EA042181B1 - ANTI-TIM-3 ANTIBODIES AND THEIR USE - Google Patents

Description

Examples of conservative amino acid substitutions

Starting amino acid residue Single letter and three letter codes conservative replacement Alanine A or Ala Gly; Ser Arginine R or Arg Lys; His Asparagine N or Asn gin; His Aspartic acid D or Asp gin; Asn Cysteine C or Cys Ser; Ala Glutamine Q or Gin Asn Glutamic acid E or Glu asp; gin Glycine G or Gly Ala G istidine H or His asn; gin Isoleucine I or Not Leu; Vai Leucine L or Leu He; val Lysine K or Lys Arg; His Methionine M or Met Leu; He; Tyr Phenylalanine Proline Serine Threonine Tryptophan F or Phe R or Pro S or Ser T or Thr W or Tgr Tyr; met; Leu Ala Thr Ser Tyr; Phe Tyrosine Y or Tug trp; Phe Valine V or Vai He; Leu

Unless specifically defined elsewhere in this document, all other technical and scientific terms used in this document have the meanings commonly understood by one of those skilled in the art to which this invention pertains.

As used herein, including the appended claims, singular forms of words include their corresponding plural references unless the context clearly dictates otherwise.

The term or is used in the meaning of and is used interchangeably with the term and/or unless the context clearly dictates otherwise.

Throughout this description and the following claims, unless the context otherwise provides, the word comprise and variants such as contains and comprising mean the inclusion of the specified amino acid sequence, DNA sequence, step or group thereof, but does not exclude any other amino acid sequences, DNA sequences, steps. As used herein, the term containing may be replaced by the term containing, including, or sometimes having.

The term Tim-3 includes various mammalian isoforms, eg, human Tim-3, human Tim-3 species homologues, and analogs containing at least one epitope in Tim-3. Both the amino acid sequence of Tim-3, eg human Tim-3, and the nucleotide sequence encoding the same are known in the art.

The terms administration, administration, treatment, and processing in this document, when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, means contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with an animal, human, subject, cell. , tissue, organ or biological fluid. Cell processing encompasses contact of a reagent with a cell as well as contact of a reagent with a liquid when the liquid is in contact with the cell. Also, the terms administration and treatment refer to in vitro and ex vivo treatments of, for example, a cell with a reagent, diagnostic, binding compound, or other cell. The term subject herein includes any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog, cat, rabbit), and most preferably a human.

An antibody or antibody molecule.

This document discloses antibody molecules that bind to Tim-3 with high affinity and specificity.

In some embodiments, the anti-Tim-3 antibody comprises at least one, two, three, four, five, or six complementarity determining regions containing the amino acid sequence of SEQ ID NOs 3-8 or 26-27, or variants thereof containing one or more conservative substitutions.

In some embodiments, the anti-Tim-3 antibody comprises at least one, two, or three CDRs from a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NOs 3-5 or 26, or variants thereof containing one or more conservative substitutions. . In some embodiments, the anti-Tim-3 antibody comprises at least one, two, or three CDRs from a light chain variable region (VL) containing the amino acid sequence of SEQ ID NOs 6-8 or 27, or variants thereof containing one or more conservative substitutions. .

In some embodiments, the anti-Tim-3 antibody comprises at least one, two, three, four, five, or six CDRs from the heavy and light chain variable region comprising the amino acid sequence of SEQ ID NOS 3-8 or 26-27, or variants thereof. containing one or more conservative substitutions.

In some embodiments, the anti-Tim-3 antibody comprises six CDRs from the heavy and light chain variable regions comprising the amino acid sequence of SEQ ID NOs 3-8 or 26-27, or variants thereof containing one or more conservative substitutions.

In some embodiments, the anti-Tim-3 antibody comprises:

(a) a heavy chain variable region (VH) comprising one, two or three CDR amino acid sequences selected from SEQ ID NOs 3, 4, 5 or 26, or variants thereof containing one or more conservative substitutions; and/or (b) a light chain variable region (VL) comprising one, two or three CDR amino acid sequences selected from SEQ ID NOs 6, 7, 8 or 27, or variants thereof containing one or more conservative substitutions.

In some embodiments, the anti-Tim-3 antibody comprises:

(a) a heavy chain variable region (VH) containing the amino acid sequence VH-CDR1 SEQ ID NO 3 or variants thereof containing one or more conservative substitutions, the amino acid sequence VH-CDR2 SEQ ID NO 4 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VH-CDR3 SEQ ID NO 5 or variants thereof containing one or more conservative substitutions; and a light chain variable region (VL) containing the amino acid sequence VL-CDR1 SEQ ID NO 6 or variants thereof containing one or more conservative substitutions, the amino acid sequence VL-CDR2 SEQ ID NO 7 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VL-CDR3 SEQ ID NO 8 or variants thereof containing one or more conservative substitutions;

(b) a heavy chain variable region (VH) containing the amino acid sequence VH-CDR1 SEQ ID NO 3 or variants thereof containing one or more conservative substitutions, the amino acid sequence VH-CDR2 SEQ ID NO 26 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VH-CDR3 SEQ ID NO 5 or variants thereof containing one or more conservative substitutions; and a light chain variable region (VL) containing the amino acid sequence VL-CDR1 SEQ ID NO 6 or variants thereof containing one or more conservative substitutions, the amino acid sequence VL-CDR2 SEQ ID NO 7 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VL-CDR3 SEQ ID NO 8 or variants thereof containing one or more conservative substitutions;

(c) a heavy chain variable region (VH) containing the amino acid sequence VH-CDR1 SEQ ID NO 3 or variants thereof containing one or more conservative substitutions, the amino acid sequence VH-CDR2 SEQ ID NO 4 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VH-CDR3 SEQ ID NO 5 or variants thereof containing one or more conservative substitutions; and a light chain variable region (VL) containing the amino acid sequence VL-CDR1 SEQ ID NO 6 or variants thereof containing one or more conservative substitutions, the amino acid sequence VL-CDR2 SEQ ID NO 7 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VL-CDR3 SEQ ID NO 27 or variants thereof containing one or more conservative substitutions; or (d) a heavy chain variable region (VH) containing the amino acid sequence VH-CDR1 of SEQ ID NO 3 or variants thereof containing one or more conservative substitutions, amino acids

- 10 042181 lot sequence of VH-CDR2 SEQ ID NO 26 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VH-CDR3 SEQ ID NO 5 or variants thereof containing one or more conservative substitutions; and a light chain variable region (VL) containing the amino acid sequence VL-CDR1 SEQ ID NO 6 or variants thereof containing one or more conservative substitutions, the amino acid sequence VL-CDR2 SEQ ID NO 7 or variants thereof containing one or more conservative substitutions, and the amino acid sequence of VL-CDR3 SEQ ID NO 27 or variants thereof containing one or more conservative substitutions.

In some embodiments, the anti-Tim-3 antibody comprises:

(a) a heavy chain variable region (VH) comprising the amino acid sequence of VH-CDR1 SEQ ID NO 3, the amino acid sequence of VH-CDR2 of SEQ ID NO 4, and the amino acid sequence of VH-CDR3 of SEQ ID NO 5; and a light chain variable region (VL) comprising the amino acid sequence of VL-CDR1 SEQ ID NO 6, the amino acid sequence of VL-CDR2 of SEQ ID NO 7, and the amino acid sequence of VL-CDR3 of SEQ ID NO 8; or (b) a heavy chain variable region (VH) comprising the amino acid sequence of VH-CDR1 SEQ ID NO 3, the amino acid sequence of VH-CDR2 of SEQ ID NO 26, and the amino acid sequence of VH-CDR3 of SEQ ID NO 5; and a light chain variable region (VL) comprising the VL-CDR1 amino acid sequence of SEQ ID NO 6, the VL-CDR2 amino acid sequence of SEQ ID NO 7, and the VL-CDR3 amino acid sequence of SEQ ID NO 27.

In some embodiments, the anti-Tim-3 antibody is a humanized antibody molecule.

In some embodiments, the anti-Tim-3 antibody is a humanized monoclonal antibody (mAb).

In some embodiments, the antibody comprises a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 9, 17, 28, or 40. In some embodiments, the antibody comprises a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 9, 17, or 28. In some embodiments, anti-Tim-3 the antibody comprises a light chain variable domain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 11, 19, 30, or 36. In some embodiments, anti-Tim -3 antibody contains:

(a) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 11;

(b) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 19;

(c) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30;

(d) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36;

(e) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 11;

(f) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 19;

(g) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30;

(h) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having a sequence identity of at least at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36;

- 11 042181 (i) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity sequences of at least 95%,

96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 11;

(j) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 19;

(k) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30;

(m) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity of at least at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36; a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO 40, and a light chain variable domain having at least 95% sequence identity , 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 11;

(n) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 40, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 19;

(o) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 40, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30; or (q) a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 40, and a light chain variable domain having sequence identity for at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36.

In some embodiments, the anti-Tim-3 antibody comprises:

(a) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 11;

(b) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 19;

(c) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30;

(d) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 9, and a light chain variable domain having a sequence identity of at least at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36;

(e) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 11;

(f) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 19;

(g) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30;

(h) a heavy chain variable domain having a sequence identity of at least

- 12 042181 at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 17, and a light chain variable domain having at least 95% sequence identity,

96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36;

(i) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 11;

(j) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 19;

(k) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity of at least least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30; or (l) a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having sequence identity for at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36.

In some embodiments, the anti-Tim-3 antibody comprises:

(a) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 9 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 11;

(b) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 17 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 19;

(c) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 28 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 30;

(d) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 28 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 36; or (e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 40 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 36.

In some embodiments, the anti-Tim-3 antibody comprises:

(a) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 9 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 11;

(b) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 17 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 19;

(c) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 28 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 30; or (d) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 28 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 36.

In some embodiments, the anti-Tim-3 antibody comprises a heavy chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 13, 22, or 32. In some embodiments, the anti-Tim-3 antibody comprises a light chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 15, 24, 34, or 38. In some embodiments, the anti-Tim-3 antibody contains:

(a) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 13, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 15;

(b) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 13, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 24;

- 13 042181 (c) a heavy chain having a sequence identity of at least 95%, 96%, 97%,

98%, 99% or 100% with the amino acid sequence of SEQ ID NO 13, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 34 ;

(d) a heavy chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 13, and a light chain having at least 95% sequence identity, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 38;

(e) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 22, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 15;

(f) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 22, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 24;

(g) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 22, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 34;

(h) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 22, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 38;

(i) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 32, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 15;

(j) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 32, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 24;

(k) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 32, and a light chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 34; or (l) a heavy chain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 32, and a light chain having a sequence identity of at least 95% , 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 38.

In some embodiments, the anti-Tim-3 antibody comprises:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO 13 and a light chain containing the amino acid sequence of SEQ ID NO 15;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO 22 and a light chain containing the amino acid sequence of SEQ ID NO 24;

(c) a heavy chain containing the amino acid sequence of SEQ ID NO 32 and a light chain containing the amino acid sequence of SEQ ID NO 34; or (d) a heavy chain containing the amino acid sequence of SEQ ID NO 32 and a light chain containing the amino acid sequence of SEQ ID NO 38.

In some embodiments, the anti-Tim-3 antibody comprises one or more of:

(a) a light chain with a mutation from aspartic acid to glutamic acid at position 1 of SEQ ID NO 24;

(b) a light chain with a leucine to methionine mutation at position 4 of SEQ ID NO 24;

(c) a light chain with a valine to isoleucine mutation at position 62 of SEQ ID NO 24;

(d) a light chain with a mutation from aspartic acid to glutamic acid at position 74 of SEQ ID NO 24;

(e) a light chain with a mutation from methionine to leucine at position 96 of SEQ ID NO 24;

(f) a heavy chain with a phenylalanine to tyrosine mutation at position 59 of SEQ ID NO 22;

(g) a heavy chain with a proline-to-valine mutation at position 60 of SEQ ID NO 22;

(h) a heavy chain with a serine to threonine mutation at position 77 of SEQ ID NO 22; or (i) a heavy chain with a cysteine to leucine mutation at position 78 of SEQ ID NO 22.

In some embodiments, the anti-Tim-3 antibody is a Fab, F(ab')2, Fv, or single chain Fv (ScFv).

In some embodiments, the aHTu-Tim-3 antibody comprises an IgG1, IgG2, IgG3, IgG4 subclass heavy chain constant region, or a variant thereof, and a kappa or lambda light chain constant region, or a variant thereof.

In some embodiments, the anti-Tim-3 antibody comprises a variant heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, wherein the variant heavy chain constant region provides reduced or eliminated effector function, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CCC). ).

In some embodiments, the anti-Tim-3 antibody comprises a human IgG1 heavy chain constant region or a variant thereof. In more preferred embodiments, the anti-Tim-3 antibody comprises a variant human IgG1 heavy chain constant region containing one or more mutations selected from the group consisting of E ₂₃₃ P, L ₂₃₄ A, L ₂₃₅ A, L ₂₃₆ A, and P329A. In some embodiments, the anti-Tim3 antibody comprises a variant human IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO 21 and a human kappa light chain constant region.

In some embodiments, the anti-Tim-3 antibody is isolated or recombinant.

In some embodiments, the anti-Tim-3 antibody contains at least one antigen-binding site or at least a variable region. In some embodiments, the anti-Tim-3 antibody comprises an antigen-binding fragment from an antibody described herein.

The term antibody is used herein in its broadest sense and specifically encompasses antibodies (including full length monoclonal antibodies) and antibody fragments if they recognize an antigen, eg Tim-3, PD-1. Typically, an antibody is monospecific, but can also be described as idiospecific, heterospecific, or polyspecific. Antibody molecules bind via specific binding sites to specific antigenic determinants or epitopes on antigens.

The term monoclonal antibody, or mAb, or Mab in this document means a population of essentially homogeneous antibodies, ie. the antibody molecules contained in a population are the same in amino acid sequence except for possible naturally occurring mutations, which may be in minor amounts. In contrast, standard (polyclonal) antibody preparations typically include many different antibodies having different amino acid sequences in their variable domains, especially their complementarity determining regions, which are often specific for different epitopes. The monoclonal modifier indicates the feature of an antibody derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody in any particular manner. Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example, Kohler G et al., Nature 1975 256:495-497; US patent No. 4376110; Ausubel FM et al., Current protocols in molecular biology 1992; Harlow E et al., Antibodies: a laboratory manual, Cold spring Harbor Laboratory 1988; and Colligan JE et al., Current protocols in immunology 1993. The mAbs disclosed herein may be of any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA, and any subclass thereof. The mAb producing hybridoma can be cultured in vitro or in vivo. High mAb titers can be obtained by in vivo production when cells from individual hybridomas are injected intraperitoneally into mice, such as originally primed Balb/c mice, to produce ascitic fluid containing high concentrations of the desired mAbs. MAbs of the IgM or IgG isotype can be purified from such ascitic fluids or from culture supernatants using column chromatography techniques well known to those skilled in the art.

Typically, the basic structural unit of an antibody contains a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair has one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Generally, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are commonly classified as α, δ, ε, γ, or μ, and antibody isotypes are defined as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a J region of about 12 or more amino acids, while the heavy chain also includes a D region of about 10 or more amino acids.

The variable regions of each light/heavy chain (VL/VH) pair form an antibody binding site. Thus, typically, an intact antibody has two binding sites. With the exception of bifunctional or bispecific antibodies, the two binding sites are generally the same.

Typically, the variable domains of both heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), which are located between the relatively conserved frame regions (FR, framework region). Typically, the CDRs are aligned with framework regions to allow binding to a specific epitope. Typically, from the N-terminus to the C-terminus, the variable domains of both the light and heavy chains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3) and FR-4 (or FR4). The assignment of amino acids to each domain is generally in accordance with the definitions from Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md. ; 5<m> ed.; NIH Publ. no. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

The term hypervariable region means the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region contains amino acid residues from the CDRs (ie VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2 and VHCDR3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (determination of CDR regions of an antibody by sequence); also see Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (determination of antibody CDR regions by structure). The term framework or FR residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

Unless otherwise indicated, an antibody fragment or antigen-binding fragment means antigen-binding antibody fragments, i.e. antibody fragments that retain the ability to specifically bind to an antigen bound by a full-length antibody, such as fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabody; linear antibodies; single chain antibody molecules, eg single chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.

An antibody that binds to a particular target protein with specificity is also described as specifically binding to a particular target protein. This means that the antibody exhibits preferential binding to this target over other proteins, but this specificity does not require absolute binding specificity. An antibody is considered specific for its intended target if its binding is indicative of the presence of the target protein in the sample, for example without producing unwanted results such as false positives. Antibodies or binding fragments thereof useful in the present invention will bind to the target protein with an affinity that is at least two times greater, preferably at least 10 times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity for non-target proteins. As used herein, an antibody is said to specifically bind to a polypeptide containing a given amino acid sequence, such as the amino acid sequence of a mature human Tim-3 molecule, if it binds to polypeptides containing that sequence but does not bind to proteins lacking that sequence.

The term human antibody as used herein means an antibody that contains only human immunoglobulin protein sequences. The human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, a mouse antibody or a rat antibody means an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

The term humanized antibody refers to forms of antibodies that contain sequences from non-human (eg, murine) antibodies as well as human antibodies. Such antibodies contain a minimal sequence derived from a non-human immunoglobulin. Typically, a humanized antibody will contain substantially all of at least one, and usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those sequences of human immunoglobulin. The humanized antibody will optionally also contain at least a portion of an immunoglobulin (Fc) constant region, typically a human immunoglobulin. The prefix hum, hu, Hu, or h is added to antibody clone designations when it is necessary to distinguish humanized antibodies from original rodent antibodies. Humanized forms of rodent antibodies will typically contain the same CDR sequences of the original rodent antibody, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

The terms cancer or tumor in this document mean or describe the physiological condition in mammals, which is usually characterized by unregulated cell growth. Examples of cancer include, but are not limited to, lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, gastric cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, cancer bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or metastatic cancer.

The term CDR refers to complementarity-determining region(s) in the variable region of an immunoglobulin, defined using the Kabat numbering system, unless otherwise indicated.

Pharmaceutical compositions and kits.

In some aspects of this invention, compositions, such as pharmaceutically acceptable compositions, are provided that comprise an anti-Tim-3 antibody as described herein combined with at least one pharmaceutically acceptable excipient. As used herein, the term pharmaceutically acceptable excipient includes any and all diluents, dispersion media, isotonic and retarding absorption agents, and the like, which are physiologically compatible. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (eg, by injection or infusion).

The compositions herein may be in various forms. They include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg injection and infusion solutions), dispersions or suspensions, liposomes and suppositories. The appropriate form depends on the intended route of administration and therapeutic application. Usual suitable compositions are in the form of injection or infusion solutions. One suitable route of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In specific embodiments, the antibody is administered by intramuscular or subcutaneous injection.

The term therapeutically effective amount, as used herein, refers to the amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of the disease or disorder, is sufficient to effect such treatment of the disease, disorder, or symptom. The therapeutically effective amount may vary depending on the antibody, the disease, disorder and/or symptoms of the disease or disorder, the severity of the disease, disorder and/or symptoms of the disease or disorder, the age of the subject being treated and/or the weight of the subject being treated. The appropriate amount in any particular case may be apparent to those skilled in the art, or may be determined by routine experimentation. In the case of combination therapy, a therapeutically effective amount refers to the total number of combined entities for effective treatment of a disease, disorder or condition.

The subject is a mammal, such as a primate, preferably a higher primate, such as a human (eg, a patient having or at risk of having the disorder described herein).

Examples.

Example 1 Generation of Anti-Tim-3 Monoclonal Antibodies.

A number of mouse anti-Tim-3 monoclonal antibodies (mAbs) are generated based on conventional hybridoma technology (de StGroth and Sheidegger, 1980, J Immunol Methods 35:1; Mechetner, 2007, Methods Mol Biol 378:1). For additional characterization, a mAb with high binding activity in ELISA and fluorescence-activated cell sorting (FACS) was selected.

Recombinant Tim-3 proteins for immunization and binding assays.

cDNA encoding full-length human Tim-3 (SEQ ID NO. 1) was synthesized based on the GenBank sequence (accession number: AF450242.1). The extracellular domain (ECD) coding region of amino acids (AMA) 1-202 Tim-3 (SEQ ID NO. 2) was amplified by PCR (polymerase chain reaction) and cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad , California, USA) with the C-terminus fused to either a mouse IgG2a Fc region (GenBank accession number: CAC20702) or a human IgG1 heavy chain Fc region (UniProtKB/Swiss-Prot accession number: P01857), resulting in two recombinant fusion protein expression plasmids, Tim-3-mIgG2a and Tim-3-huIgG1, respectively. A schematic representation of Tim-3 fusion proteins is shown in FIG. 1. To produce the recombinant fusion protein, Tim-3-mIgG2a and Tim-3-huIgG1 plasmids are transiently transfected into a mammalian cell expression system based on HEK293 (self-developed) and cultured for 5-7 days in a CO ₂ incubator equipped with a rotating shaker . The supernatant containing the recombinant protein is collected and clarified by centrifugation. Tim-3-mIgG2a and Tim-3-huIgG1 are purified using a Protein G Sepharose Fast Flow column (catalog number: 17061805, GE Life Sciences). Both Tim-3-mIgG2a and Tim-3-huIgG1 proteins are dialyzed against phosphate buffered saline (PBS) and stored in a freezer at -80° C. in small aliquots.

Stable cell lines of expression.

To generate stable cell lines that express full-length human Tim-3 (huTim-3) or monkey Tim-3 (mkTim-3, gene commercially available from ZYAGE, catalog number: KD-17 042181

702), the Tim-3 genes were cloned in the pFB-Neo retroviral vector (catalog number: 217561, Agilent, USA). Dual tropism retroviral vectors were generated according to the previous protocol (Zhang T, et al., 2005, Blood 106:1544-51). Vectors containing huTim-3 and mkTim-3 were transduced into HuT78 and NK92MI cells (ATCC (American Type Culture Collection), Manassas, VA, USA), respectively, to generate HuT78/huTim-3 and NK92MI/mkTim-3. High-expressing cell lines are harvested by culturing in RPMI1640 complete medium containing 10% FBS with G418 and FACS binding assay.

Immunization, hybridoma fusion and cloning.

Eight to twelve week old Balb/c mice (HFK BIOSCIENCE CO., LTD, Beijing, China) are immunized intraperitoneally (ip) with 100 µl of an antigen solution containing 10 µg of Tim-3mIgG2a and a water-soluble adjuvant (catalogue number: KX0210041 , KangBiQuan, Beijing, China). The procedure is repeated after three weeks. Two weeks after the 2nd immunization, mouse sera are assessed for Tim-3 binding by ELISA and FACS. Ten days after screening, sera from mice with the highest serum anti-Tim-3 antibody titers are stimulated ip with 50 μg Tim-3 mIgG2a. Three days after stimulation, splenocytes are isolated and hybridized to the mouse myeloma cell line, SP2/0 cells (ATCC) using standard techniques (Colligan JE, et al., Current protocols in immunology, 1993).

Assessment of the binding activity of Tim-3 antibodies during ELISA and FACS.

Hybridoma clone supernatants are first tested by ELISA as described in Methods in Molecular Biology (2007) 378:33-52 with some modifications. Briefly, 96-well plates were coated with Tim-3-huIgG1 protein. HRP (horseradish peroxidase) coupled anti-mouse IgG antibody (cat. no.: 7076S, Cell Signaling Technology, USA) and substrate (cat. no.: 00-4201-56, eBioscience, USA) were used for development and the signal was measured absorption at 450 nm using a spectrophotometer (SpectraMax Paradigm, Molecular Devices, USA). ELISA positive clones are further screened by FACS using either HuT78/huTim3 cells or NK92mi/mkTim-3 cells described above. Tim-3 expressing cells (105 cells/well) are incubated with supernatants of ELISA-positive hybridomas followed by binding to goat anti-mouse IgG labeled with Alexa Fluro-647 (catalogue number: A0473, Beyotime Biotechnology, China). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte 8HT, Merck-Millipore, USA).

Conditioned media from hybridomas that showed positive signals in both ELISA and FACS screens are subjected to functional assays to recognize antibodies with good functional activity in human immune cell-based assays (see following sections). Antibodies with the desired functional activities are further subcloned and characterized.

Subcloning and adaptation of hybridomas to serum-free or low-serum media

After screening mainly with ELISA, FACS and functional assays (described in examples 7 and 8), positive hybridoma clones are subcloned by limiting dilution. The best antibody subclones tested by functional assays are adapted for growth in CDM4MAb medium (catalog number: SH30801.02, Hyclone, USA) with 3% FBS.

Expression and purification of monoclonal antibodies.

Hybridoma cells are cultured in CDM4MAb medium (catalog number: SH30801.02, Hyclone) and incubated in a CO ₂ incubator for 5-7 days at 37°C. The conditioned medium is collected by centrifugation and filtered through a 0.22 μm membrane before purification. Supernatants containing mouse antibodies are applied and bound to a Protein A column (catalog number: 17127901, GE Life Sciences) following the manufacturer's instructions. Usually, antibodies with a purity greater than 90% were obtained during the procedure. Protein A affinity purified antibodies are either dialyzed against PBS or further purified using a HiLoad 16/60 Superdex200 column (catalog number: 17531801, GE Life Sciences) to remove aggregates. Protein concentrations are determined by measuring absorbance at 280 nm. The final antibody preparations were stored in aliquots in a freezer at -80°C.

Example 2 Cloning and sequence analysis of anti-Tim-3 antibodies.

Mouse hybridoma cells are selected to obtain total RNA using the Ultrapure RNA kit (cat. no.: 74104, QIAGEN, Germany) according to the manufacturer's protocol. 1st strand cDNA was synthesized using the Invitrogen cDNA synthesis kit (catalog number: 18080-051) and PCR amplification of the Vh and Vk genes of mouse mAbs was performed using the PCR kit (catalog number: CW0686, CWBio, Beijing, China). The oligoprimers used to clone the variable heavy chain (Vh) and variable kappa light chain (Vk) cDNA antibodies are synthesized based on previously reported sequences (Brocks et al., 2001 Mol Med 7:461). The PCR products are then subcloned into the pEASY-Blunt cloning vector (catalog number: CB101-02,

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TransGen, China) and sequenced. The amino acid sequences of the Vh and Vk regions are deduced from the results of DNA sequencing.

Mouse mAbs are analyzed by comparing sequence homology and grouped based on sequence similarity (FIG. 2). Complementarity determining regions are established based on the Kabat system (Wu and Kabat, 1970, J. Exp. Med. 132:211-250) and IMGT (ImMunoGeneTics information system) (Lefranc, 1999, Nucleic Acids Research 27:209-212) by annotation sequences and sequence analysis on the Internet (http://www.imgt.org/IMGT_vquest/share/textes/index.html). The amino acid sequences of a representative mu425 best clone (Vh and Vk) are listed in Table 1. 1 (SEQ ID NO. 9 and 11). The mu425 CDR sequences are listed in Table 1. 2 (SEQ ID NOS 3-8).

Table 1

Amino acid sequences of the VH and VK regions of mu425

Subsequence SEQID NO VH mu425 EVKLVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQIP EKRLEWVAAISSGGSLYFPDSVKGRFTISRDNARNICYLQMN SLRSDDT AMYYC ARGREAD GGYFDYWGQGTTLT VS S 9 VK mu425 DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWYQQ KPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEED DIAMYFCQQSMKVPLTFGAGTKLELK eleven

table 2

CDR sequences (amino acids) of VH and VK regions of mu425

CDR1 SEQ ID NO CDR2 SEQ ID NO CDR3 SEQ ID NO mu425, vh RYAMS 3 AISSGGSLYF PDSVKG 4 GREADGG YFDY 5 mu425, VK RASESVEY YGTSLMQ 6 AASNVES 7 QQSMKVP LT 8

Example 3 Affinity Determination of Purified Mouse Anti-Tim-3 Antibodies by NIR (Surface Plasmon Resonance).

Anti-Tim-3 antibodies with high binding activities in ELISA and FACS, as well as strong functional activities in cell assays (described in Examples 7 and 8) are characterized by their binding kinetics in SPR assays using BIAcore™ T-200 (GE Life sciences). Briefly, an anti-human IgG antibody is immobilized on an activated CM5 biosensor chip (catalogue number: BR100530, GE Life Sciences). The Fc-labeled Tim-3 human IgV domain is passed over the chip surface and captured by the anti-human IgG antibody. Subsequently, serially diluted (0.36 nM to 90 nM) purified mouse antibodies are passed over the chip surface and changes in SPR signals are analyzed to calculate association rates (ko _n ) and dissociation rates (ko _ff ) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (KD) is calculated as the ratio k _off /k _on . The binding affinity profiles of the best mAbs, including mu425, mu44, mu 225, and mu411, are shown in Table 1. 3.

Table 3

Comparison of binding affinities of hybridoma antibodies using SPR Antibodies to _op (M'Ch' ¹ ) koff (c' ¹ ) KD (nM)

mu425 1.59*10° 9.3340 ^-5 0.058 mu44 1.2440 ⁶ 1.8640 ⁴ 0.150 mu255 5.5240 ⁵ 7.8440 ⁴ 1.42 mu411 1.4440 ⁶ 3.3640 ⁴ 0.234

Example 4 Humanization of the mouse anti-human mAb mu425 to Tim-3.

Humanization and engineering mAb.

To humanize mu425, human germline IgG genes are examined for sequences that have high homology to mu425 variable region cDNA sequences using BLAST (Basic Local Alignment Search Tools) for the human immunoglobulin gene database at the IMGT websites (http://www.imgt .org/IMGT_vquest/share/textes/index.html) and NCBI (http://www.ncbi.nlm.nih.gov/igblast/). Human IGVH and IGVK genes that are present in the spectra

- 19 042181 human antibodies with high frequencies (Glanville, 2009, PNAS 106:20216-20221) and are highly homologous to mu425 are selected as templates for humanization. Prior to humanization, the mu425 heavy and light chain variable domains are fused to a modified human IgG1 constant region termed human IgG1mf (SEQ ID NO. 21) and a human kappa chain constant region, respectively. IgG1mf (SEQ ID NO: 21) is an IgG1 mutant containing a combination of mutations, E ₂₃₃ P, L ₂₃₄ A, L ₂₃₅ A, L ₂₃₆ A and P ₃₂₉ A (amino acid numbering based on the EU system), compared to wild-type IgG1 type of person.

Humanization is carried out by CDR grafting (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols, Humana Press) and humanized antibodies (hu425) are constructed in human IgG1 format. In the initial humanization cycle, mutations of murine to human amino acid residues in the framework regions are carried out with the 3D modeled structure, and murine framework residues structurally relevant to maintain canonical CDR structures are retained in version 1 of the humanized 425 antibody, hu425-1-1 ( the amino acid sequences of the heavy chain and light chain are given in SEQ ID NOs 22 and 24). Namely, the Vk mu425 CDRs are grafted into human germline variable IGVK3-15 gene frameworks with 4 conserved murine framework residues (D1, L4, V62 and D74) (light chain variable domain amino acid sequences are given in SEQ ID NO. 19). Vh mu425 CDRs are grafted into human germline variable IGVH3-7 gene frameworks with 1 conserved murine framework (C ₇₈ ) residue (heavy chain variable domain amino acid sequences are given in SEQ ID NO. 17).

Hu425-1-1 is constructed as a full-length human antibody format using self-developed expression vectors that contain the constant regions of a human IgG1 variant termed IgG1mf (SEQ ID NO. 21) and the kappa chain, respectively, with easily adaptable subcloning sites. . Expression and production of the hu425-1-1 antibody is achieved by co-transfection of the above two constructs into 293G cells and by purification using a Protein A column (catalog number: 17543802, GE Life Sciences). Purified antibodies are concentrated to 0.5-5 mg/ml in PBS and stored in aliquots in a freezer at -80°C.

Based on the hu425-1-1 template, the authors make a number of single mutations converting conserved murine residues in the Vk framework regions into the corresponding human germline residues, which include DIE, L4M, V62I and D74D into Vk. The resulting hu425-1-2a (DIE), hu425-1-2b (L4M), hu425-1-2c (V62I), and hu425-1-2d (D74E) all have binding and functional activities similar to hu425-1 -1. To further improve the level of humanization of the heavy chain, we also change the conserved C78 residue and the C-terminal portion of H-CDR2 (Kabat definition) from the mouse sequence to the corresponding human germline residues. The specifications of the 3 humanized antibodies are hu425-2A-1 (F59Y in Vh), hu4252B-1 (P60V in Vh) and hu425-2C-1 (C78L in Vh). All humanization mutations are performed using primers containing mutations at specific positions and a site-directed mutagenesis kit (catalog number FM111-02, TransGen, Beijing, China). Required mutations are verified by sequence analysis. These hu425 variant antibodies are tested in binding assays and functional assays as previously described. Compared to hu425-1-1, hu425-2B-1 has significantly reduced binding affinities and functional properties (data not shown), while the remaining versions of the humanized variants of hu425 have binding activities and functional activities similar to hu425-1-1.

The hu425 antibodies are further engineered by introducing mutations in the CDRs and framework regions to improve molecular biochemical and biophysical properties for human therapeutic applications. Factors considered include amino acid compositions, thermal stability (T _m ), surface hydrophobicity, and isoelectric points (pI, isoelectronic points) while maintaining functional activities.

Taken together, two well-engineered versions of humanized monoclonal antibodies, hu425-2-2 (heavy chain and light chain variable domain amino acid sequences are given in SEQ ID NOs. 28 and 30) and hu425-2-3b (heavy chain and light chain variable domain amino acid sequences the chains are given in SEQ ID NOs 28 and 36) are generated by the mutation process described above and have been characterized in detail. Both hu425-2-2 and hu425-2-3b contain a mutation in H-CDR2 (SEQ ID NO: 26) and in L-CDR3 (SEQ ID NO: 27). The amino acid sequences of the heavy/light chain variable regions and the six CDRs of hu425-2-2 and hu425-2-3b are listed in Table 1. 4 and table. 5 below. The results showed that both hu425-2-3b and hu425-2-2 are very similar in binding affinity and functional activities such as inhibition of Tim-3 mediated downstream signaling.

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Table 4

Amino acid sequences of the VH and VK regions of hu425-2-2 and hu425-2-3b

Subsequence SEQID NO VH hu425-2-2 VK hu425-2-2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAP GKGLEWVAAISSGGSLYYPDSVKGRFTISRDNAKNTLYLQM NSLRAEDTAVYYCARGREADGGYFDYWGQGTLVTVSS EIVMTQSPATLSVSPGERATLSCRASESVEYYGTSLMQWYQ QKPGQAPRLLIYAAGSPLVESGIPARFSIKAVGTEFTLTISSLQSE 28 thirty VH hu425-2-3b EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAP GKGLEWVAAISSGGSLYYPDSVKGRFTISRDNAKNTLYLQM NSLRAEDTAVYYCARGREADGGYFDYWGQGTLVTVSS 28 VK hu425-2-3b EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAP GKGLEWVAAISSGGSLYYPDSVKGRFTISRDNAKNTLYLQM NSLRAEDTAVYYCARGREADGGYFDYWGQGTLVTVSS 36

Table 5

The CDR (amino acid) sequences of the VH and VK regions of hu425-2-2 and . hu425-2-3b

CDR1 SEQ ID NO CDR2 SEQ ID NO CDR3 SEQ ID NO VH hu425-2-2 RYAMS 3 AISSGGSLYY PDSVKG 26 GREADGGYF DY 5 VK hu425-2-2 RASESVEYY GTSLMQ 6 AASNVES 7 QQSLKVPLT 27 VH hu425-2-3b RYAMS 3 AISSGGSLYY PDSVKG 26 GREADGGYF DY 5 VK hu425-2-3b RASESVEYY GTSLMQ 6 AASNVES 7 QQSLKVPLT 27

For affinity determination, Fabs derived from the hu425 mAb group are prepared using the Pierce Fab Preparation Kit (catalog number 44985, ThermoFisher Scientific) and used in an affinity assay based on Surface Plasmon Resonance (SPR) technology. The results of anti-Tim-3 Fab antibody binding profiles determined by SPR are summarized in Table 1. 6. Fab hu425-22 and hu425-2-3b have very similar binding profiles with an average dissociation constant at 0.419 nM and 0.361 nM, respectively, which is close to that of hu425-1-1.

Table 6

Comparison of hu425 Fab binding affinities by SPR

Anti- Tim-3 fab Test 1 Test 2 Average meaning k _O n (M'Ch ¹ ) ΜΛ Kd(hM) k _O n (M'Ch ¹ ) Wc ¹ ) _KD (hM) K _D (nM) ^ch425a l,67*10 ⁶ 2.04* ^IO'4 0.122 n.d.? hu425-l-l 6.79* ¹⁰⁵ l,08* ^10'4 0.159 l,26*10 ⁶ 4.35* ^IO'4 0.346 0.253 hu425-2-2 l,71*10 ⁶ 7.40* ^IO'4 0.434 l,97*10 ⁶ 7.95* ^IO'4 0.404 0.419 hu425-2- 3b l,60*10 ⁶ 5.70* ^IO'4 0.356 l,75*10 ⁶ 6.40* ^IO'4 0.366 0.361

^and ch425 consists of mu425 variable domains fused to human/mouse kappa IgGlmf constant regions (heavy chain and light chain amino acid sequences are given in SEQ ID NOs. 13 and 15) ^b n.d.: not available.

All of the humanized antibodies shown above also confirm functional activities on human primary immune cells isolated from healthy donors (described in Example 8).

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Example 5 Affinity Comparison of Anti-Tim-3 Antibodies by SPR.

Hu425-2-3b and two known anti-Tim-3 antibodies, Ab1 (containing the heavy chain variable region of SEQ ID NO: 40 and the light chain variable region of SEQ ID NO: 41) and Ab2 (containing the heavy chain variable region of SEQ ID NO: 42 and light chain variable region SEQ ID NO: 43) were generated in human IgG4 format with the S228P mutation and characterized by their binding kinetics in SPR assays using BIAcore™ T-200 (GE Life Sciences).

Briefly, an anti-human Fab antibody is immobilized on an activated CM5 biosensor chip (catalogue number: BR100530, GE Life Sciences). Anti-Tim-3 is passed over the surface of the chip and captured by the anti-human Fab antibody. Serially diluted (0.12 nM to 30 nM) Tim-3-his is then passed over the chip surface and changes in SPR signals are analyzed to calculate association rates (k _on ) and dissociation rates ( _koff ) using the binding model Langmuir one to one (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (K _D ) is calculated as the ratio ko _ff /ko _n . Hu425-2-3b, Ab1 and Ab2 show comparable binding affinities. Antibodies in particular mistake! Reference source not found. Hu425-2-3b binds Tim-3 in a dose dependent manner with a nanomolar KD.

Table 7

Comparison of Anti-Tim-3 Binding Affinities by SPR

Anti-Tim-j Ka (1/Mc) Kd(i/c) Κ _υ (M) Hu425-2-3b 5.28E+05 7.78E-04 l,47E-09 Abi 4.44E-05 l,20E-04 2.71 E-l 0 Ab2 6.27EH)5 0.0122 1.94E-08

Example 6 Contribution of mu425/hu425 CDRs to Tim-3 binding.

During the humanization process, several variants with a single amino acid mutation from hu425-1-1 are generated. Two of these mutant variants almost completely abolish binding to Tim-3, as seen in FIG. 3, but each has only a neutral (chemically similar) amino acid substitution, D102E (mAb named hu425-2E-1) and G103A (named hu425-2F-1), in the heavy chain CDR3. These findings support the notion that the heavy chain CDR3 in humanized mu425 antibodies contributes significantly to the functionality of Tim-3 binding.

On the other hand, the mu425/hu425 light chain CDR1 and CDR2 are identical to the mouse germline IGKV3-1 gene. Several mouse antibodies have also been found containing the same mouse germline CDR1 and CDR2 sequences in their light chain variable regions, such as Ab2000 (US Pat. No. 7,989,597) and mAb 1G5 (US Pat. No. 7,563,874). The authors are investigating whether these antibodies can also bind to Tim-3. To this end, Ab2000 and 1G5 are generated with human IgG1mf format and evaluated for Tim-3-mIg binding by ELISA. As shown in FIG. 4, neither Ab2000 nor 1G5 are able to produce any Tim-3 binding signal, unlike hu4252-3b. Therefore, CDR1 and CDR2 of the mouse germline IGKV3-1 gene are not sufficient for Tim-3 binding.

To further evaluate the contribution of CDRs to Tim-3 binding, two hybrid antibodies were generated by exchanging the heavy chain and light chain of the hu425-2-3b antibody and the Ab1 antibody with each other. Both hu425-23b and Ab1 are mouse anti-Tim-3 derivatives and have the same L-CDR1 and L-CDR2 as those of mouse germline IGKV3-1. The hu425-2-3b heavy chain and Ab1 light chain are co-expressed to generate the 425 HC/Ab1 LC hybrid antibody, while the Ab1 HC/425 LC hybrid antibody is generated by co-expression of the Ab1 heavy chain and hu425-2-3b light chain. The binding activities of hybrid antibodies are analyzed by biolayer interferometry (ForteBio Octet). The fusion antibodies are captured at the ends of the A protein and then immersed in a Tim-3-his solution for BLI (BioLayer Interferometry) binding signal analysis. Ab1 HC/425 LC was found to retain the ability to bind Tim-3, while captured 425 HC/Ab1 LC did not produce a significant binding signal. It is suggested that hu425-2-3b LC-CDR3 is required for its binding to Tim3, while its L-CDR1 and L-CDR2 are insufficient for binding Tim3, or may not be required for binding to Tim3.

Example 7 Blockade of Tim-3-mediated phagocytosis by anti-Tim-3 antibodies.

Tim-3 has been shown to bind to phosphatidylserine (PS) via its IgV domain and mediate phagocytosis of apoptotic cells (DeKruyff et al., J. Immunol, 2010, 184:1918-1930; Nakayama et al., Blood, 2009, 113 : 3821-3830). PS is a phospholipid that is restricted to the inner leaflet of the cytoplasmic membrane in normal mammalian cells but becomes exposed on the outer surface of apoptotic cells. PS is involved in immunosuppression in the tumor microenvironment by interfering with immune responses (Fadok et al., J Clin Invest, 1998, 101:890-898; Frey et al., Semin Immunopathol, 2011, 33:497-516). In vivo administration of anti-Tim-3 antibodies results in less clearance of apoptotic cells, increased local inflammation, and impaired immunological tolerance, suggesting that the PS-Tim-3 axis is involved in in vivo immunosuppression (Chabtini et al., J.

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Immunol 190:88-96, 2013; Nakayama et al., Blood 113: 3821-30, 2009).

To determine if anti-Tim-3 antibodies can block Tim-3-mediated phagocytosis, cell analysis was performed using a sensory and functional readout cell line, THP-1/Tim-3, ie. THP-1 (ATCC, human monocyte cell line) stably transfected with the full-length Tim-3 gene. In this assay, HuT78 (ATCC) undergoes limited apoptosis when treated overnight with 2% ethanol followed by CFSE (carboxyfluorescein succinimidyl ester) dye labeling (Invitrogen, 1 μM) according to the manufacturer's instructions. THP-1/Tim-3 cells are then co-cultured with CFSE-labeled apoptotic HuT78 cells for 6 hours in the presence of anti-Tim-3 humanized antibodies. Tim-3-mediated phagocytosis is defined as the percentage of CFSE ⁺ THP-1/Tim-3 to all THP-1/Tim-3 cells (gated on the CD3 ⁻ population). As shown in FIG. 5, hu425-2-3b dose-dependently inhibits phagocytosis of apoptotic HuT78 cell by THP1/Tim-3 cells.

Example 8 Activation of IFN-γ secretion by anti-Tim-3 antibodies in primary human PBMC co-cultured with tumor cells expressing a T-cell recruiting activator.

Both Tim-3 and PD-1 have been reported to be inhibitory receptors expressed on activated T cells that can act to cause T cell depletion (Anderson AC. et al., 2016, Immunity 44:989-1004). Tim-3 ⁺ CD4 and CD8 T cells from cancer patients indeed secrete much less Thl cytokine, IFN-γ, than Tim-3 ^- T cells (Arai Y. et al., 2012, Yonago Acta medica 55:1-9 ; Xu B, et al., 2015, Oncotarget 6:20592-603).

The authors investigated the function of Tim-3 and anti-Tim-3 antibodies using anti-CD3 mAb OKT3 activated T cells in human PBMC. Peripheral blood mononuclear cells (PBMCs) are isolated from healthy donors using a Histopaque-1077 density gradient centrifugation protocol (catalog number: 10771, Sigma). Three days prior to analysis, PBMCs are stimulated with 40 ng/ml anti-CD3 mAb OKT3 (catalog number: 16-0037, eBioscience, USA) to amplify CD3 ⁺ T cells, which are used as effector cells. The target cell, designated A549/OS8, is the A549 lung cancer cell line (ATCC) stably transfected with a T-cell recruiting activator (OS8) according to the method described in US Patent No. 8,735,553. -terminal domains that interact directly with the TCR/CD3 complex and activate T cells. Effector cells, PBMCs, are co-cultured with rapidly mitomycin C-treated A549/OS8 target cells to mimic the response of activated T cells to tumor cells involving the TCR/CD3 complex. The assay is performed in the presence or absence of anti-Tim-3 antibodies in 96-well flat bottom plates. After 15-18 hours of co-culture, culture supernatants are analyzed for IFN-γ levels by ELISA using Ready-Set-Go! (part number: 88-7316, eBiosciences). As shown in FIG. 6, when A549/OS8 target cells are co-cultured with effector cells, anti-Tim-3 antibodies (ch425 and hu425-1-1) cause increased secretion of IFN-γ in effector cells.

Example 9 Activation of CMV-specific human T cells with anti-Tim-3 antibodies.

Anti-Tim-3 antibody performance was further assessed using naturally derived T cells that recognize the human CMV peptide PP65 (NLVPMVATV, 495503, HLA-A2.1-restricted) (Boeckh M, Boeckh M and Geballe AP, 2011, J Clin Invest 121:167380). Briefly, PBMC from healthy donors are first screened by FACS using an anti-HLA-A2 mAb. HLA-A2+ PBMCs are then modeled with PP65 peptide (greater than 98% pure, synthesized by GL Biochem, Shanghai) in complete RPMI with 10% FBS for a week.

The target cell line A549/A2.1 is established by stable transfection with HLA-A2.1. After 30 minutes of treatment with mitomycin C (100 μg/ml) and pp65 peptide (5 μg/ml), A549/A2.1 cells (10 ⁴ ) were co-cultured with an equal number of activated pp65 PBMCs in 96-well plates overnight in the presence of or absence of anti-Tim-3 antibodies or controls. IFN-γ in the culture supernatant is determined by ELISA. All conditions are fulfilled in triplicate. As shown in FIG. 7, hu425-2-3b stimulates pp65-specific T cells to secrete IFNγ in the cell culture supernatant.

Example 10 Anti-Tim-3 antibodies increase NK cell mediated cytotoxicity.

Tim-3 is known to be constitutively expressed on natural killer (NK) cells at relatively high levels (Ndhlovu LC, et al., 2012, Blood 119:3734-43; da Silva IP, et al., 2014, Cancer Immunol Res 2:410-22). Higher expression of Tim-3 on NK cells in melanoma was found to be associated with progressive tumor stages and poor prognosis. In addition, NK cell function appears to be affected by Tim-3 activity (da Silva IP, et al., 2014, Cancer Immunol Res. 2:41022).

To confirm whether humanized anti-Tim-3 antibodies can promote NK

- 23 042181 mediated cytotoxicity, primary NK cells are isolated from healthy donor PBMCs using an NK cell isolation kit from Miltenyi Biotec (Germany) according to the manufacturer's instructions. After one day stimulation with human IL-2 (1000 U/ml), NK cells are co-cultured with K562 cells in the presence of anti-Tim-3 antibodies, brefeldin A and anti-CD107a-APA (eBioscience) at 37°C for 5 hours . Expression of CD107a on CD3-CD56+ NK cells is quantified by flow cytometry. The results show that the hu425-2-3b anti-Tim-3 antibody increases CD107a expression as measured by mean fluorescence intensity (MFI) and cell percentage (FIG. 8).

Example 11 Anti-Tim-3 antibodies reduce surface expression of the Tim-3 receptor.

To address the possibility of hu425-2-3b-inclusive Tim-3 internalization, NK cells from healthy donors are first incubated with hu425-2-3b (10 μg/mL) in complete RPMI1640 medium at either 37°C or 4°C. C within 1 hour. Surface expression of the Tim-3 receptor is determined by staining with non-competing Ab mu420 for Tim-3 (self-produced) followed by staining with goat anti-mouse IgG APC (Biolegend). As shown in FIG. 9, hu425-2-3b at 37° C. causes a significant decrease in surface expression of Tim-3 compared to IgG-treated negative control human cells. The decrease appears to be the result of anti-Tim-3 antibody induced receptor internalization, as a clear temperature dependence was found over a short period of time. The results clearly show that hu4-25-2-3b downregulates the Tim-3 receptor, probably due to internalization of Tim-3. By inducing Tim-3 internalization, hu425-2-3b is expected to reduce interactions of Tim-3 with its multiple ligands (such as galectin-9 and PS).

Example 12 Anti-Tim-3 antibodies show an increased rate of internalization.

To further investigate antibody internalization induced by anti-Tim-3 antibody, internalization was determined using a Tim-3 expressing NK cell line (NK92MI/huTim-3) at different time points (1, 3, 5 and 18 hours). Briefly, anti-Tim-3 antibodies (10 μg/ml), including hu425-23b, Ab1 and Ab2, are incubated with NK92MI/huTim-3 (5*10 ⁴ ) at either 37°C or 4°C for 18 hours. Surface expression of the Tim-3 receptor is determined by staining with goat anti-human IgG-FITC (fluorescein isothiocyanate). Calculate % internalization as the reduction (%) of surface expression of Tim-3 at 37°C compared to the level of expression at 4°C. As shown in FIG. 10, during a shorter incubation (1-5 hours), hu425-2-3b induces comparable levels of Tim-3 internalization as Ab1 and Ab2. It shows significantly more robust internalization than both Ab1 and Ab2 after 18 hours of incubation, suggesting consistent hu4252-3b Tim-3 internalization activity.

Example 13 Humanized anti-Tim-3 mAbs stimulate immune cells alone and in combination with an anti-PD-1 mAb.

The immune inhibitory receptors PD-1 and Tim-3 have been reported to be upregulated in dysfunctional tumor antigen-specific CD8+ T cells in patients with advanced tumors and chronic viral infections (Fourcade J, et al., 2010, J Exp Med. 207: 2175-86; Thommen DS, et al., 2015, Cancer Immunol Res. 3:1344-55; Jin HT, et al., 2010, Proc Natl Acad Sci USA. 107:14733-8). Simultaneous blockade of both PD-1 and Tim-3 receptors can spread vaccine-induced NY-ESO-1-specific CD8 ⁺ T cells (Fourcade 1, et al., 2014, Cancer Res. 74:1045-55). The traditional T-cell response assay, the mixed lymphocyte response (MLR), is being developed to characterize the possible co-stimulatory effects of anti-Tim-3 and anti-PD-1 antibodies. Briefly, stimulating PBMCs are pretreated with mitomycin C (100 μg/mL, Sigma) and co-cultured with reactive PBMCs from another donor in a one-to-one ratio in RPMI1640 complete medium with 10% AB serum (Sigma) plus anti-Tim-3 and/or anti-PD-1 mAb 317-4B6 (also referred to as hu317-4B6, 317-4B6/IgG4mt10, described in US Pat. No. 8,735,553). Reactions were run in 96-well flat-bottomed plates for 4 days with a triple measurement point set for each condition. The secretion of IFN-γ in the cell culture supernatant was analyzed as a data readout.

The results show that hu425-2-3b significantly enhances IFN-γ production in RSL in a dose dependent manner. Coupling hu425-2-3b with 317-4B6 (50 ng/mL) results in a greater increase in IFN-γ production than either hu425-2-3b or anti-PD-1 antibody alone, demonstrating costimulatory effects of anti-Tim-3 mAb with anti-PD-1 mAb (Fig. 11).

Mitomycin C pretreated stimulatory PBMCs were co-cultured with reactive PBMCs in the presence of anti-Tim-3 mAb, either hu425-2-3b or hu425-2-3b, plus anti-PD-I Ab hu317-4b6 (50 ng/mL) in 96-well flat-bottomed plates for 4 days. IFN-γ in the supernatant is determined by ELISA. All conditions were met in triplicate. Results are shown as mean plus RMS (standard deviation).

Example 14 Hu425-2-3b lacks ADCC and CDC effector functions. The ability of hu425-23b to induce ADCC and CDC is determined using an in vitro assay as described below. IgG1mf (SEQ ID NO:

- 24 042181

21) is an IgG1 mutant containing a combination of mutations, E ₂₃₃ P, L ₂₃₄ A, L235A, L23A and P329A (amino acid numbering is based on the EU system). These mutations are designed to eliminate Fc binding to all FcyRs as well as C1q.

ADCC (antibody-dependent cell-mediated cytotoxicity).

The classic ADCC assay is performed to determine if hu425-2-3b can cause ADCC. The effector cell line for analysis, NK92MI/CD16V cells, was generated from NK92MI cells (ATCC) by co-transduction of expression plasmids containing CD16 _V158 (V158 allele) and FcRy cDNA. A Tim-3 expressing T cell line, HuT78/Tim-3, was used as target cells. Effector cells (4*10 ⁴ ) are co-cultured with an equal number of target cells for 5 hours in the presence of hu425-2-3b or control antibodies, or anti-MHC-IA, B, C positive control (Biolegend), or human IgG negative control. Cytotoxicity was determined in a lactate dehydrogenase (LDH) release assay using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Specific lysis is determined by the following equation.

% Specific Experimental - Spontaneous with effector - Spontaneous with target * θθ of lysis Maximum with target - Spontaneous with target

The results confirm that hu425-2-3b has the same baseline level of ADCC as the negative control antibody, while anti-MHC-I A,B,C induces ADCC in a dose-dependent manner (FIG. 12A).

CPC (complement dependent cytotoxicity).

Whether hu425-2-3b will initiate CSC is determined using pre-activated human PBMCs and fresh autologous sera from healthy donors. Cell lysis through CSC was determined using the Celltiter glo assay kit (Promega, Beijing, China). Briefly, PBMCs from healthy donors were pre-activated with PHA (phytohemagglutinin) (10 µg/mL, Sigma) for 3 days and then incubated in RPMI1640 plus autologous serum (15%) and hu425-2-3b or control antibodies (0.04- 30 µg/ml) overnight at 37°C. Cell death due to CSC is analyzed by the decrease in ATP (adenosine triphosphate) released from viable cells after cell lysis at the end of the reaction. Anti-MHC-I A,B,C is used as a positive control. Fluorescence readings were performed using a 96-well fluorimeter (PHERA Star FS, BMG LABTECH) and CSC activities are calculated from relative fluorescence units (RFU) readings as follows: % CSC activity = [(RFU test - RFU background)/(RFU at full cell lysis - RFU background)] * 100. Experimental results show that hu425-2-3b has no detectable CSC with PBMC isolated from two different donors. Conversely, the positive control antibody, anti-MHC-I, elicited significant CSC activity (FIG. 12B).

The results showed that hu425-2-3b eliminated the effector functions of ADCC and CDC while maintaining optimal physicochemical properties.

Example 15 An anti-Tim-3 antibody in combination with an anti-PD-1 antibody inhibits tumor growth in a mouse xenograft cancer model.

The potential anti-cancer activity of an anti-Tim-3 antibody is evaluated in combination with an anti-PD-1 antibody in a cancer xenograft model in which immunocompromised mice are implanted with human cancer cells and allogeneic PBMCs. Briefly, NOD/SCID mice are pre-treated with cyclophosphamide (150 mg/kg) 2 days prior to tumor inoculation. Human PBMCs are isolated from the peripheral blood of healthy volunteers, mixed with A431 squamous cell carcinoma cells (catalog number CRL-1555, ATCC) in matrigel, and injected subcutaneously into animals. Starting from day 0, animals are randomly assigned to 4 groups of 5-10 mice per group. Mice are given once a week (QW, once a week) injection of ip vehicle (PBS), 5 mg/kg anti-Tim-3 mAb chimeric 425 (ch425), anti-PD-1 antibody 317-4B6 (1 mg/kg). kg) or combination therapy (5 mg/kg ch425 plus 1 mg/kg 317-4B6) for 4 weeks. The tumor size of an individual mouse is recorded twice a week, while the mice are checked daily for clinical signs of toxicity during the study. Tumor volumes are calculated using the formula: [D * (d ² )]/2, in which D is the long diameter of the tumor and d is the short diameter. All animal studies are conducted in accordance with Beigene's Animal Care and Use Procedure.

As shown in FIG. 13, treatment with ch425 alone at 5 mg/kg had little or no effect on tumor growth. 317-4B6 at a dose of 1 mg/kg shows a trend towards slower tumor growth without statistical significance (P>0.05). However, the combined treatment with ch425 and 317-4B6 shows significant synergistic effects, inhibiting tumor growth by more than 60% compared to the vehicle treated group.

The results show that combination therapy of ch425 with 317-4B6 can activate human immune cells to inhibit tumor growth in an in vivo mouse cancer model, consistent with the in vitro data described in Example 11.

Claims (19)

1. A humanized antibody capable of binding to human Tim-3 (T-cell immunoglobulin domain and mucin 3 domain) containing: a heavy chain variable region (VH) comprising the VHCDR1 amino acid sequence of SEQ ID NO 3, the VH-CDR2 amino acid sequence of SEQ ID NO 26, and the VH-CDR3 amino acid sequence of SEQ ID NO 5; and a light chain variable region (VL) comprising the VL-CDR1 amino acid sequence of SEQ ID NO 6, the VLCDR2 amino acid sequence of SEQ ID NO 7, and the VL-CDR3 amino acid sequence of SEQ ID NO 27. 2. An antibody according to claim 1, containing: (i) a heavy chain variable domain having a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having a sequence identity of at least at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 36; or (ii) a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO 28, and a light chain variable domain having sequence identity for at least 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of SEQ ID NO 30. 3. Antibody according to claim 1, containing: (i) a heavy chain variable domain containing one or more conservative substitutions in SEQ ID NO 28 and a light chain variable domain containing one or more conservative substitutions in SEQ ID NO 36; or (ii) a heavy chain variable domain containing one or more conservative substitutions in SEQ ID NO 28 and a light chain variable domain containing one or more conservative substitutions in SEQ ID NO 30. 4. Antibody according to claim 1, containing: (i) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO 28 and a light chain variable domain containing the amino acid sequence of SEQ ID NO 36; or (ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 28 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 30. 5. An antibody according to claim 1, which is a Fab, F(ab')2, Fv or single chain Fv (ScFv). 6. An antibody according to claim 1, comprising an IgG1 (immunoglobulin G), IgG2, IgG3 or IgG4 subclass heavy chain constant region or variant thereof, and a kappa or lambda light chain constant region or variant thereof. 7. An antibody according to claim 6, containing a variant heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass, providing a reduced or eliminated effector function. 8. An antibody according to claim 7, wherein the effector function is antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity. 9. The antibody of claim 6, wherein the antibody comprises a human IgG1 heavy chain constant region or a variant thereof. 10. The antibody of claim 9, wherein the variant human IgG1 heavy chain constant region contains one or more mutations selected from the group consisting of E233P, L ₂₃₄ A, L ₂₃₅ A, L ₂₃₆ A, and P ₃₂₉ A. 11. The antibody of claim 10, wherein the antibody comprises a variant human IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO 21 and a human IgG1 kappa light chain constant region. 12. A pharmaceutical composition containing an antibody according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient. 13. A method of treating cancer, comprising administering to a subject in need thereof an antibody according to any one of claims 1 to 11 in an amount effective to treat cancer. 14. The method of claim 13 wherein the cancer is selected from lung cancer, liver cancer, stomach cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, lymphoma, ovarian cancer, head and neck cancer, or metastatic cancer lesions. 15. The method of claim 13 wherein the antibody is administered in combination with a second therapeutic agent or procedure, wherein the second therapeutic agent or procedure is selected from chemotherapy, targeted therapy, oncolytic drug, cytotoxic agent, immune therapy, cytokine, surgical procedure, radiation procedure , an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or cellular immunotherapy. 16. The method of claim 13, wherein the antibody is administered in combination with an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR. - 26 042181 17. The method of claim 13 wherein the antibody is administered in combination with anti-PD-l mAb 317-4B6 or 3174B6/IgG4mt10. 18. A method of treating an infectious disease comprising administering to a subject in need thereof an antibody according to any one of claims 1 to 11 in an amount effective to treat the infectious disease. 19. The method of claim 18, wherein the infectious disease is a chronic viral infection selected from HIV (human immunodeficiency virus) infection and HCV (hepatitis C virus) infection.