the anti-PD-1 antibody D-S228P IgG4 disclosed in WO2017025016A1, which is also referred to as PD-1 antibody IBI308 in this application or the anti-PD-1 antibody disclosed in WO2017133540A1 C-S228P IgG4, also referred to as PD-1 antibody 11430 in this application.

IX. Uses and methods

One aspect of the present invention provides a method for preventing or treating tumors (e.g. cancer) in a subject, comprising administering to the subject an effective amount of the anti-TIM-3 antibody or antigen-binding fragment or immunoconjugate thereof of the present invention , Pharmaceutical composition, pharmaceutical combination or kit.

In some embodiments, the tumor (e.g., cancer) patient has (e.g., elevated levels, e.g., nucleic acid or protein levels) TIM-3. In some embodiments, the tumor (e.g., cancer) patient has (e.g., elevated levels, e.g., nucleic acid or protein levels) PD-L1 or PD-1 or PD-L2. In some embodiments, the tumor (e.g. cancer) patient has both (e.g. elevated levels, e.g. nucleic acid or protein levels) TIM-3 and (e.g. elevated levels, e.g. nucleic acid or protein levels) PD -L1 or PD-1 or PD-L2.

In some embodiments, the cancer is a cancer that is resistant to PD-1 pathway antibody treatment.

In some embodiments, the tumor, such as cancer, includes solid tumors and hematological tumors, as well as metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be early, middle or late or metastatic cancer. In some embodiments, the tumor is a tumor that requires activation of T cells, such as cancer, such as a tumor or cancer with T cell dysfunction.

In some embodiments, the tumor treatment will benefit from PD-L1 or PD-1 or PD-L2 that inhibits nucleic acid or protein levels. In some embodiments, the tumor treatment benefits from blocking the binding of PD-1 to PD-L1, or the binding of PD-1 to PD-L2.

In some embodiments, the tumor treatment will benefit from TIM-3 that inhibits nucleic acid or protein levels. In some embodiments, the tumor treatment benefits from blocking the binding of TIM-3 to its ligand, such as phosphatidylserine.

In a specific embodiment, the tumor treatment will benefit from

(i) PD-L1 or PD-1 or PD-L2 that inhibits nucleic acid or protein levels;

(ii) Block the binding of PD-1 and PD-L1, or PD-1 and PD-L2;

(iii) TIM-3 that inhibits nucleic acid or protein levels;

(iv) Blocking the binding of TIM-3 to its ligand, such as phosphatidylserine;

(v) A combination of one or more of the above.

In a specific embodiment, the anti-TIM-3 antibody of the present invention can activate T cells (eg CD4+ T cells) or NK cells.

In a specific embodiment, the anti-TIM-3 antibody of the present invention enhances CD4+ T cell function, for example, by increasing CD4+ T cell proliferation and/or increasing CD4+ T cell cytokine production. In some embodiments, the cytokine is an interleukin, such as IL-2.

In some embodiments, the tumor is tumor immune escape. In some embodiments, the tumor is cancer.

The subject may be a mammal, for example, a primate, preferably a higher primate, for example, a human (e.g., an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject has or is at risk of suffering from a disease described herein (e.g., cancer). In certain embodiments, the subject has received or has received other treatments, such as chemotherapy treatments and/or radiation therapy. In some embodiments, the subject has previously received or is receiving immunotherapy, for example with PD-1 pathway antibodies.

In other aspects, the present invention provides the use of antibody molecules or fragments or immunoconjugates or pharmaceutical compositions or pharmaceutical combinations or kits in the production or preparation of drugs for the purposes described herein, for example, For the prevention or treatment of related diseases or disorders mentioned herein.

In some embodiments, the antibody molecules or fragments or immunoconjugates or pharmaceutical compositions or drug combinations or kits of the present invention will delay the onset of the disorder and/or symptoms related to the disorder.

In some embodiments, the antibody molecules of the present invention or fragments or immunoconjugates or pharmaceutical compositions thereof can also be combined with PD-1 pathway antibodies, and optionally one or more other therapies such as treatment modalities and/or Other therapeutic agents are administered in combination for the purposes described herein, for example for the prevention and/or treatment of related diseases or disorders mentioned herein.

In some embodiments, the treatment modality includes surgery; radiation therapy, localized irradiation or focused irradiation, and the like.

In some embodiments, the therapeutic agent is selected from chemotherapeutics, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulators.

Exemplary immunomodulators include immunosuppressive agents or anti-inflammatory agents.

In some embodiments, the antibody combinations described herein may be administered separately, for example, as separate antibodies, or when linked (for example, as a bispecific or trispecific antibody molecule).

Such combination therapies encompass combined administration (for example, two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case, additional therapeutic agents and/or agents may be administered. The administration of the antibody of the invention occurs before, at the same time, and/or afterwards.

The route of administration of the pharmaceutical composition is according to known methods, for example, oral, intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Approach; through a sustained release system or through an implanted device. In certain embodiments, the composition may be administered by bolus injection or by continuous infusion or by implantation device.

The composition may also be applied topically via an implanted membrane, sponge, or another suitable material on which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implanted device is used, the device can be implanted into any suitable tissue or organ, and the desired molecule can be delivered via diffusion, timed release bolus, or continuous administration.

X. Methods and compositions for diagnosis and detection

In certain embodiments, any anti-TIM-3 antibody or antigen-binding fragment thereof provided herein can be used to detect the presence of TIM-3 in a biological sample. When the term "detection" is used herein, it includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule complexed magnetic beads, ELISA assays Method, PCR-technology (for example, RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a lesion associated with a hyperproliferative or cancerous lesion.

In one embodiment, an anti-TIM-3 antibody or antigen-binding fragment thereof for use in a diagnosis or detection method is provided. In another aspect, a method of detecting the presence of TIM-3 in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of TIM-3 protein in a biological sample. In certain embodiments, TIM-3 is human TIM-3 or cynomolgus TIM-3. In certain embodiments, the method includes subjecting a biological sample to an anti-TIM-3 antibody or antigen-binding fragment thereof as described herein under conditions that allow the anti-TIM-3 antibody or antigen-binding fragment thereof to bind to TIM-3 Contact and detect whether a complex is formed between the anti-TIM-3 antibody or its antigen-binding fragment and TIM-3. The formation of the complex indicates the presence of TIM-3. The method can be an in vitro or in vivo method. In one embodiment, the anti-TIM-3 antibody or antigen-binding fragment thereof is used to select subjects suitable for treatment with the anti-TIM-3 antibody or antigen-binding fragment thereof, for example, where TIM-3 is used to select the The subject's biomarker.

In one embodiment, the antibodies of the invention can be used to diagnose tumors, such as cancer, for example to evaluate (eg, monitor) the treatment or progression, diagnosis, and/or staging of the diseases described herein in an individual. In certain embodiments, a labeled anti-TIM-3 antibody or antigen-binding fragment thereof is provided. Labels include, but are not limited to, labels or parts that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and parts that are indirectly detected, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.

In some embodiments provided herein, the sample is obtained prior to treatment with the anti-TIM-3 antibody or antigen-binding fragment thereof. In some embodiments, the sample is obtained before other therapies are used. In some embodiments, the sample is obtained during or after treatment with other therapies. In some embodiments, the sample is obtained before, during, or after treatment with the PD-1 pathway antibody or antigen-binding fragment thereof.

In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

In some embodiments, TIM-3 is detected before treatment, for example, before initiating treatment or before some treatment after the treatment interval.

In some embodiments, there is provided a method of treating the disease of the present invention, the method comprising: testing a subject (e.g., sample) (e.g., subject sample) for the presence of TIM-3, thereby determining TIM- 3 value, compare the TIM-3 value with the control value, and if the TIM-3 value is greater than the control value, then administer a therapeutically effective amount of an anti-TIM-3 antibody, optionally in combination with one or more other therapies, to the subject Or an antigen-binding fragment thereof (for example, an anti-TIM-3 antibody or antigen-binding fragment thereof as described herein), thereby treating the disease.

These and other aspects and embodiments of the invention are described in the accompanying drawings (the brief description of the drawings immediately follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative rather than a limiting manner, and various modifications can be made by those skilled in the art.

Example

Example 1. Preparation of hybridoma cells

Hybridoma technology is to fuse two kinds of cells while maintaining the main characteristics of both. These two kinds of cells are mouse spleen cells immunized with antigen and mouse myeloma cells. The main feature of mouse spleen cells (B lymphocytes) immunized with specific antigens is its antibody secretion function, but they cannot be continuously cultured in vitro. Mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, they have The so-called immortality. Under the action of the selective medium, only hybrid cells fused with B cells and myeloma cells can have the ability to continue culture, forming cell clones that have both the function of antibody secretion and the maintenance of cell immortality. In this experiment, mice were immunized with human Tim-3 protein, and then spleen cells of the mice were fused with myeloma cells to obtain hybridoma cells capable of expressing positive antibodies.

Hybridoma fusion

Laboratory animal and immune information

Preparation of the electrofusion dish: Thoroughly soak the electrofusion dish with 70% ethanol, and dry it in an ultra-clean table for later use.

Isolation of spleen cells: the mice were sacrificed by cervical dislocation, the body surface was disinfected with 75% alcohol for 5 minutes, and then placed on the mouse dissecting board in the ultra-clean bench, lying on the left side, and fixing the limbs with a 7-gauge needle. Open the abdominal cavity aseptically and take out the spleen, wash it with basal medium (the configuration method is as follows), and carefully remove the surrounding connective tissue. The spleen was then transferred to another petri dish containing basal medium. Press the spleen with an elbow needle, insert a hole on the spleen with a small needle, and squeeze with tweezers to fully release the spleen cells to make a spleen cell suspension. The cell suspension was filtered through a 70μM cell sieve and washed with 30ml basal medium, and centrifuged at 1200rpm for 6min.

Lysis of red blood cells: Remove the supernatant, and resuspend the cells with 10ml RBC Lysis Buffer (GIBCO, A10492-01). Then add 20ml RBC Lysis Buffer. The suspension was allowed to stand for 5 minutes and then centrifuged at 1100 rpm for 6 minutes. After removing the supernatant, resuspend the cells in 10ml basal medium, then add 30ml basal medium and centrifuge at 1100rpm for 6min. After removing the supernatant, the cells were resuspended in 20 ml of basal medium and counted.

Electrofusion: Resuspend mouse myeloma SP2/0 cells (ATCC, CRL-1581) in 20ml basal medium and count them. The SP2/0 and spleen cells were mixed at a ratio of 1:2 to 1:1, and centrifuged at 1000 rpm for 6 min. After removing the supernatant, the mixed cells were resuspended in 10 ml fusion buffer (BTXpress, 47-0001). Then add 15 ml of fusion buffer, centrifuge at 1000 rpm for 5 min, and remove the supernatant. After repeating the above steps, reselect the cells with an appropriate amount of fusion buffer, and adjust the mixed cell density to 1×10 ⁷ cells/ml. The parameters of the electrofusion instrument are set as follows. Add 2ml of cell suspension to each electrofusion dish for electrofusion.

Plating after electrofusion: cells are allowed to stand for 5 min at room temperature in an electrofusion dish. Transfer the cells to a centrifuge tube, and dilute the cells to 1 to 2×10 ⁴ cells/ml with the selection medium (the configuration method is as follows). Add 100μl of cell suspension to each well of a 96-well plate. The selection medium was replaced on the 7th day after fusion. Screening is performed after the 10th day of culture (or longer, depending on the cell growth status). FACS (C6 (BD Biosciences)) was used to screen out hybridoma cells expressing specific anti-Tim-3 antibodies.

Cryopreservation of cells: Observe the cell status, and when the cells grow well and the viability is >90%, centrifuge at 1000 rpm for 5 minutes to remove the supernatant. Resuspend the cells with cryopreservation solution (45.5% FBS (Hyclone), 44.5% RPMI-1640 (Hyclone), 10% DMSO (SIGMA)) to 1×10 ⁷ cells/ml, aliquot into cryopreservation tubes, and put Store in a program cooling box at -80°C.

Example 2. Production and purification of chimeric antibodies

The present invention uses molecular biology technology to obtain the antibody sequence of the anti-Tim-3 antibody produced by the hybridoma cell in Example 1, and uses it to construct a human-mouse chimeric antibody.

Hybridoma sequencing

RNA extraction: Centrifuge the fresh cells obtained in Example 1 at 300g for 5min, remove the supernatant, add 500μl LY buffer (Biomiga, R6311-02) to the pellet (add 20μl β-mercaptoethanol per 1ml before use), and mix well To clarify. Add to the DNA removal tube, centrifuge at 13000 rpm for 2 min, and collect the flow-through. Add 100% ethanol to the flow-through liquid at a ratio of 1/2, and mix 5 times until it is clear. Add the clear solution to the RNA collection tube, centrifuge at 13000 rpm for 1 min to remove the liquid, add 500 μl RB (Recovery Buffer) (Takara), centrifuge at 13000 rpm for 30 seconds, and then add 500 μl RNA washing buffer (Biomiga) (add before use Ethanol), centrifuge for 30 seconds, repeat the above process, after centrifugation to completely volatilize the ethanol, add 30μl DEPC water to the collection column, centrifuge at 12000g for 2min, and collect the eluate. Determine the RNA concentration.

Use PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, 6210A) to obtain cDNA by reverse transcription:

Configure reaction system I as follows:

After incubating at 65°C for 5 min, quickly place on ice to cool. Add the following reverse transcription system to reaction system I, the total amount is 20μl:

Slowly mix and perform reverse transcription and translation according to the following conditions: 42℃60min→95℃5min, then cool on ice to obtain cDNA.

Use TaKaRa EX Tag kit to connect cDNA to T vector:

PCR amplifies the variable regions of the heavy chain and light chain respectively, and the PCR reaction system is as follows:

The heavy chain variable region (VH) primers of the mouse anti-Tim-3 antibody (Primer Mix 1, mixed in the following proportions to obtain Primer Mix 1 for subsequent VH PCR amplification):

The light chain variable region (VL) primers of the mouse anti-Tim-3 antibody (Primer Mix 2, after mixing in the following proportions, obtain Primer Mix 2 for subsequent PCR amplification of VL.):

The PCR reaction conditions are as follows:

Take 4.5μl of the PCR product obtained from the above PCR reaction, add 0.5μl pMD20-T vector (Clontech), 5μl Ligation Mighty Mix (Takara, 6028), mix gently, and react at 37°C for 2h to obtain the ligation product.

Transformed cells:

Take out TOP10 competence (Tiangen Biochemical Technology (Beijing) Co., Ltd., CB104-02) at -80℃, melt on ice, take 5μl of the ligation product obtained above and add to the melted TOP10 competence, mix well and incubate on ice for 30min . After heat shock at 42°C for 90s, quickly cool it on ice for 2 minutes, add 900μl of LB medium (Sangong Bioengineering (Shanghai) Co., Ltd.) to the EP tube, and incubate for 1 hour at 37°C in a shaker at 220 rpm. Centrifuge at 3000g for 2min, aspirate 800μl of supernatant, resuspend the bacteria with the remaining medium and spread it on an ampicillin-resistant plate. Incubate overnight at 37°C and pick clones for sequencing.

Construction of chimeric antibodies

The VH and VL regions of the murine anti-Tim-3 antibody produced by the hybridoma in Example 1 that have been sequenced were amplified by PCR.

The PCR system is as follows:

*For VH chain amplification, Primer Mix 1 is used; for VL chain amplification, Primer Mix 2 is used.

The gel is cut to recover the PCR amplified product.

Homologous recombination reaction:

The homologous recombination system is as follows:

React at 37°C for 30 min to obtain a recombinant product. The recombinant product was transformed into TOP10 (Tiangen, CB104-02) competent, and a single clone was selected for sequencing, the clone containing the plasmid with the correct insertion direction was selected as the positive clone, and the positive clone was saved.

Expression and purification of chimeric antibodies

A plasmid containing anti-Tim-3 antibody was extracted from the positive clone obtained above.

Passage 293F cells (Invitrogen) according to the required transfection volume, and adjust the cell density to 1.5×10 ⁶ cells/ml the day before transfection. The cell density on the day of transfection was approximately 3×10 ⁶ cells/ml. Take 1/10 of the final volume of Opti-MEM medium (Gibco, 31985-070) as the transfection buffer, add the plasmid obtained above to the transfected cells at 1.0 μg/ml, and mix. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI is 1:3 in 293F cells), mix and incubate at room temperature for 20 minutes to obtain a DNA/PEI mixture. Slowly add the DNA/PEI mixture to the cells, gently shake the flask while adding, and incubate at 36.5°C, 120 rpm, and 8% CO ₂ conditions. After 18 hours, add 2M sodium valproate solution (Sigma) with a transfection volume of 0.1% and 2.5% homemade feed, and culture _{at 36.5°C, 120 rpm, 8% CO 2.} When the culture is continued to the 6th day or when the cell viability is ≤60%, the cell supernatant is collected for purification.

The chromatography column used for purification was treated with 0.1M NaOH for 2 hours, and the glass bottles were washed with distilled water and then dried at 180°C for 4 hours to obtain a purification column. Before purification, centrifuge the collected cell material at 4500 rpm for 30 min, and discard the cells. The supernatant was filtered with a 0.22 μm filter. Use 10 column volumes of binding buffer (sodium phosphate 20mM.NaCl 150mM, pH7.0) to equilibrate the Protein A column (Hitrap Mabselect Sure 5*5ml, GE, 11-0034-95). Add the filtered supernatant to the purification column and equilibrate with 10 column volumes of binding buffer. Add 5ml of elution buffer (citric acid+sodium citrate 0.1M, pH3.5), collect the eluate, add 80μl of 2M Tris-HCl for every 1ml of eluate. Tris-HCl. The collected antibodies were concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the concentration was detected.

For the two chimeric antibodies (CH4-3 and CH5-17) obtained in the present invention, please refer to the CDR sequence, the variable region of the light chain and the variable region of the heavy chain, the amino acid sequence of the light chain and the heavy chain, and the sequence numbers. Attached sequence list.

The control antibody used in the present invention comes from the TIM-3 antibody of the patent WO2016161270 of Tesaro, Inc., hereinafter referred to as TSR-022, the negative control application antibody IgG1, the sequence of which is shown in the attached sequence table. The expression and purification method of the control is the same as the antibody of the present invention.

Example 3 Determination of the binding kinetics between the chimeric antibody of the present invention and the antigen by the biofilm thin-layer interference technique

_{The equilibrium dissociation constant (K D} ) of the antibody of the present invention bound to human Tim-3 is determined by the biofilm thin-layer interferometry (BLI) technique. The BLI method affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p.270-8).

Half an hour before the start of the experiment, according to the number of samples, take an appropriate number of AHC (Foertbio, 18-5060) sensors and soak them in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

Take 100μl of SD buffer, antibody (CH4-3, CH5-17 and TSR-022, the same concentration), antigen (including human Tim-3, and cynomolgus Tim-3, all purchased from Novoprotein) were added to 96 Holes in a black polystyrene half-volume microplate (Greiner, 675076). Fortebio Octet Red96 is used for detection, and the sensor position is selected according to the sample position layout. The instrument setting parameters are as follows: Operating steps: Baseline, Loading～1nm, Baseline, Association and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the speed is 1000rpm, and the temperature is 30℃. _{The K D} value was analyzed using ForteBio Octet analysis software.

In the experiment described in the above assay, the affinities of antibodies CH4-3 and CH5-17 are shown in Table 1:

Table 1. Affinity constants (equilibrium dissociation constants) for detecting antigen-antibody binding by ForteBio

_{In the above experiment, the K D} values of the chimeric antibodies CH4-3, CH5-17 and human Tim-3 were 2.02E-09M and 5.36E-09M, respectively. Compared with the control group TSR-022, the antibody in this study Have similar or better K _D values.

Example 4 Binding experiment of chimeric antibody and CHO-S cells overexpressing human Tim-3

In this study, a flow cytometer was used to detect the binding of the serially diluted chimeric antibody of the present invention to the stable CHO-S cell line overexpressing human Tim-3 on the surface.

The cDNA encoding human Tim-3 (SEQ ID NO: 34) was cloned into the pCHO1.0 vector (Invitrogen) and transfected into CHO-S cells (Invitrogen) to produce CHO-S cells overexpressing human Tim-3 ( CHOS-hTim-3).

The CHOS-hTim-3 cells were counted and diluted to 2×10 ⁶ cells/ml, and 100 μl/well was added to the U-shaped bottom 96-well plate. Centrifuge at 300g for 5 minutes to remove the cell culture medium. The samples (respectively chimeric antibodies CH4-3, CH5-17, and positive control antibody TSR-022) (antibody dilution method: the highest antibody concentration is 400nM, three-fold dilution in PBS, a total of 12 concentrations were tested ) Add the U-shaped plate and resuspend the cells, 100 μl/well, and let stand on ice for 30 min. Centrifuge at 400g for 5 min to remove the supernatant, and wash the cells twice with PBS. Centrifuge at 300 g for 5 min, remove PBS, add 100 μl anti-human Fc PE-labeled secondary antibody (SoutherBiotech; 2040-09) (diluted in PBS 1:100) to each well, and incubate for 30 min on ice in the dark. The supernatant was removed by centrifugation at 400g for 5 min, and the cells were washed 3 times with PBS. Resuspend the cells with 200μl 1×PBS and FACS detection.

In the experiment described in the above assay, the binding of antibodies CH4-3, CH5-17 and CHOS-hTim-3 cells is shown in Figure 1.

In the experiment described in the above assay, the test results are shown in Figure 1. The antibodies CH4-3 and CH5-17 both bind to the human Tim-3 molecule overexpressed on CHO-S cells, and the EC50 is 0.7943nM and 0.6521nM, respectively. Compared with the control antibody TSR-022, the binding ability is similar.

Example 5 Humanization of Chimeric Antibodies

The antibody CH4-3 chimeric antibody obtained in Example 2 was humanized. And go through the following steps for humanization:

①Determine the structure of the CDR loop;

②Find the closest homologous sequence for each V/J region of the heavy chain and light chain in the human germline sequence database;

③ Screen the human germline that best matches the heavy chain and light chain and the lowest number of back mutations;

④ Construct the CDR region of the chimeric antibody onto the human framework region;

⑤Using sequence and structural features, determine the amino acid positions in the framework region that maintain the CDR function;

⑥ Make back mutations at the sequence positions that are determined to be important (return to the input amino acid type);

⑦ Optimize amino acids at risk sites.

For the CDR, light chain variable region and heavy chain variable region of a humanized antibody Hz4-3.6 obtained in the present invention, please refer to the attached sequence table for the amino acid sequences of the light chain and the heavy chain.

Example 6 Production and Purification of Humanized Antibody

The humanized antibody Hz4-3.6 sequence obtained above was cloned into pcDNA3.1 (Invitrogen) to obtain plasmid DNA.

The ExpiCHO ^TM Expression system (Gibco, A29133) was used to produce protein. Specifically: ExpiCHO cells (Gibco) were passaged according to the required transfection volume, and the cell density was adjusted to 3.5×10 ⁶ cells/ml the day before transfection. . On the day of transfection, the cell density was adjusted to 6×10 ⁶ cells/ml. Take a 50ml centrifuge tube, add the transfection buffer reagent OptiPRO SFM (Gibco, 12309019) at 8% of the transfected cell volume, and calculate the total amount of plasmid needed for the transfected cells at 0.8μg/ml (the ratio of light to heavy chain is 1: 1) Use a 0.22μm filter membrane to filter the transfection buffer containing the obtained plasmid DNA into another new 50ml centrifuge tube, add ExpiFectamineTMCHO reagent (Gibco ,100033022), mix thoroughly, mix the transfection reagent with the plasmid DNA and slowly add it to the cells immediately, gently shake the flask while adding, and control the incubation time of the transfection reagent with the plasmid not to exceed 5min; 36.5℃, 8% CO ₂ , shaker culture. After 18-22 hours, Enhancer (Gibco, 100033019) was added at a cell volume of 6 μl/ml, and Feed (Gibco, A29101-01) was added to a cell volume of 300 μl/ml. The cells were cultured at 36.5° C., 120 rpm, and 8% CO ₂ . When the culture is continued to the 6th day or when the cell viability is ≤60%, the cell supernatant is collected for purification.

The chromatography column used for purification was treated with 0.1M NaOH for 2 hours, and the glass bottles were washed with distilled water and then dried at 180°C for 4 hours to obtain a purification column. Before purification, centrifuge the collected cell material at 4500 rpm for 30 min, and discard the cells. Filter the supernatant with a 0.22μl filter. Use 10 column volumes of binding buffer (sodium phosphate 20mM.NaCl 150mM, pH7.0) to equilibrate the pre-packed Protein A column (Hitrap Mabselect Sure 5*5ml, GE, 11-0034-95). Add the filtered supernatant to the purification column and equilibrate with 10 column volumes of binding buffer. Add 5ml of elution buffer (citric acid+sodium citrate 0.1M, pH3.5), collect the eluate, add 80μl of 2M Tris-HCl for every 1ml of eluate. The collected antibodies were concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the concentration was detected.

Example 7 Determination of the binding kinetics of the humanized antibody of the present invention with the cynomolgus monkey antigen by the biofilm thin-layer interference technique

_{The equilibrium dissociation constant (K D} ) of the antibody of the present invention bound to the cynomolgus monkey Tim-3 was determined by the biofilm thin-layer interferometry technique (BLI). The BLI method affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p.270-8).

Take 100μl of SD buffer, antibody (Hz4-3.6 and TSR-022 at the same concentration), antigen (cynomolgus Tim-3, NOVOPROTEIN, CU83) and add them to a 96-well black polystyrene half-well microplate (Greiner, 675076). Fortebio Octet Red96 is used for detection, and the sensor position is selected according to the sample position layout. The instrument setting parameters are as follows: Operating steps: Baseline, Loading～1nm, Baseline, Association and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the speed is 400rpm, and the temperature is 30℃. _{The K D} value was analyzed using ForteBio Octet analysis software.

In the experiment described in the above assay, the affinity of the humanized antibody Hz4-3.6 is shown in Table 2:

Table 2. Affinity constants (equilibrium dissociation constants) for detecting antigen-antibody binding by ForteBio

In the above experiment, the K _D value of the humanized antibody Hz4-3.6 is 1.04E-08M. Compared with the control antibody TSR-022, the antibody in this study has a similar K _D value.

Example 8 SPR Determination of Affinity of Humanized Antibodies

The surface plasmon resonance method (SPR) was used to determine the equilibrium dissociation constant (K _D ) of the antibody of the present invention binding to human Tim-3. Based on the SPR principle, when a beam of polarized light is incident on the end face of the prism at a certain angle, a surface plasmon wave will be generated at the interface between the prism and the gold film, causing free electrons in the metal film to resonate, that is, surface plasmon resonance. When analyzing, first fix a layer of biomolecular recognition film on the surface of the sensor chip, and then flow the sample to be tested across the chip surface. If there are molecules in the sample that can interact with the biomolecular recognition film on the chip surface, it will cause the gold film surface The change of refractive index eventually leads to the change of SPR angle. By detecting the change of SPR angle, information such as affinity and kinetic constant of the analyte can be obtained.

In this example, Biacore (GE Healthcare, T200) was used to determine the K _{D of a} humanized antibody. The specific method is as follows: After the antibody (Hz4-3.6 and TSR-022 at the same concentration) is captured on the chip by the anti-human Fc antibody, the antigen is detected The binding and dissociation with the captured antibody obtains affinity and kinetic constants. The method includes chip preparation and affinity detection. In the measurement process, 10×HBS-EP+(BR-1006-69, GE Healthcare) diluted by 10 times was used as the experimental buffer. The chip preparation process uses an amino coupling kit (BR-1006-33, GE Healthcare), and the anti-human Fc antibody is coupled to the surface of the CM5 chip (29-1496-03, GE Healthcare). Then, the anti-human Fc antibody was diluted in 10mM Acetate (pH 5.0) and injected into the dual channel of the CM5 chip to make the protein covalently coupled to the surface of the chip channel with a coupling height of about 6000RU. Finally, 1M ethanolamine was injected to block the remaining activated sites. Each cycle of affinity detection includes capturing antibody, binding a concentration of antigen and chip regeneration. First, the diluted antibody was captured on the fourth channel of the CM5 chip at a flow rate of 10μl/min, and the capture time was 60s. Channel 3 was the blank control channel. Then, according to the optimal concentration range after method development, the antigen (human Tim-3 (SINO, 10390-H08H-50)) after gradient dilution, from low concentration to high concentration, is injected into the dual channel of the chip with a binding time of 180s , The dissociation time is 600s. Finally, 10mM Glycine pH 1.5 (BR-1003-54, GE Healthcare) was used to regenerate the chip. The data results use Biacore T200 analysis software, and the analysis model used is a 1:1 combination model for kinetic analysis.

In the experiment described in the above determination method, the affinity of the antibody Hz4-3.6 is shown in Table 3:

Table 3. Affinity constants (equilibrium dissociation constants) for detecting antigen-antibody binding by Biacore

In the above experiments, the K _D values of the humanized antibody Hz4-3.6 were 3.768E-09M. Compared with the control antibody TSR-022, the antibody in this study has a better K _D value.

Example 9 Binding experiment of humanized antibody and cells expressing human Tim-3

In this study, a flow cytometer was used to detect the binding of the serially diluted humanized antibody of the present invention to the stable CHO-S cell line overexpressing human Tim-3 on the surface.

Detection steps: 400g, 5min, centrifugation, remove the cell culture medium, resuspend CHOS-hTim-3 cells in PBS, after counting, adjust the cell density to 2×10 ⁶ cells/ml, add 100μl/ml to the U-shaped bottom 96-well plate hole. Add the antibody to be tested, three-fold gradient dilution, and let stand on ice for 30 min. 300g, 5min remove the supernatant, wash the cells with PBS once. 300g, 5min remove PBS, add 100μl 1:200 dilution of PE-anti-human Fc antibody (SOUTHERN BIOTECH, 2040-09) to each well. Incubate on ice for 30 min in the dark. 400g, 5min, remove the supernatant, and wash the cells twice with PBS. Resuspend the cells with 100 μl PBS and detect by flow cytometry (BD, FACSCELESTA).

In the experiment described in the above assay, the test results are shown in Figure 2. The humanized antibody Hz4-3.6 binds to the human Tim-3 over-expressed on CHO-S cells. Compared with the control antibody TSR-022, it has Considerable combination ability.

Example 10 Combination experiment of humanized antibody and CD4+ T cells

In this study, a flow cytometer was used to detect the binding of the humanized antibody of the present invention to CD4+T in serial dilutions.

Resuscitate human PBMC cells (ALLCELLS, PB005F), and use Human T cell enrichment kit (STEMCELL, 19052) to isolate CD4+ T cells.

Culture in Medium CTS (GIBCO, A3021002) medium, add Dynabeads Human T-Activator CD3/CD28Beads (Gibco, 11131D) according to CD4+:anti-CD3/CD28Beads=1:1, and stimulate for 4 days.

Detection steps: 400g, 5min, centrifugation, remove the cell culture medium, resuspend the cells in PBS, after counting, adjust the cell density to 2×10 ⁶ cells/ml, and add 100 μl/well to the U-shaped bottom 96-well plate. Add the antibody to be tested, three-fold gradient dilution, and let stand on ice for 30 min. 300g, 5min remove the supernatant, wash the cells with PBS once. 300g, 5min remove PBS, add 100μl 1:200 dilution of PE-anti-human Fc antibody (SOUTHERN BIOTECH, 2040-09) to each well. Incubate on ice for 30 min in the dark. 400g, 5min, remove the supernatant, and wash the cells twice with PBS. Resuspend the cells with 100μl PBS and detect by flow cytometry (BD, ACCURIC6plus).

In the above experiment, the results are shown in Figure 3. The humanized antibody Hz4-3.6 has the same ability to bind CD4+ T cells to the control antibody TSR-022.

Example 11 MOA method to detect the biological activity of antibodies

One of the ligands of TIM-3 is PtdSer on the surface of apoptotic cells. In this experiment, apoptotic L363 cells were used to detect the ability of anti-TIM-3 antibodies to block the binding of TIM-3 to PtdSer. First, H ₂ O _{2 was used to} induce apoptosis of L363 cells (Kebai, CBP60240), and then flow cytometry was used to detect the ability of anti-Tim-3 antibody to block hTim-3 protein (R&D, 2365-TM-050) from binding to apoptotic cells , So as to determine the ability of the anti-Tim-3 antibody to block the binding of Tim-3 to the ligand PtdSer.

Take the L363 cells in the logarithmic growth phase, centrifuge at 400g for 5 min, and discard the culture supernatant. Use apoptosis induction medium (see table below) to resuspend to 2×10 ⁶ cells/ml, 37°C, 5% CO ₂ incubator, and culture overnight.

Centrifuge at 400g, discard the supernatant, resuspend in 1×binding buffer (10×Annexin V Binding Buffer, BD, 51-66121E) to 2×10 ⁶ /ml; add 50μl/well to 1×binding buffer diluted hTim-3 protein (R&D, 2365-TM-050) Concentration 8μg/ml, add the initial concentration of the antibody to be tested 200nM, double dilution, a total of ten concentration points, add 50μl of resuspended cells to each well, incubate at room temperature for 30 minutes, 200μl/ Add 1×binding buffer to the hole and wash it twice. Centrifuge at 400 g for 5 min to remove PBS, add 100 μl of anti-human Fc PE-labeled secondary antibody (SoutherBiotech; 2040-09) (diluted in PBS 1:200) to each well, and incubate for 30 min on ice in the dark. Centrifuge at 400g for 5min to remove the supernatant, and wash the cells with PBS once. Resuspend the cells with 100μl 1×PBS and FACS detection.

In the above experiments, the experimental results are shown in Figure 4. The antibody Hz4-3.6 can effectively block the binding of Tim-3 to the ligand PtdSer, and has a similar blocking ability compared with the control antibody TSR-022.

Example 12 CD4+T activated cell experiment

In this study, antibodies and CD4+ T cells were incubated with induced maturation DC cells, and the expression of IL-2 in the system was detected to reflect the activation effect of different antibodies on CD4+ T cells. The detailed experimental process is as follows:

Resuscitate human PBMC cells (ALLCELLS, PB005F), stand for 3 hours to adhere to the wall and become monocytes, add 10ml AIM

Medium CTS (GIBCO, A3021002) medium, add IL4 (20ng/ml) (R&D, 204-IL), GM-CSF (10ng/ml) (R&D, 215-GM) to induce monocytes to differentiate into DC cells, culture By day 5, add TNFα (1000U/ml, 10ng/ml) (R&D, 210-TA), RhIL-1β (5ng/ml) (R&D, 201-LB), RhIL-6 ( 10ng/ml) (R&D, 206-IL), 1μM PGE (Tocris, 2296), in a carbon dioxide incubator at 37°C and 5% CO ₂ for 2 days, as a mature DC for mixed lymphocyte reaction (MLR) cell;

Human PBMC cells (ALLCELLS, PB005F) were resuscitated, and CD4+ T cell isolation was performed according to the instructions of Human CD4+ T cell enrichment kit (STEMCELL, 19052). In short, the suspension cell liquid drawn from the PBMC cultured for 2 hours is placed in a 20ml centrifuge tube, centrifuged at 300g for 10 minutes, 500μl of separation solution and 100μl of the purified antibody provided in the kit are added to the cell pellet. Incubate for 20 minutes at 4°C, wash once with the separating solution, add 500μl bead buffer and incubate for 15 minutes, remove the beads by magnetic field, and use AIM

Medium CTS (GIBCO, A3021002) medium wash once, use 8ml AIM

Medium CTS (GIBCO, A3021002) culture medium cells, CD4+ T cells obtained by culture;

Mix the above-mentioned induced mature DC cells with CD4+ T cells, with a volume of 200μl per well, 12,000 DC cells and 120,000 CD4+ cells, add antibodies (gradiently diluted antibodies Hz4-3.6, TSR-022, IBI308 and IgG1), and mix Incubate for 4 days, use Human IL-2 Uncoated ELISA Kit (2nd Gen) detection kit (EBIOSCIENCE, 88-7025-77) to detect the expression of IL2 in each sample. The expression of IL2 of different antibodies reflects the activation of T cells ability.

The experimental results are shown in Figure 5. The combination of Hz4-3.6 and anti-PD-1 antibody IBI308 (sintilimab) can effectively activate T cells in vitro, and its activation effect is stronger than the anti-PD-1 antibody IBI308 antibody alone.

Example 13 NK cell activation experiment

Anti-Tim-3 antibodies can bind to Tim-3 molecules expressed on the surface of NK cells, and then mediate the activation of NK cells. In this study, in a mixed system of NK cells and tumor cells K562 (ATCC), the activation of NK cells was reflected by detecting the activation of NKG2D and CD107a on the surface of NK cells to detect the activation activity of antibodies on NK cells.

Take fresh NK cells (ALLCELLS, PB012-C), adjust the cell density to 1.6x10 ⁶ /ml, plate 50μl per well, add the antibody to be tested, incubate at 37°C, 5% CO _{2 for} 30 minutes, add the target cell K562 (ATCC) (Density 4x10 ⁵ /ml), 50μl per well, incubate at 37°C and 5% CO _{2 for} 5 hours. Centrifuge at 400g for 5 minutes, resuspend the cells in PBS, add fluorescent antibodies FITC anti-human CD314 (NKG2D) Antibody (BIOLEGEND, 320820), PerCP/Cy5.5 anti-human CD56 (NCAM) (BIOLEGEND, 318322) and Brilliant Violet 421 ^TM anti-human CD107a (BIOLEGEND, 328626), incubate at 4°C for 30 minutes, wash once with PBS, and read on a flow cytometer (BD, FACSCELESTA).

In the above experiments, the experimental results are shown in Figure 6. The antibody Hz4-3.6 can effectively enhance the expression of NKG2D and CD107a on the cell surface characterized by NK activation, and the enhancement effect is similar to the control antibody TSR-022.

Example 14. Anti-tumor efficacy test

In this experiment, MC38 cells were used to inoculate hTim-3 gene knock-in mice to determine the anti-tumor effect of the anti-Tim-3 antibody combined with the anti-PD-1 antibody 11430 (WO2017/133540) of the present invention.

Human Tim-3 gene knock-in mice:

The female C57BL/6 background human Tim-3 knock-in mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd., and the grade was SPF. The quality inspection unit was Suzhou Xishan Biotechnology Co., Ltd., and the certificate number was NO.20170010001249. The mice were acclimated for 7 days after arrival, and then the study began.

cell:

Mouse MC38 cells were purchased from Shanghai Heyuan Biotech (CAT#:HYC3401), and routinely subcultured in strict accordance with the instructions for subsequent in vivo experiments. Collect the cells by centrifugation, resuspend the cells in sterile PBS and adjust the cell density to 5×10 ⁶ cells/ml. On day 0, 0.2ml of the cell suspension was subcutaneously inoculated into the right abdominal region of human Tim-3 knock-in mice to establish the MC38-hTim-3 tumor-bearing mouse model.

Administration:

After 7 days of tumor cell inoculation, the tumor volume of each mouse was measured. The mice with tumor volume ^{ranging from 25.23mm 3} to 108.66mm ^{3 were} selected and divided into groups according to tumor volume (7 mice per group), dosage and method of administration As shown in Table 4, h-IgG (purchased from EQUITECH-BIO) was used as a negative control and was administered on the 7, 10, 14, 17, and 21 days after vaccination, and the tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration, and the relative tumor inhibition rate (TGI%) was calculated on the 25th day after inoculation. The calculation formula is as follows:

TGI%=100%*(control group tumor volume—treatment group tumor volume)/(control group tumor volume—control group tumor volume before administration).

Tumor volume measurement: the largest long axis (L) and largest wide axis (W) of the tumor are measured with a vernier caliper, and the tumor volume is calculated according to the following formula: V=L×W ² /2. An electronic balance is used to determine body weight.

Table 4. Experimental design

The results of tumor suppression rate are shown in Figure 7 and Table 5. On the 25th day after vaccination, neither TSR-022 nor Hz4-3.6 single drugs showed tumor suppression effects. The anti-PD-1 antibody 11430 single-agent tumor inhibition rate was 41%, and the tumor inhibition rates of TSR-022 and Hz4-3.6 combined with the anti-PD-1 antibody 11430 were 60% and 73%, respectively. The tumor suppressive effect of TSR-022 and Hz4-3.6 combined with anti-PD-1 antibody 11430 is stronger than that of anti-PD-1 antibody 11430, TSR-022, Hz4-3.6, and h-IgG monotherapy group. At the same time, we tested the body weight of the mice, and the results are shown in Figure 8. There was no significant difference in the body weight of the mice. Therefore, the combination of the antibody against the Tim-3 molecule and the PD-1 antibody of the present invention has a significant inhibitory effect on tumors.

Table 5. Tumor inhibition rate on day 25

Example 15 Detection of Antibody Thermal Stability

Differential scanning fluorimetry (DSF) can provide information about the stability of the protein structure according to the fluorescence change process in the protein map, detect the configuration change of the protein, and obtain the melting temperature (Tm) of the protein. In this example, the DSF method was used to determine the T _m value of the antibody of the present invention.

The antibody Hz4-3.6 of the present invention was diluted to 1 mg/ml with PBS solution.

Add 196μl PBS to 4μl SYPRO Orange Protein Gel Stain (Gibco, catalog number: S6650), and dilute SYPRO Orange Protein Gel Stain 200 times.

To 50μl of the antibody with the above concentration of 1mg/ml, add 10μl of the above-mentioned 50-fold diluted SYPRO Orange Protein Gel Stain, then add 40μl of water, mix and add 50μl to a 96-well PCR plate (Applied Biosystems life technologies, 4306737) . The PCR plate was placed in the 7500 Real Time PCR system (Applied Biosystems, AB/7500) for detection. Set the system temperature to increase by 0.5°C per minute, and the temperature corresponding to the peak of the absolute value of the fluorescence curve is the T _{m of} the protein.

The experimental results are shown in Table 6 and Figure 9 below. _{The T m} value of the antibody of the present invention is >65°C, therefore, it has better thermal stability.

Table 6. Differential scanning fluorescence method to detect antibody T _m

In the above experiments, the humanized antibody Hz4-3.6 described herein has a T _m greater than 65°C, and has good thermal stability.

Example 16 Accelerated Stability Test of Antibody

In order to confirm the stability of the antibody, this example evaluated the long-term thermal stability of the antibody by detecting the changes in purity and cell binding activity of a batch of antibodies prepared after being placed at 40°C for 0, 7, and 14 days.

Concentrate the antibody Hz4-3.6 sample obtained above to 10 mg/ml (dissolved in PBS), and distribute it in EP tubes, 200 μl/tube, and store at 40°C in the dark.

On the 0th, 7th, and 14th days, one tube was taken to use size exclusion chromatography (SEC) to detect the purity of the main monomer peak. The ability of the humanized antibody of the present invention to bind to human Tim-3 cells was determined by flow cytometry, and the determination method was the same as in Example 9. In the experiment described in the above determination method, the purity of the main peak of antibody Hz4-3.6 is shown in Table 7. The cell binding activity test is shown in Figure 10:

Table 7. Changes in the ratio of the main monomer peaks when the antibody is incubated at 40°C

放置40℃(天)Place at 40°C (days)	主峰比例(％)Proportion of main peak (%)
00	98.6298.62
77	98.1198.11
1414	97.9797.97

In the above experiment, the humanized antibody Hz4-3.6 described herein was placed at 40°C for 14 days, and its monomer main peak ratio decreased by only 0.65%, and the ability to bind to cells expressing human Tim-3 did not change significantly. The results show that the humanized antibody Hz4-3.6 described herein has better accelerated stability.

Sequence Listing

Claims

An antibody or antigen-binding fragment thereof that binds to TIM-3, which comprises

The three complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO: 8, 15 or 26, and the 3 complementarity determining regions of the light chain variable region shown in SEQ ID NO: 9, 16 or 27 Area LCDR.
The antibody or antigen-binding fragment thereof of claim 1, which comprises

(i) The 3 complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO: 8 and the 3 complementarity determining regions LCDR of the light chain variable region shown in SEQ ID NO: 9;

(ii) The 3 complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO: 15 and the 3 complementarity determining regions LCDR of the light chain variable region shown in SEQ ID NO: 16; or

(iii) The three complementarity determining regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 26, and the three complementarity determining regions LCDR of the light chain variable region as shown in SEQ ID NO: 27.
An antibody or antigen-binding fragment thereof that binds to TIM-3, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR1 includes or consists of the amino acid sequence of SEQ ID NO:1;

Under the Kabat rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 2 or 12;

Under the Kabat rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3; and

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 5 or 14;

Under the Kabat rule, LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 6;

Under the Kabat rule, LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 7;

or

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 19;

Under the Kabat rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 20;

Under the Kabat rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21; and

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 23;

Under the Kabat rule, LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 24;

Under the Kabat rule, LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25;

or

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR1 includes or consists of the amino acid sequence of SEQ ID NO:1;

Under the Abm rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 4 or 13;

Under the Abm rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3;

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 5 or 14;

Under the Kabat rule, LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 6;

Under the Kabat rule, LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 7;

or

(a) The VH comprises HCDR1, HCDR2 and HCDR3, wherein

Under the Abm rules, HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 19;

Under the Abm rules, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 22;

Under the Abm rules, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21;

(b) The VL includes LCDR1, LCDR2 and LCDR3, wherein

Under the Kabat rule, LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 23;

Under the Kabat rule, LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 24;

Under the Kabat rule, LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises a light chain variable region and/or a heavy chain variable region, wherein,

(a) Heavy chain variable region

(i) The amino acid sequence comprising SEQ ID NO: 8, 15 or 26 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% The identical amino acid sequence or consists of said amino acid sequence; or

(ii) It comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, 15 or 26; or

(iii) Comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared with the amino acid sequence shown in SEQ ID NO: 8, 15 or 26; and/or

(b) Light chain variable region

(i) The amino acid sequence comprising SEQ ID NO: 9, 16 or 27 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% The identical amino acid sequence or consists of said amino acid sequence; or

(ii) It comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, 16 or 27; or

(iii) An amino acid sequence comprising or consisting of 1-10 amino acid substitutions, insertions or deletions compared with the amino acid sequence shown in SEQ ID NO: 9, 16 or 27.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises

(i) Contains amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 8 Sequence or heavy chain variable region consisting of the amino acid sequence, and/or comprising the amino acid sequence shown in SEQ ID NO: 9 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or light chain variable regions consisting of said amino acid sequences;

(ii) Contains amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 15 Sequence or heavy chain variable region consisting of the amino acid sequence, and/or comprising the amino acid sequence shown in SEQ ID NO: 16 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or light chain variable region consisting of said amino acid sequence; or

(iii) Contains amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 26 Sequence or heavy chain variable region consisting of the amino acid sequence, and/or comprising the amino acid sequence shown in SEQ ID NO: 27 with at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence with 96%, 97%, 98% or 99% identity or a light chain variable region composed of said amino acid sequence.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises

(i) Containing the amino acid sequence shown in SEQ ID NO: 8 or the heavy chain variable region composed of the amino acid sequence, and/or containing the amino acid sequence shown in SEQ ID NO: 9 or consisting of the amino acid sequence Light chain variable region;

(ii) Containing the amino acid sequence shown in SEQ ID NO: 15 or the heavy chain variable region composed of the amino acid sequence, and/or containing the amino acid sequence shown in SEQ ID NO: 16 or consisting of the amino acid sequence Light chain variable region; or

(iii) Containing the amino acid sequence shown in SEQ ID NO: 26 or the heavy chain variable region composed of the amino acid sequence, and/or containing the amino acid sequence shown in SEQ ID NO: 27 or consisting of the amino acid sequence Light chain variable region.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which comprises

(a) Heavy chain

(i) The amino acid sequence comprising SEQ ID NO: 10, 17 or 28 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity or consisting of said amino acid sequence; or

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 10, 17 or 28; or

(iii) Comprising or consisting of an amino acid sequence having 1-20 amino acid changes compared with the amino acid sequence shown in SEQ ID NO: 10, 17 or 28;

and / or

(b) Light chain

(i) The amino acid sequence comprising SEQ ID NO: 11, 18 or 29 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity or consisting of said amino acid sequence; or

(ii) It comprises or consists of an amino acid sequence selected from SEQ ID NO: 11, 18 or 29; or

(iii) An amino acid sequence having 1-20 amino acid changes compared with the amino acid sequence shown in SEQ ID NO: 11, 18, or 29, or consisting of the amino acid sequence.
The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which comprises a heavy chain and/or a light chain, wherein

(i) The heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 10 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 11 , 95%, 96%, 97%, 98%, or 99% identical amino acid sequence or consist of said amino acid sequence;

(ii) The heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 17 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 18 , 95%, 96%, 97%, 98%, or 99% identical amino acid sequence or consist of said amino acid sequence;

(iii) The heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 28 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 29 , 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consist of said amino acid sequence.
The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which comprises a heavy chain and/or a light chain, wherein

(i) The heavy chain contains or consists of the amino acid sequence shown in SEQ ID NO: 10, and/or the light chain contains or consists of the amino acid sequence shown in SEQ ID NO: 11;

(ii) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 18;

(iii) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29.
The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1 or IgG2 or IgG3 or IgG4.
The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody.
The TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (such as scFv) Or (Fab') 2 , single domain antibody, double antibody (dAb) or linear antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody is a bispecific or multispecific antibody molecule, preferably, the bispecific antibody molecule is also combined with PD-1, PD-L1 or PD-L2 binding.
An isolated nucleic acid that encodes the light chain variable region or the heavy chain variable region, or the light chain or the heavy chain in the TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.
A vector comprising the nucleic acid of claim 15, preferably the vector is an expression vector.
A host cell comprising the nucleic acid of claim 15 or the vector of claim 16, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO cells, For example, 293F cells or CHO-S cells or ExpiCHO cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
A method for preparing an antibody or antigen-binding fragment thereof that binds TIM-3, the method comprising under conditions suitable for expressing a nucleic acid encoding the TIM-3-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 14 Culturing the host cell of claim 17, optionally isolating the antibody or antigen-binding fragment thereof, and optionally the method further comprises recovering the antibody or antigen-binding fragment thereof that binds to TIM-3 from the host cell.
An immunoconjugate comprising the TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 and other substances, such as a cytotoxic agent.
A pharmaceutical composition comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of claims 1 to 14 or the immunoconjugate of claim 19, and optionally one or more other therapeutic agents, For example, chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.
A pharmaceutical combination comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of claims 1 to 14 or the immunoconjugate of claim 19, and a PD-1 pathway antibody such as an anti-PD-1 antibody or anti PD-L1 antibody or anti-PD-L2 antibody, and optionally one or more other therapeutic agents, such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the TIM-3 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, or claims The immunoconjugate of 19, or the pharmaceutical composition of claim 20.
The method of claim 22, further comprising co-administering a PD-1 pathway antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, to the subject.
A method of preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical combination of claim 21.
The method of any one of claims 22-24, wherein the tumor is a cancer, more preferably, the cancer has elevated levels (e.g., nucleic acid or protein levels) of TIM-3 and/or elevated levels of ( For example, at the nucleic acid or protein level) PD-L1 or PD-1 or PD-L2.
The method of any one of claims 22-25, wherein the tumor is a tumor that is resistant to treatment with a PD-1 pathway antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.
The method according to any one of claims 22-26, wherein the method further comprises administering to the patient one or more therapies, such as treatment modalities and/or other therapeutic agents, preferably, the treatment modalities include surgical treatment and/or radiation Therapy, other therapeutic agents are selected from chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
A method for detecting TIM-3 in a sample, the method comprising

(a) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14; and

(b) Detect the formation of a complex between the antibody or antigen-binding fragment thereof and TIM-3; optionally, the antibody is detectably labeled.