WO2019174637A1

WO2019174637A1 - Completely humanized antibody molecule against tim-3, antigen-binding fragment and medical use thereof

Info

Publication number: WO2019174637A1
Application number: PCT/CN2019/078313
Authority: WO
Inventors: 刘佳建
Original assignee: 珠海市丽珠单抗生物技术有限公司
Priority date: 2018-03-15
Filing date: 2019-03-15
Publication date: 2019-09-19
Also published as: CN110272489A; CN110272489B

Abstract

Disclosed are a TIM-3 antibody, an antigen-binding fragment thereof and a medical use thereof. Specifically, disclosed are a murine-derived antibody, a chimeric antibody, a humanized antibody, a completely humanized antibody, a fast-binding and slow-dissociation antibody containing CDR regions of the TIM-3 antibody, a TIM-3 antibody which is excellent in activating the functional activity of human blood cells, a pharmaceutical composition containing the human TIM-3 antibody and antigen-binding fragment thereof, and the use of same as an anti-cancer drug. In particular, disclosed are a pharmaceutical composition of the TIM-3 antibody in combination with a PD-1 antibody, and a use of same in treating tumors.

Description

Fully humanized antibody molecule, antigen-binding fragment for TIM-3 and medical use thereof

Cross-reference to related applications

This application claims the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit.

Technical field

The present invention relates to a TIM-3 antibody, a TIM-3 antigen-binding fragment, a murine antibody comprising the CDR region of the TIM-3 antibody, a chimeric antibody of a murine antibody variable region and a human antibody constant region, and a human source Antibody, fully humanized antibody, and the use of the antibody and antigen-binding fragment thereof to activate human blood cell PBMC, NK cells and killing of tumor cells, and their individual or pharmaceutical compositions, particularly combined with PD-1 antibody Use as an anticancer drug.

Background technique

Tumor immunotherapy is a major breakthrough in the field of cancer treatment in recent years. Anti-drugs against the immune checkpoint PD-1/PD-L1 Since the beginning of 2014, five drugs have been approved for marketing, but have not yet been listed in China. The total anti-drug sales of PD-1/PD-L1 is close to the $10 billion mark in 2017. Unfortunately, PD-1/PD-L1 antibody drugs are only effective in some patients, and a large number of patients have no way to benefit from this huge advancement in tumor immunotherapy. T cells are the center of immunotherapy, and whether T cells can effectively regulate T cells is one of the key factors for the effectiveness of immunotherapy. The PD-1/PD-L1 antibody drug is one of the immunological checkpoint antibody drugs against T cells. The idea of combining immune checkpoint antibodies is to expect better use of T cells as a central role in tumor immunotherapy and is one of the ways to improve the efficiency of cancer patients. In fact, the combination of anti-PD-1 antibody Opdivo and anti-CTLA-4 antibodies has been approved for the treatment of melanoma. Other immunological checkpoint antibodies and PD-1/PD-L1 antibody combinations are also considered to be effective ways to increase the effectiveness of PD-1/PD-L1 antibodies, and some are in clinical trials. Recently (2017 ASCO Annual Meeting), new immunological checkpoint antibody combination therapy has been used to improve the dissemination of effective clinical data.

In immunological checkpoints other than PD-1/PD-L1, the T cell immunoglobulin domain and mucin domain 3 (TIM-3) are immunologically compatible with PD-1/PD-L1 to regulate T cell activity. Checkpoint one of the molecules. TIM-3 is a transmembrane receptor protein expressed on IFN-γ secreting Th1 (T helper 1) CD4+ cells and cytotoxic CD8+ T (Tc1) cells. In addition to the expression of Th1 and Tc1 secreting INF-γ, it is expressed in regulatory T cells (Treg), innate immune cells, including dendritic cells DC, natural killer cells NK, monocyte monocytes, etc. (Anderson ACet Al, Immunity 44, 2016, p989-1004). TIM-3 has multiple ligands, including galectin-9, phosphatidylserine phosphatidylserine, HMGB1, CEACM-1 and the like. TIM-3 is generally not expressed on naive T cells, but is up-regulated on activated effector T cells and has a role in regulating immunity and tolerance in vivo. Unlike some immunological checkpoint molecules, TIM-3 is not only highly expressed after T cell activation, such as Th1, Tc1, participates in synergistic inhibition, inhibits T cell activity, causes tolerance, and is exhausted. High expression in cells inhibits the function of T cells. TIM-3 is highly expressed in an animal model of PD-1 antibody treatment, and combined with TIM-3 antibody significantly improves therapeutic efficacy. Nigiow SF et al. (Cancer Res. 71 (10): 3540-51, 2011) also found that patients treated with PD-1 antibody developed drug resistance, and their expression of CD4+ and CD8+ cells was significantly increased. Consistent with what is seen on animal models (Koyama S et al. Nat Communication. 2016 Feb 17). Therefore, the combination of antibody TIM-3 antibody and PD-1 antibody may not only be one of the effective methods for increasing PD-1 antibody, but also may be an effective choice for patients with drug resistance after PD-1 antibody treatment. The current clinical trial of the TIM-3 antibody is Tesaro's TSR-022 for the treatment of advanced or metastatic solid tumors and Novartis MGB-453, either alone or in combination with PD-1, for use alone or in combination with PD-1 antibodies. Treatment of advanced malignant tumors. There is no more clinical treatment information for antibodies to the TIM-3 target.

There is a need in the art for better anti-TIM-3 antibodies, including fully humanized antibodies or as few humanized antibodies with murine antibody sequences, either alone or in combination with PD-1 antibodies for tumor immunotherapy. To improve the effectiveness of tumor patients in the treatment of PD-1 antibodies.

The present invention obtains an antibody against TIM-3 by a large number of optimized innovation screens. The present invention provides murine antibodies, humanized, fully humanized antibodies, and the like that bind human Tim-3. They have better binding activity, fast binding, slow dissociation, and better activity on human blood cells to activate human blood cells to kill tumor cells, either alone or in combination with PD-1 antibody to increase PD-1 antibody activation. Cellular activity. And the unique site of the human Tim-3 protein should be related to its function. These characteristics make the antibody of the present invention more suitable for drug development, have small immunogenicity, and have long-lasting binding target. The PD-1 antibody alone or in combination can effectively exert the killing effect of human blood cells, including NK cells on tumor cells, to achieve better treatment of tumors. The patient's therapeutic effect and improved PD-1 treatment efficiency.

Summary of the invention

The present invention provides an antibody molecule or a binding fragment thereof which specifically binds to a human T cell immunoglobulin domain and a mucin domain 3 (TIM-3), comprising at least one CDR region sequence selected from the group consisting of Mutation sequence:

a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and the heavy chain variable region (VH) Which comprises a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 8, a VHCDR2 amino acid sequence of SEQ ID NO: 9, and a VHCDR3 amino acid sequence of SEQ ID NO: 10;

VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: The VHCDR2 amino acid sequence of SEQ ID NO: 12 and the VHCDR3 amino acid sequence of SEQ ID NO: 10;

VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: The VHCDR2 amino acid sequence of SEQ ID NO: 14 and the VHCDR3 amino acid sequence of SEQ ID NO: 10;

VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: The VHCDR2 amino acid sequence of SEQ ID NO: 9 and the VHCDR3 amino acid sequence of SEQ ID NO: 10;

VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 15, the VLCDR2 amino acid sequence of SEQ ID NO: 16 and the VLCDR3 amino acid sequence of SEQ ID NO: 17; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: The VHCDR2 amino acid sequence of SEQ ID NO: 19 and the VHCDR3 amino acid sequence of SEQ ID NO: 20.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR1 set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR3 set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR1 set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR3 set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR1 as set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR3 as set forth in SEQ ID NO: Amino acid or its mutated sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising a light chain variable region (VL) comprising the VLCDR1 amino acid of SEQ ID NO: 5, is provided. The sequence or variant thereof, the VLCDR2 amino acid sequence of SEQ ID NO: 6, or a variant thereof, and the VLCDR3 amino acid sequence of SEQ ID NO: 7, or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 15 or a variant thereof, SEQ ID NO: The VLCDR2 amino acid sequence of 16 or a variant thereof and the VLCDR3 amino acid sequence of SEQ ID NO: 17 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising a heavy chain variable region (VH) comprising a SEQ ID NO: 8 The VHCDR1 amino acid sequence or a variant thereof, the VHCDR2 amino acid sequence of SEQ ID NO: 9 or a variant thereof, and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 11 or a variant thereof The VHCDR2 amino acid sequence of SEQ ID NO: 12 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 13 or a variant thereof The VHCDR2 amino acid sequence of SEQ ID NO: 14 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 11 or a variant thereof The VHCDR2 amino acid sequence of SEQ ID NO: 9 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 18 or a variant thereof The VHCDR2 amino acid sequence of SEQ ID NO: 19 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 20 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 8 or a variant thereof, SEQ ID The VHCDR2 amino acid sequence of NO: 9 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 11 or a variant thereof, SEQ ID NO: The VHCDR2 amino acid sequence of 12 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 13 or a variant thereof, SEQ ID NO: The VHCDR2 amino acid sequence of 14 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 11 or a variant thereof, SEQ ID The VHCDR2 amino acid sequence of NO: 9 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 15 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 16 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 17 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 18 or a variant thereof, SEQ ID NO: The VHCDR2 amino acid sequence of 19 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 20 or a variant thereof.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or binding fragment thereof is a murine antibody molecule or a binding fragment thereof.

In a preferred embodiment of the invention, a TIM-3 antibody or a binding fragment thereof, wherein the TIM-3 antibody or binding fragment thereof is a murine antibody molecule or a binding fragment thereof, The murine TIM-3 antibody molecule or a binding fragment thereof is increased in affinity by affinity by a factor of 3-10 times, preferably 10 times.

In a preferred embodiment of the present invention, a TIM-3 antibody or a binding fragment thereof as described above, wherein the mouse antibody molecule or binding fragment thereof has a light chain variable region amino acid sequence of SEQ ID NO: 1 or its variant sequence.

In a preferred embodiment of the present invention, a TIM-3 antibody or a binding fragment thereof as described above, wherein the human antibody molecule or binding fragment thereof has a heavy chain variable region amino acid sequence of SEQ ID NO: 2 or its variant sequence.

In a preferred embodiment of the present invention, a TIM-3 antibody or a binding fragment thereof, wherein the mouse antibody light chain variable region amino acid sequence is SEQ ID NO: 1 or a variant thereof And the heavy chain variable region amino acid sequence is SEQ ID NO: 2 or a variant thereof.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or binding fragment thereof is a variable of a murine antibody molecule or a binding fragment thereof The region and the human antibody constant region include a chimeric antibody molecule or a binding fragment thereof comprising a human antibody heavy chain constant region IgG1, IgG2, IgG4, IgG4 and a light chain constant region kappa, a lambda chain or the like.

In a preferred embodiment of the invention, a TIM-3 antibody or a binding fragment thereof, wherein the chimeric antibody molecule or binding fragment thereof has the light chain amino acid sequence of SEQ ID NO: 25 or Variation sequence.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, wherein the chimeric antibody molecule or binding fragment thereof has the heavy chain amino acid sequence of SEQ ID NO: 26 or Variation sequence.

In a preferred embodiment of the invention, a TIM-3 antibody or a binding fragment thereof, wherein the chimeric antibody molecule or binding fragment thereof has the light chain amino acid sequence of SEQ ID NO: 25 or A variant sequence, and the heavy chain amino acid sequence is SEQ ID NO: 26 or a variant thereof.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof is a chimeric antibody, and the TIM-3 is chimeric The antibody or its binding fragment is increased in affinity by affinity by a factor of 3-10 times, preferably 10 times.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or binding fragment thereof is a humanized antibody molecule or a binding fragment thereof.

In a preferred embodiment of the invention, a TIM-3 antibody or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof, the light chain variable region framework FR sequence is selected from the group consisting of Germline light chain sequence, including IGKV1D-39*01(F), IGKV1-12*01(F), IGKV1-39*01(F), IGKV1-12*02(F), IGKV1-17*01(F ), IGKV1-27*01(F), IGKV1-39*02(P), IGKV1-6*01(F), IGKV1-NL1*01(F), IGKV1D-12*01(F), etc., preferably the same The source, and the preferred frequency of the germline, etc., preferably IGKV1-39*01(F) is a germline light chain for humanization. The light chain J gene hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4, hJK3 and the like have high homology, preferably hJK2.1. The FR sequence region preferably has a 0-10 amino acid back mutation.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein said humanized antibody molecule or binding fragment thereof has a light chain variable region CDR sequence according to Kabat, respectively Each CDR sequence as defined by the numbering rules of Chothia, AbM, Contact, or CCG, the CDR sequences comprising the light chain CDR sequences set forth in Tables 3-6, Table 2b, or variants thereof.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof has a light chain variable region sequence selected from the group consisting of SEQ ID NO: 27-33 or its variant sequence.

In a preferred embodiment of the present invention, a TIM-3 antibody or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof, the heavy chain variable region framework FR sequence is selected from the group consisting of Germline heavy chain sequence, including IGHV3-7*01(F), IGHV3-7*02(F), IGHV3-7*03(F), IGHV3-48*01(F), IGHV3-48*02(F ), IGHV3-48*03 (F), IGHV3-21*01 (F), IGHV3-21*02 (F), IGHV3-21*03 (F), etc., preferably IGHV3-21*01 (F). Antibody J gene and human antibody germline hJH1, hJH2, hJH3.1, hJH3.2, hJH4.1, hJH4.2, hJH4.3, etc., preferably hJH4.1. The FR sequence region preferably has a 0-10 amino acid back mutation.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein said humanized antibody molecule or binding fragment thereof has a heavy chain variable region CDR sequence according to Kabat, respectively Each CDR sequence defined by a numbering rule, such as Chothia, AbM, Contact or CCG, comprising the heavy chain CDR sequences listed in Tables 3-6, Table 2, or variants thereof.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof has a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 34-37 or its variant sequence.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody comprises a sequence selected from the group consisting of SEQ ID NO: 27-33 or variants thereof A light chain variable region and a combination of a heavy chain variable region selected from the group consisting of SEQ ID NO: 34-37 or variants thereof.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof has a light chain comprising a human κ or λ chain constant Zone or its variant.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment heavy chain thereof comprises a human antibody IgG1, IgG2, IgG4, IgG4 heavy chain constant region or variant thereof.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof has a light chain comprising SEQ ID NO: 38, SEQ ID NO: 40, or SEQ ID NO: 43 or a full length light chain sequence having at least 85% sequence homology thereto.

In a preferred embodiment of the invention, the TIM-3 antibody or binding fragment thereof, wherein the humanized antibody molecule or binding fragment heavy chain thereof comprises SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 42 or a full length heavy chain sequence having at least 85% sequence homology thereto.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Preferably, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 38 and SEQ ID NO: 39.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 40 and SEQ ID NO: 39.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 38 and SEQ ID NO: 41.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 40 and SEQ ID NO: 41.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 38 and SEQ ID NO: 42.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 43 and SEQ ID NO: 42.

In a preferred embodiment of the invention, a TIM-3 antibody or binding fragment thereof, as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 43 and SEQ ID NO: 39.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof is a humanized antibody, the TIM-3 human source The affinity of the antibody or its binding fragment is increased by a factor of 3-10 times, preferably 10 times, by affinity.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the anti-TIM-3 antibody or a binding fragment thereof is a murine antibody, a humanized antibody, The humanized antibody, the anti-IM-3 murine antibody, the humanized antibody, the fully humanized antibody antibody or the binding fragment thereof biacore detection shows the characteristics of fast binding and slow dissociation; preferably, the dissociation rate kd is 10 ^-4 magnitude.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof comprises an antigen-binding fragment of a half antibody or a half antibody, preferably , comprising Fab, F(ab")2, Fv or a single-chain Fv fragment (scFv).

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof binds to a human blood cell PBMC antigen, enhancing PBMC against tumor cells The killing effect.

In a preferred embodiment of the invention, there is provided a TIM-3 antibody or binding fragment thereof as described above, wherein said TIM-3 antibody or binding fragment thereof enhances growth of human blood T cells against tumor cell growth Inhibition.

In a preferred embodiment of the present invention, a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof enhances secretion of INF-γ by activated human blood T cells .

In a preferred embodiment of the invention, a TIM-3 antibody or a binding fragment thereof, as described above, wherein the TIM-3 antibody or a binding fragment thereof synergizes with a PD-1 antibody enhances activated human blood T Inhibition of tumor cell growth by cells.

In a preferred embodiment of the invention, a TIM-3 antibody or a binding fragment thereof, as described above, wherein the TIM-3 antibody or a binding fragment thereof synergizes with a PD-1 antibody enhances activated human blood T The cells secrete INF-γ.

In a preferred embodiment of the present invention, there is provided a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof is the same as human Tim-3, macaque (Macaca) Tim-3 Marmoset Tim-3 protein binding activity is different.

In a preferred embodiment of the invention, there is provided a TIM-3 antibody or binding fragment thereof as described above, wherein said TIM-3 antibody or binding fragment thereof has a unique binding region to human Tim-3.

The invention further provides a DNA molecule encoding the above TIM-3 antibody molecule or a binding fragment thereof.

The present invention further provides an expression vector for expressing a DNA molecule of the above TIM-3 antibody molecule or a binding fragment thereof.

The invention further provides a method of producing an antibody comprising transforming, using an expression vector, a host cell, preferably a mammalian cell, more preferably a CHO cell, expressing the TIM-3 antibody molecule or binding fragment thereof.

The invention further provides a pharmaceutical composition comprising, in a preferred embodiment of the invention, any one of the TIM-3 antibodies or binding fragments thereof and a pharmaceutically acceptable carrier, excipient or stabilizer.

The invention further provides a method of treating cancer comprising administering to a subject in need thereof in an amount effective to treat cancer, in a preferred embodiment of the invention, providing any of the TIM-3 antibody molecules described above or a combination thereof Fragment or pharmaceutical composition, preferably, the method comprises any combination comprising an antibody molecule of the invention or a binding fragment thereof, more preferably an antibody or antibody drug combination of any of the above antibody molecules or binding fragments thereof and PD-1 .

The invention further provides a method of treating cancer comprising a method of treatment in combination with an antibody of the invention.

According to a further preferred embodiment of the present invention, there is provided a method of treating cancer comprising a TIM-3 antibody as described above, the cancer being preferably lung cancer, melanoma, renal cancer, colorectal cancer of the breast, liver cancer, Metastatic lesions of pancreatic cancer, bladder cancer, leukemia, etc. or cancer.

DRAWINGS

Figure 1 Binding activity (ELISA) of the anti-TIM-3 antibody mab15-c of the present invention and human TIM-3.

Figure 2a shows the specific binding activity of the anti-TIM-3 humanized, fully humanized antibody and human Tim-3 of the present invention.

Figure 2b shows the specific binding activity of the anti-TIM-3 humanized, fully humanized antibody and human Tim-3+ cells of the invention.

Figure 3. The anti-TIM-3 fully humanized, fully humanized antibody of the invention activates human blood cells to kill tumor cells (NK cells).

Figure 4a shows the binding activity of the anti-TIM-3 humanized, fully humanized antibody and macaque (Tim-3) protein of the present invention.

Figure 4b shows the binding activity of the anti-TIM-3 humanized, fully humanized antibody and marmoset Tim-3 protein of the invention.

The best way to implement the invention

In order to more easily understand the present invention, certain technical and scientific terms of the present invention will be described before describing the embodiments.

Unless otherwise expressly defined in the present invention, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The three-letter code and the one-letter code for amino acids used in the present invention are as described in J.biol.chem, 243, p3558 (1968).

The term "TIM-3" includes isoforms, mammalian (eg, human) TIM-3, human TIM-3 species homologs, and analogs comprising at least one co-epitope with TIM-3. The amino acid sequence of TIM-3 (e.g., human TIM-3) is known in the art, for example, Sabatos et al, 2003. Nat Immunol, 4(11): 1102.

The antibody of the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain γ, α, respectively. Chain, ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either a kappa chain or a lambda chain by the constant region. Each type of Ig in the five classes of Ig may have a kappa chain or a lambda chain.

The antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof. The antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgGl, 2, 3, 4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to VLCDR1, VLCDR2, and VLCDR3; the three CDR regions of the heavy chain refer to VHCDR1, VHCDR2 and VHCDR3.

The CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (VLCDR1-3, VHCDR1-3), Chothia, AbM, Contact, and CCG in number and position. Numbering rules (see Table 2, CDR definition methods; Table 2b, CGG definitions). The CDR regions of the light chain and heavy chain of the antibody of the present invention define respective CDR sequences according to the numbering rules of Kabat, Chothia, AbM, or Contact, respectively, as shown in Table 2, Table 2b, and Table 3-6.

The term "murine antibody" is in the present invention a monoclonal antibody to human TIM-3 prepared according to the knowledge and skill in the art. The test subject is injected with the TIM-3 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated. In a preferred embodiment of the invention, the murine TIM-3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or IgG4 or a variant thereof.

The term "chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody. To establish a chimeric antibody, a hybridoma that secretes a murine monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the mouse variable region gene is The human constant region gene is ligated into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic cell, industrial system or prokaryotic system. In a preferred embodiment of the invention, the antibody light chain variable region of the TIM-3 chimeric antibody further comprises a light chain FR region of a murine kappa, a lambda chain or a variant thereof, an antibody light chain variable region The amino acid sequence is shown in SEQ ID NO: 3. The antibody heavy chain variable region of the TIM-3 chimeric antibody further comprises a heavy chain FR region of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the antibody heavy chain variable region amino acid sequence is SEQ ID NO: 4 is shown. The constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) after amino acid mutation Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) IgG1. The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc fragment on IgG. The modification refers to mutations in the heavy chain constant region of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutation.

The term "humanized antibody", also referred to as CDR-grafted antibody, refers to the CDR sequence of a mouse, in particular, the CDR of the TIM-3 antibody of the present invention is according to Kabat , CDR sequences defined by the numbering rules of Chothia, AbM, Contact or CGG (see Table 2, Table 2b, Tables 3-6), antibodies produced by transplantation into the variable region framework of human antibodies. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr. In a preferred embodiment of the invention, the CDR sequences of the TIM-3 humanized antibody mouse are selected from the group consisting of the light chain SEQ ID NO: 5, 6, 7; the heavy chain SEQ ID NO, 8, 9, 10 . Alternatively the sequence is selected from the light chain SEQ ID NO: 5, 6, 7; the heavy chain SEQ ID NO, 11, 9, 10. The human antibody variable region framework is designed and selected, wherein the light chain FR region sequence on the antibody light chain variable region is derived from the combined sequence of human germline light chain IGKV1-39*01(F) and hJK2.1 SEQ ID NOs: 27-33, comprising the FR1, FR2, FR3 region of human germline light chain IGKV1-39*01 (F) and the FR4 region of hJK2.1; wherein the heavy chain on the heavy chain variable region of the antibody FR region sequence, derived from the human germline heavy chain IGHV3-21*01 (F) and hJH4.1 combination sequence SEQ ID NO: 34-37, comprising FR1 of human germline heavy chain IGHV3-21*01 (F) , FR2, FR3 area and FR4 area of hJH4.1. In order to avoid a decrease in the activity caused by a decrease in immunogenicity, the human antibody variable region may be subjected to minimal back mutation, and the site of the back mutation may be 10 or more, preferably 0-10, to maintain activity. .

The "fully humanized antibody" of the present invention means that the antibody has a human antibody germline sequence other than the CDR sequence derived from the murine antibody. The antibody obtained by the above humanization method has a number of back-mutation sites of light and heavy chains of 0 in the process of humanization, that is, the light-weight chain FR region includes the J region, which is a complete human antibody germline sequence. Also included is that the FR region of the light and heavy chain, including the J region, is the majority of the CDR regions, even 1-6 intact CDRs, preferably one complete CDR sequence and the corresponding CDRs of the human germline antibody, in addition to the entire human antibody germline sequence. The sequence is 100% consistent.

The "antigen-binding fragment" as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab')2 fragment, and an Fv fragment sFv fragment which binds to human TIM-3. The Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site. In general, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding. The two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (sFv). Single-chain antibodies can also be used for bispecific antibodies. The term "in combination with TIM-3" of the present invention means that it is capable of interacting with human TIM-3. The term "antigen binding site" as used in the present invention refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.

Antibody molecules comprise diabodies and single-stranded molecules as well as antigen-binding fragments of antibodies (eg, Fab, F(ab')2 and Fv). The antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody in the present invention). Fab', F(ab')2, Fc, Fd, Fv, single chain antibodies (eg, scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (eg, , humanized) antibodies, which can be produced by modifying intact antibodies, or those that are synthesized de novo using recombinant DNA techniques. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any antibody class including, but not limited to, IgG, IgA, IgM, IgD, and IgE and from any antibody subclass (eg, IgGl, IgG2, IgG3, and IgG4). Preparation of antibody molecules can be monoclonal or polyclonal. The antibody may also be a human antibody, a humanized antibody, a CDR-grafted antibody, or an antibody produced in vitro. The antibody may have, for example, a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3 or IgG4. The antibody may also have, for example, a light chain selected from kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" in the present invention.

The antibodies disclosed herein may also be single domain antibodies. Single domain antibodies can include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack light chains, single domain antibodies derived from conventional four chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. A single domain antibody can be any antibody of the prior art, or any single domain antibody of the future. Single domain antibodies can be derived from any species including, but not limited to, mice, humans, camels, alpacas, fish, heavy fish, goats, rabbits, and cattle. According to some aspects, a single domain antibody is a naturally occurring single domain antibody, referred to as a heavy chain antibody lacking a light chain. For clarity reasons, such a variable domain derived from a heavy chain antibody that naturally lacks a light chain is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in Camelidae species such as camels, alpacas, dromedaries, llamas and guanaco. Other species other than camels can produce heavy chain antibodies that naturally lack light chains, and such VHHs are also considered. The VH and VL regions can be subdivided into hypervariable regions, referred to as "complementarity determining regions" (CDRs), which are interspersed with more conserved regions called "framework regions" (FR). The range of frame regions and CDRs has been defined in a number of ways.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody or mAb according to the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain. The host cell of the vector of the present invention may be, but not limited to, a eukaryotic cell, a bacterial cell, an insect cell or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, suitable insect cells including, but not limited to, Sf9 cells.

Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques. Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor Antibody Testing Technical Guide. The antigen-binding fragment can also be prepared by a conventional method.

"Administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid. "Administration" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells. "Administering" and "treating" also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.

"Treatment" means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect. Generally, a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement. The amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.

"Conservatively modified" or "conservative substitution or substitution" refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein.

The term "consisting essentially of" or variations thereof, as used throughout the specification and claims, includes all such elements or groups of elements, and optionally includes other elements that are similar or different in nature to the elements, the other The element does not significantly alter the essential or novel properties of a given dosage regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to permit or facilitate the diagnosis. The effective amount for a particular patient can vary depending on such factors as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to a substance that is produced outside of a living organism, cell, or human body depending on the background. "Endogenous" refers to a substance produced in a cell, organism or human body depending on the background.

"Homology", "variant sequence" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position . The percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared × 100. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.

The expression "cell", "cell line" and "cell culture" as used herein are used interchangeably and all such names include progeny. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a small portion of a particular portion of nucleic acid, RNA, and/or DNA is amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. The PCR used in the present invention is regarded as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify Or produce a specific portion of a nucleic acid.

"Optional," "optionally," "arbitrary," or "any" means that the subsequently described event or environment may, but need not, occur, including where the event or environment occurs or does not occur. For example, "optionally comprising 1 antibody heavy chain variable region" means that the antibody heavy chain variable region of a particular sequence may, but need not, be present.

As used herein, "a" and "an" are used to mean one or more than one grammatical object. The term "or" is used in the present invention to mean the term "and/or" and is used interchangeably unless the context clearly dictates otherwise. "About" and "about" shall generally mean the degree of acceptable error of the measured quantity in view of the nature or precision of the measurement. Exemplary degrees of error are generally in the range of 10% thereof and more generally in the range of 5% thereof. The methods and compositions disclosed herein encompass polypeptides and nucleic acids having a specified sequence, a variant sequence or a sequence substantially identical or similar thereto, for example, at least 85%, 90%, 95% or more with the sequence designation The same sequence. In the context of an amino acid sequence, the term "substantially identical" is used in the present invention to refer to a first amino acid sequence.

"Pharmaceutical composition" means a mixture comprising one or more compounds of the invention or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carrier and excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity. Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high antibody concentrations. A sterile injectable solution can be prepared by combining the active compound (i.e., antibody or antibody portion) in the required amount in combination with one of the ingredients or ingredients listed above, in a suitable solvent, and, if necessary, followed by filtration and disinfection. .

The methods, compositions, combination therapies of the invention may be combined with other active agents or treatments, the methods comprising administering to the subject an amount of an anti-TIM of the invention effective to treat or prevent a disease (eg, cancer) -3 antibody molecule, optionally in combination with one or more inhibitors of PD-1, PD-L1, PD-L2, LAG-3, CEACAM-1 and/or CEACAM-5 or CTLA-4 antibodies Also included is administration of an anti-TIM-3 antibody molecule, additional active agents or all may be administered in an amount or dose that is higher, lower or equal to each of the individual (eg, as monotherapy) The amount or dose of active agent. The anti-TIM-3 antibody, the additional active agent or the total application amount or dose is lower than the amount or dose of each active agent used alone (eg, as a monotherapy) (eg, at least 20%, at least 30%, at least 40) % or at least 50%).

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains has been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), with uncharged polar side Chain amino acids (eg glycine, aspartame, glutamic acid, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg alanine, arginine) , leucine, isoleucine, valine, phenylalanine, methionine, tryptophan), amino acids with self-branched side chains (eg threonine, tyrosine, isoleucine) An acid), and an amino acid having an aromatic side chain (for example, tyrosine, phenylalanine, tryptophan, histidine). The terms "polypeptide", "peptide" and "protein" (if single stranded) are used interchangeably in the present invention. The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. The term "isolated" refers to a material that is removed from its original or original environment (eg, if the official naturally exists, the natural environment).

The term "competition" or "cross-competition" is used interchangeably in the present invention to mean the ability of an antibody molecule to interfere with the binding of an anti-TIM-3 antibody molecule to a target (eg, human TIM-3). Interference with binding can be direct or indirect (eg, by allosteric modulation of the antibody molecule or target). A competitive binding assay (eg, FACS assay, ELISA, or BIACORE assay) can be used to determine the extent to which an antibody molecule can interfere with the binding of another antibody molecule to its target and whether it can therefore be said to be competitive. The term "epitope" refers to a portion of an antigen (eg, human TIM-3) that specifically interacts with an antibody molecule.

Furthermore, as described in the Examples of the present invention, the anti-TIM-3 antibody can stimulate NK cell-mediated killing of target cells and can enhance IFN-γ secretion and proliferation of CD4+ T cells. Thus, in certain embodiments, the anti-TIM-3 antibody molecules described in the present invention are useful for stimulating a desired immune response, such as an immune response against a cancer cell or pathogen. The anti-TIM-3 antibodies described in the present invention can be used to treat immune disorders, particularly those associated with T lymphocytes, including but not limited to chronic inflammatory diseases and cancer. And a method of inhibiting tumor cell growth comprising administering to a subject a therapeutically effective amount of an anti-TIM-3 antibody molecule of the invention. This method is suitable for in vivo treatment of cancer. In order to obtain an antigen-specific enhancement of immunity, an anti-TIM-3 antibody molecule can be administered together with other antibodies. Where a TIM-3 antibody is administered in combination with one or more active agents, the combination can be administered in any order or simultaneously. In certain aspects, methods of treating a subject (eg, reducing or alleviating) a hyperproliferative condition or disease (eg, cancer), eg, a solid tumor, a hematological cancer, a soft tissue tumor, or a metastatic lesion, are provided in a subject. The method comprises administering to the subject one or more anti-TIM-3 antibody molecules of the invention, alone or in combination with other active agents or treatment modalities.

As used herein, the terms "cancer", "cancer", "cancer patient" are intended to include all types of cancerous growth or tumorigenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of histopathology. What is the type or invasiveness phase. Examples include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems (including adenocarcinoma and squamous cell carcinoma), such as invasive liver, lung, breast, lymph, gastrointestinal tract (eg, colon), genitourinary Road (eg, kidney, bladder epithelial cells), prostate and pharynx. Adenocarcinomas include malignant tumors such as most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma in lung cancer, small bowel cancer, and esophageal cancer. Squamous cell carcinomas include malignant tumors, such as in the lungs, esophagus, skin, head and neck regions, the mouth, anus, and cervix. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions described herein. Antibody molecules directed against TIM-3 can be combined with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and sugar molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines.

The combination further includes an inhibitor or activator of an immunological checkpoint modulator (eg, a Tim-3 inhibitor (eg, an anti-TIM-3 antibody molecule), a PD-L1 inhibitor (eg, an anti-PD-L1 antibody molecule), PD -1 inhibitor (eg, anti-PD-1 antibody molecule), or CTLA-4 inhibitor (eg, anti-CTLA-4 antibody), or any combination thereof. TIM-3 blocking may also be combined with standard cancer treatment. TIM- 3 Blocking can be effectively combined with a chemotherapeutic regimen. In these cases, the dose of chemotherapeutic agent administered can be reduced. The composition can be administered in combination with one or more, immunomodulatory agents (eg, activators of costimulatory molecules or Inhibitors of inhibitory molecules); vaccines or other forms of cellular immunotherapy.

Exemplary, non-limiting combinations and uses of anti-TIM-3 antibody molecules include administration of an anti-TIM-3 antibody molecule in combination with a modulator of a co-stimulatory molecule or an inhibitory molecule (eg, a co-inhibitory ligand or receptor). The anti-TIM-3 antibody molecule is administered in combination with a modulator (eg, an agonist of a costimulatory molecule). And agonists of co-stimulatory molecules are selected from the group consisting of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD13 7), GITR, CD30, CD40, An agonist of a BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand (eg, an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion). The anti-TIM-3 antibody molecule is administered in combination with an inhibitor of an immunological checkpoint molecule (or immunosuppressive molecule). The term "immunization checkpoint" refers to a group of molecules on the cell surface of an immune cell that can act as a "gate" to down-regulate or suppress an immune response, such as an anti-tumor immune response. Immunological checkpoint molecules include, but are not limited to, PD-1, PD-L1, cytotoxic T lymphocyte antigen 4 (CTLA-4), B7-H1, B7-H3, OX-40, 4-1BB (CD137), CD40 , TIM-3 and lymphocyte activation gene 3 (LAG-3), and so on. Immunotherapeutic agents that can be used in combination with the anti-TIM-3 molecules described in the present invention as inhibitors of immunological checkpoint molecules include, but are not limited to, PD-L1, PD-L2, CTLA-4, LAG-3, VISTA, Inhibitors of BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (eg CEACAM-1, CEACM-3 and/or CEACAM-5) and/or TGFR beta. Immunosuppressive molecules can be inhibited by inhibition at the DNA, RNA or protein level. Immunological checkpoint molecules (eg, PD-1, LAG-3, TIM-3, CEACAM-1/-5) can modulate T cell function and promote tumor immune evasion. The anti-TIM-3 antibody molecule is administered in combination with an anti-TIM-3 antibody or antigen-binding fragment thereof, and the anti-TIM-3 antibody molecule is administered in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. The anti-TIM-3 antibody molecule is administered in combination with an anti-TIM-3 antibody and an anti-PD-1 antibody or antigen-binding fragment thereof. A bispecific antibody is administered comprising an anti-TIM-3 antibody molecule and an anti-PD-1 or anti-TIM-3 antibody or antigen-binding fragment thereof, or a TIM-3 antibody and a LAG-3 antibody or antigen-binding fragment thereof.

Treatment of kidney cancer, such as renal cell carcinoma (RCC), with an anti-TIM-3 antibody molecule, alone or in combination with another immunomodulatory agent (eg, an anti-LAG-3, anti-PD-1 or anti-PD-L1 antibody molecule) (eg, clear cell renal cell carcinoma (CCRCC) or metastatic RCC. Anti-TIM-3 antibody molecules can be administered in combination with one or more of: an immune-based strategy (eg, interleukin 2 or interferon alpha), Targeted drugs (eg, VEGF inhibitors such as monoclonal antibodies against VEGF); VEGF tyrosine kinase inhibitors such as sulphonic acid, sorafenib, axidini and paclitaxel; RNAi inhibitors) An inhibitor of a downstream mediator of VEGF signaling, for example, an inhibitor of a mammalian target of rapamycin (mTOR).

Exemplary cancers that can inhibit their growth using the antibody molecules disclosed in the present invention include cancers that are typically responsive to immunotherapy. Non-limiting examples of cancers suitable for treatment include melanoma (eg, metastatic malignant melanoma), renal cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone-refractory prostate adenocarcinoma), breast cancer, colon Cancer and lung cancer (eg non-small cell lung cancer). In addition, antibody molecules described in the present invention can be used to treat refractory or recurrent malignancies. Cancer includes but is not limited to basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, primary CNS lymphoma, central nervous system (CNS) tumor, breast cancer, cervical cancer, choriocarcinoma, Colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intradermal tumor, kidney cancer, laryngeal cancer, blood disease (including acute marrow) Leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic or acute leukemia), liver cancer, lung cancer (eg small cell and non-small cell carcinoma), lymphoma, including He Jiejin And non-Hodgkin's lymphoma, lymphocytic lymphoma, melanoma, such as malignant melanoma in the skin or eye, myeloma, neuroblastoma, oral cancer (eg lip, tongue, oral cancer); Ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, sarcoma: skin cancer, stomach cancer, thyroid cancer, uterine cancer, urinary system cancer, liver cancer, monthly work Cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, small intestine cancer, endocrine system cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumor, spinal axis tumor, brain stem glioma Pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally-induced cancer, including those induced by asbestos, and combinations of other cancers and sarcomas and said cancers.

Specific implementation example

The invention is further described in the following examples, which are not intended to limit the scope of the invention. The experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions, such as the cold spring harbor antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific source are routine reagents purchased from the market.

Example 1: Cloning, expression and purification of antigen, antibody

The antigen used in the present invention was purchased from different companies, TIM-3-his (Cat. No.: TM3-H5229), TIM-3-hFc (Cat. No.: TM3-H5258) was purchased from Beijing Baipusisi Biotechnology Co., Ltd.; TIM -3-his-hFc was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., article number: 10390-H03H; or purified by expression of the present invention. The expressed human Tim-3 protein sequence was UniProtKB/Swiss-Prot: Q8TDQ0.3, the extracellular (ECD) region of HAVR2_HUMAN (amino acids 22-199). The antibodies used in the present invention, including the positive control antibody ABTIM3 (sequence from WO2015117002A1), and the PD-1 antibody nivolumab (sequence from WO2013019906) were all purified by expression of the present invention.

The pTT5 vector (Biovector, Cat#: 102762) used for expression cloning was transformed into a suitable cloning site by the present invention and designated as pJX5. All protein expression, including recombinant protein, antibody light and heavy chain, was expressed by transient transfection of HEK293E cells (Life Technologies Cat. No. 11625019) with pJX5 vector, and purified.

Specifically, 293 cells were expanded in Gibco FreeStyle 293 Expression Medium (Gibco, Cat #12338018) medium. Before the start of the transient, adjust the cell concentration to 6 ~ 8 × 10 ⁵ cell / ml, 1% FBS (Aus Gene X FBS Excellent supplier: AusGeneX, China, Cat # FBSSA500-S), 37 ° C 8% CO2 shaker culture At 24h, the microscopic examination survival rate was >95%, and the cell concentration was 1.2×10 ⁶ cell/ml.

300 ml of culture system cells were prepared, and 15 ml of Opti-MEM (Gibco, Cat# 31985070) was dissolved in 150 ug of each of the heavy chain and light chain plasmids (in the case of recombinant protein, the amount of a single plasmid was 300 ug), and 0.22 um was sterilized by filtration. 15 ml of Opti-MEM was dissolved in 1 mg/ml PEI (Polysciences, Inc, Cat# 23966-2) 600 ul and allowed to stand for 5 min. The PEI was slowly added to the plasmid, incubated at room temperature for 10 min, and the plasmid PEI mixed solution was slowly dropped while shaking the culture flask, and the mixture was cultured for 5 days at 37 ° C in an 8% CO 2 shaker, and the supernatant was taken at 3300 G for 10 min for purification.

Antibody or -Fc fusion protein purification: The sample was centrifuged at high speed to remove impurities, and the gravity column containing Protein A (Mabselect, GE Healthcare Life Science, Cat #71-5020-91AE) was equilibrated with PBS pH 7.4 (Bio-Bio, Cat# F506606-0001), 2-5 column volume rinse. Pass the sample through the column. The column was washed with 5-10 column volumes of PBS (Biotech, Cat# B548117-0500). The target protein was eluted with pH 3.5 0.1 M acetic acid, then adjusted to neutral with Tris-HCl at pH 8.0, and the concentration was determined by a microplate reader, and the mixture was stored and stored for use.

Purification of His Tagged protein: The sample is centrifuged at high speed to remove impurities. Balanced nickel column (Ni smart beads 6FF Changzhou Tiandi Renhe Biotechnology Co., Ltd. Cat#SA036010): The nickel column was equilibrated with a PBS pH 7.4 solution containing 10 mM imidazole 0.5 M NaCl, and washed in 2-5 column volumes. The sample was supernatanted through the column. Rinsing the heteroprotein: The column was washed with a PBS pH 7.4 solution containing 10 mM imidazole 0.5 M NaCl to remove non-specifically bound heteroprotein and the effluent was collected. The protein of interest was eluted with PBS pH 7.4 containing 250 mM imidazole 0.5 M NaCl. Buffer replacement: The eluted target protein was centrifuged at 12000 g for 10 min in an ultrafiltration tube (ultrafiltration tube Merck Millipore Cat #UFC500308), and then added with 1 ml of PBS, and the concentration was measured, and the mixture was stored and stored for use.

Example 2: Construction and binding activity (ELISA) of human Tim-3 high expression cell line (hTim-3+ cells)

The human Tim-3 high expression cell line used in the present invention is completed by the company stable cell line construction platform. The specific steps are as follows: On the first day of the experiment, 293T cells ( Catalogue of the Culture Collection Committee of the Chinese Academy of Sciences, Cat# GNHu17 ) were inoculated into two 6 cm culture dishes, and the number of cells in each dish reached 7.5×10 ⁵ . On the second day, 4 μg each of the plasmid (pGag-pol, pVSV-G, etc. BioVector Plasmid Vector Culture Cell Collection) and the plasmid pBabe-hTim-3 cloned with the Tim-3 gene were added to OPTI-MEM (Thermofisher Scientific Cat#) 31985070), the final volume is 200μl, and another 200μl OPTI-MEM is added to 36μl transfection reagent fectin (Shanghai Yuanpei Biotechnology Co., Ltd. Cat#F210), the two are mixed, left at room temperature for 5min, then the mixture (each dish) 200 μl of each was added dropwise to the cultured 293T cells. On the third day, the 293T cell culture medium was changed to 4 ml DMEM high-sugar medium (Shanghai Yuanpei Biotechnology Co., Ltd. / Yuanpei Bio: Cat# L130KJ). On the fourth day, CHO-K1 cells (Chinese Academy of Sciences, Culture Collection Committee Cell Bank Cat# SCSP-507) were inoculated into a 10 cm culture dish to bring the number of cells to 5 × 10 ⁵ . On day 5, 293T cell supernatant (virus) was collected, filtered through a 0.45 μm filter to cultured CHO-K1 cells, and 10 ug/ml polybrene (Shanghai Shengsheng Biotechnology Co., Ltd. Cat#40804ES76) was added and mixed. The incubator was changed to DMEM/F12 10% FBS medium (Source Pei Bio, Cat# L310KJ) after 3 to 4 hours. CHO-K1 cells were passaged on day 7, and cells passaged on day 8 were initially screened by adding 10 ug/ml puromycin (Source Culture, Cat# S250J0). After 2-3 days, the cells died in large amounts, and the medium was changed to continue the culture until the cells no longer died. The cells were expanded in a large amount, and the monoclonal cell strains were screened, expanded, and stored frozen.

hTim-3+ cell binding activity assay:

The cell line with high expression of human human Tim-3 obtained in the above example was expanded, and then incubated at 6× ^{10 4} /well 96-well plate, incubating at 37 ° C overnight, and then removing the supernatant, and using immunostaining fixative ( Shanghai Biyuntian Biotechnology Co., Ltd. Cat#P0098) 100 ul / hole fixed at room temperature for half an hour. After washing with PBS (source culture, Cat# B320), 5% milk was blocked at 37 ° C for 2 hours, and PBST was washed 3 times. Add the sample to be tested (human or murine antibody, Jackson Immuno Research). Incubate for 1 hour at 37 ° C and wash 3 times with PBST. Add Anti-human or mouse HRP 1:2500 50ul/well for 1 hour at 37°C, then wash 3 times with PBST, develop color with TMB (Surmodic Cat#TTMB-1000-01), and stop by adding 50μl/well 1M H ₂ SO ₄ . The microplate reader (MultiskanGO Thermo model 51119200) was read and the Graphpad prism 5 was used for data analysis.

The binding activity of the screening optimized cell strain and ABTIM3 expressed in Example 1 of the present invention was: EC50 = 0.15 nM - 0.23 nM.

The amino acid sequence of human Tim-3 (pBabe-hTim-3) used in this example is UniProtKB/Swiss-Prot: Q8TDQ0.3, the full sequence of HAVR2_HUMAN is 1-301 amino acids, wherein the position 1-21 is a signal peptide sequence, ie, 22-301 is a protein sequence expressed by the CHO-K1hTim-3+ cell line of the present invention.

Example 3: Anti-TIM-3 antibody binding ELISA assay

The goat-anti-hFc (Jackson, 109-005-008) was diluted to a concentration of 1 ug/ml with PBS buffer pH 7.4, and added to a 96-well microtiter plate (Corning, CLS3590-100EA) at a volume of 50 ul/well. Place in an incubator at 37 ° C for 2 hours. (Or directly with the antigen Tim-3-his, 0.5ug / ml plate, then directly add the test antibody). After discarding the liquid, a 5% skim milk (bright skimmed milk powder) blocking solution diluted with PBS was added at 200 ul/well, and incubated at 37 ° C for 2.5 hours or at 4 ° C overnight (16-18 hours) for blocking. Discard the blocking solution and wash the plate 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tweeen-20), then add 50 ul / well, 0.5 ug / ml of TIM-3-hFc (Example 1), set at 37 ° C Incubate for 2 hours in an incubator. After the incubation, wash the plate with PBST 6 times, add 50 ul / well supernatant (containing detection antibody) or different concentrations of the test antibody, incubate at 37 ° C for 2 hours, wash the plate 5 times with PBST, add 50ul / hole 1: 2500 dilution HRP-labeled secondary antibody (Jackson Immuno Research, 115-035-003) was incubated for 1 hour at 37 °C. After washing the plate with PBST 5 times, add 50 μl/well TMB chromogenic substrate (KPL, 52-00-03), incubate for 10-15 min at room temperature, and add 50 μl/well 1 M H ₂ SO _{4 to} stop the reaction with MULTIYSAN Go The instrument (ThermoFisher, 51119200) reads the absorbance at 450 nm, calculates the EC50 from the OD value, or selects a clone with high binding activity.

Example 4: Anti-human TIM-3 antibody human blood cell killing (NK) activity against tumor cells

Human blood cells (PBMC) are derived from peripheral blood donated by healthy people. The PBMC Press 2.5x10 ⁵ cells / well, chronic myelogenous leukemia cell line K562 (ATCC NO: CCL-243 ^TM, Agent: Shanghai Co., Ltd. of Seoul prime) according to 5x10 ⁴ cells / well in a 96 well plate (Corning3599). The hybridoma supernatant antibody to be tested, or the purified antibody is added to a 96-well cell plate, and incubated in a 37 ° C incubator for 6 hours, and then used LDH detection kit (Shanghai Tongren Biotechnology Co., Ltd., catalog number: CK12), and tested according to the instructions. The 490 nm absorption value (OD) was read by a MULTISKAN Go microplate reader, and the percentage change of the release amount of LDH was calculated, and the activity of the test sample in the human blood cell killing tumor cell (NK) was compared.

Example 5: Discovery of anti-human TIM-3 antibodies

The invention uses human TIM-3 as an antigen, immunizes mice, screens fusion hybridomas, screens from hundreds of thousands of hybridoma clones, optimizes, and unexpectedly finds that the mouse antibodies obtained by the invention are optimized by computer, and the human source design is fully obtained. The humanized antibody (that is, the antibody has no mouse sequence except the CDR region) can maintain a good binding activity, and more surprisingly, the human blood cell killing activity and the antigen-binding dissociation rate are excellent. As a drug development, it provides a very good advantage in treating tumors.

Specifically, the experimental SJL white mice, female, 4 weeks old were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2016-0011. After the mice were purchased, the laboratory environment was kept for 1 week, and the daylight/night dark cycle was adjusted at a temperature of 20-25 ° C; the humidity was 40-60%. The mice were divided into 3 groups/groups/cage. The antigen prepared in Example 1 was used for immunization. The adjuvant is Quickantibody (Beijing Boaolong Immunotechnology Co., Ltd., item number KX0210041). The ratio of antigen to adjuvant is 1:1. 100ul/10ug/first exemption, calf muscle injection. Three days before the fusion, 100ul/25ug/only boosted. Immunization time was 0, 14, 28, 42, 56, and 59 days (enhanced immunization). On the 22nd, 36th, 50th, and 64th day, the serum antibody titer of the mouse was detected by the ELISA method of the above Example 3, and the mouse with high antibody titer and titer in the plateau was selected for spleen cell fusion, and the spleen was obtained. Lymphocytes and myeloma cells Sp2/0 cells (

The CRL-8287 ^(TM ) is fused to obtain a hybridoma cell-packed 96-well plate, followed by screening, preferably cloning.

The primary screening of the hybridoma cell line includes the detection of the binding activity of the antibody in the secretory supernatant of the hybridoma cell line and the human TIM-3 by the ELISA method of Example 3, and selecting the cloned supernatant having good activity to carry out the method of Example 4 The method detects the activity of the secreted antibody in human blood cells, preferably cloned, and the results of partial preferred cloning are shown in Table 1.

Table 1 Monoclonal cell cloning activity of murine hybridoma screening

The initial clones in Table 1 were further subjected to limiting dilution, and after 7-10 days after each dilution, the NK method was used to re-detect the binding and NK activity of the secreted antibody (supernatant) of each clone by the ELISA method described above. After several times of limiting dilution, it was unexpectedly found that the secreted supernatant of the monoclonal cell line 3.13H9G6A6D8 obtained from the actual clone of 3.13H9 maintained a good binding activity and human blood cell NK activity. The antibody sequence is extracted from this monoclonal to obtain the preferred murine mab15 antibody of the present invention.

Example 6: Screening and identification of murine anti-human TIM-3 antibody

The process of extracting antibody sequences from monoclonal cell lines preferably obtained from hybridomas is a method commonly used by those skilled in the art. Specifically, the above monoclonal cell strain is collected, and after expansion and culture, 1×10 ⁶ cells are taken, and RNA is extracted with Trizol (Invitrogen, 15596-018) (according to the kit instructions), and the extracted RNA is reverse-transcribed into cDNA. The reverse transcription kit was purchased from Biotech Biotechnology (Shanghai) Co., Ltd., Cat# B532435. The cDNA obtained by reverse transcription was used as a template to carry out PCR amplification. The amplified product was sequenced to obtain the light/heavy chain variable region base/coding sequence of the mab15 antibody (see below). The primers used are referred to the manual TB326 Rev. C0308 published by Novagen.

The monoclonal light chain variable region amino acid coding sequence (lined portion) obtained in the preferred hybridoma cell line of the invention:

Monoclonal heavy chain variable region amino acid coding sequence (lined portion) obtained in a preferred hybridoma cell line of the invention:

The amino acid sequence encoded by the base sequence of the light heavy chain variable region of the above monoclonal antibody obtained by the present invention is SEQ ID NO: 3 and SEQ ID NO: 4 below.

Monoclonal antibody mab15 light chain variable region protein sequence obtained in a preferred hybridoma monoclonal cell line of the invention:

The monoclonal antibody mab15 heavy chain variable region sequence obtained in the preferred hybridoma cell monoclonal strain of the present invention:

CDR definition of antibodies There are several different methods in the art, including methods such as Kapat, as shown in the following table.

Table 2 Antibody CDR definition method*

LoopLoop	Kabat定义Kabat definition	AbM定义AbM definition	Chothia定义Chothia definition	Contact定义Contact definition
轻链CDR1Light chain CDR1	L24-L34L24-L34	L24-L34L24-L34	L24-L34L24-L34	L30-L36L30-L36
轻链CDR2Light chain CDR2	L50-L56L50-L56	L50-L56L50-L56	L50-L56L50-L56	L45-L55L45-L55
轻链CDR3Light chain CDR3	L89-L97L89-L97	L89-L97L89-L97	L89-L97L89-L97	L89-L96L89-L96
重链CDR1Heavy chain CDR1	H31-35H31-35	H26-35H26-35	H26-32H26-32	H30-35H30-35
重链CDR2Heavy chain CDR2	H50-65H50-65	H50-58H50-58	H52-56H52-56	H47-H58H47-H58
重链CDR3Heavy chain CDR3	H95-H102H95-H102	H95-H102H95-H102	H95-H102H95-H102	H93-H101H93-H101

*For more information, please visit the website: http://www.bioinf.org.uk/abs/#cdrdef

The light and heavy chain CDR region sequences of the present invention are determined as follows according to the definition/annotation of the above antibody CDRs.

Table 3 Anti-human TIM-3 antibodies of the invention define CDR sequences according to Kabat

抗体antibody	mab15CDRsmab15CDRs
轻链CDR1Light chain CDR1	RASENIYSYLT(SEQ ID NO:5)RASENIYSYLT (SEQ ID NO: 5)
轻链CDR2Light chain CDR2	NAKTLAE(SEQ ID NO:6)NAKTLAE (SEQ ID NO: 6)
轻链CDR3Light chain CDR3	QQHYGTPLT(SEQ ID NO:7)QQHYGTPLT (SEQ ID NO: 7)
重链CDR1Heavy chain CDR1	DYYMT(SEQ ID NO:8)DYYMT (SEQ ID NO: 8)
重链CDR2Heavy chain CDR2	SINYDGRNTYYLDSLKS(SEQ ID NO:9)SINYDGRNTYYLDSLKS (SEQ ID NO: 9)
重链CDR3Heavy chain CDR3	GYYYYGSSPNYFDY(SEQ ID NO:10)GYYYYGSSPNYFDY (SEQ ID NO: 10)

Table 4 Anti-human TIM-3 antibodies of the invention define CDR sequences by AbM

antibody

mab15CDRs

轻链CDR1Light chain CDR1	RASENIYSYLT(SEQ ID NO:5)RASENIYSYLT (SEQ ID NO: 5)
轻链CDR2Light chain CDR2	NAKTLAE(SEQ ID NO:6)NAKTLAE (SEQ ID NO: 6)
轻链CDR3Light chain CDR3	QQHYGTPLT(SEQ ID NO:7)QQHYGTPLT (SEQ ID NO: 7)
重链CDR1Heavy chain CDR1	GFTFSDYYMT(SEQ ID NO:11)GFTFSDYYMT (SEQ ID NO: 11)
重链CDR2Heavy chain CDR2	SINYDGRNTY(SEQ ID NO:12)SINYDGRNTY (SEQ ID NO: 12)
重链CDR3Heavy chain CDR3	GYYYYGSSPNYFDY(SEQ ID NO:10)GYYYYGSSPNYFDY (SEQ ID NO: 10)

Table 5 Anti-human TIM-3 antibodies of the invention are defined by Chothia CDR sequences

抗体antibody	mab15CDRsmab15CDRs
轻链CDR1Light chain CDR1	RASENIYSYLT(SEQ ID NO:5)RASENIYSYLT (SEQ ID NO: 5)
轻链CDR2Light chain CDR2	NAKTLAE(SEQ ID NO:6)NAKTLAE (SEQ ID NO: 6)
轻链CDR3Light chain CDR3	QQHYGTPLT(SEQ ID NO:7)QQHYGTPLT (SEQ ID NO: 7)
重链CDR1Heavy chain CDR1	GFTFSDY(SEQ ID NO:13)GFTFSDY (SEQ ID NO: 13)
重链CDR2Heavy chain CDR2	NYDGRN(SEQ ID NO:14)NYDGRN (SEQ ID NO: 14)
重链CDR3Heavy chain CDR3	GYYYYGSSPNYFDY(SEQ ID NO:10)GYYYYGSSPNYFDY (SEQ ID NO: 10)

Table 6 Anti-human TIM-3 antibodies of the invention are defined by Contact CDR sequences

抗体antibody	mab15CDRsmab15CDRs
轻链CDR1Light chain CDR1	YSYLTWY(SEQ ID NO:15)YSYLTWY (SEQ ID NO: 15)
轻链CDR2Light chain CDR2	LLVYNAKTLA(SEQ ID NO:16)LLVYNAKTLA (SEQ ID NO: 16)
轻链CDR3Light chain CDR3	QQHYGTPL(SEQ ID NO:17)QQHYGTPL (SEQ ID NO: 17)
重链CDR1Heavy chain CDR1	SDYYMT(SEQ ID NO:18)SDYYMT (SEQ ID NO: 18)
重链CDR2Heavy chain CDR2	WVASINYDGRNTY(SEQ ID NO:19)WVASINYDGRNTY (SEQ ID NO: 19)
重链CDR3Heavy chain CDR3	ARGYYYYGSSPNYFD(SEQ ID NO:20)ARGYYYYGSSPNYFD (SEQ ID NO: 20)

In addition to the above CDR definition/annotation methods, the variable region CDRs of the light and heavy chains of the antibodies of the present invention are as follows, according to the CCG antibody CDR definition method in the art.

Table 2b Anti-human Tim-3 antibodies of the invention define CDR sequences by CCG

抗体antibody	mab15CDRsmab15CDRs
轻链CDR1Light chain CDR1	RASENIYSYLT(SEQ ID NO:5)RASENIYSYLT (SEQ ID NO: 5)

轻链CDR2Light chain CDR2	NAKTLAE(SEQ ID NO:6)NAKTLAE (SEQ ID NO: 6)
轻链CDR3Light chain CDR3	QQHYGTPLT(SEQ ID NO:7)QQHYGTPLT (SEQ ID NO: 7)
重链CDR1Heavy chain CDR1	GFTFSDYYMT(SEQ ID NO:11)GFTFSDYYMT (SEQ ID NO: 11)
重链CDR2Heavy chain CDR2	SINYDGRNTYYLDSLKS(SEQ ID NO:9)SINYDGRNTYYLDSLKS (SEQ ID NO: 9)
重链CDR3Heavy chain CDR3	GYYYYGSSPNYFDY(SEQ ID NO:10)GYYYYGSSPNYFDY (SEQ ID NO: 10)

Human antibody light chain constant region kappa chain:

Heavy chain constant region of human IgG4:

Heavy chain constant region of human IgG1:

Heavy chain constant region of human IgG2:

The antibody of the present invention can be combined with the above-mentioned light and heavy chain constant regions, and the chimeric antibody can be obtained by the method of expression and purification of Example 1. The chimeric antibody may be a chimeric antibody of human IgG4-kappa chain (hIgG4), and the present invention is represented by the IgG4-k. This chimeric antibody is hereinafter referred to as mab15-c.

The amino acid sequence of the mab15-c light chain of the present invention:

The amino acid sequence of the mab15-c heavy chain of the present invention:

The mab15-c antibody was cloned, expressed and purified as in Example 1. The binding activities of mab15-c and human TIM-3 were examined as described in Example 3, and the results are shown in Table 7 below and Figure 1.

Table 7 Anti-binding activity of antibody mab15-c and human TIM-3 of the present invention (ELISA)

抗体antibody	EC50(nM)EC50(nM)
Mab15-cMab15-c	0.1580.158
对照(Reference)#Comparison (Reference)#	0.3760.376

# The control (positive control, control molecule) antibody of the present invention uses ABTIM3, see patent WO2015117002A1

This result indicates that the ELISA-binding activity of the antibody mab15-c of the present invention is superior to that of the control antibody, which is more than 1 time (0.376/0.158 = 2.38) than the control antibody.

Example 7 Determination of affinity (KD) of antibody mab15-c and human TIM-3 of the present invention

The affinity of the antibodies of the invention and human TIM-3 was determined using a Biacore T200, GE Healthcare instrument. Protein A (Thermo Pierce, Cat #21181) was first coupled to the biosensing chip CM5 (Cat.) using pH 7.4 running buffer HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P20). #BR-1005-30, GE), the chip was activated with freshly prepared 50 mM NHS (N-hydroxysuccinimide) and 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and then injected at pH 4.0 10 mM. 10 ug/ml of Protein A formulated in NaAC. The anti-test concentration was 5 ug/ml, the antigen TIM-3 concentration gradient was 0 nM, 1.875 nM, 3.75 nM, 7.5 nM, 15 nM, and 30 nM, the flow rate was 30 ul/min, the binding time was 180 seconds, and the dissociation time was 300 seconds. After the experiment, the chip was washed with 10 mM Glycine-HCl, pH 1.5, 30 ul/min, 30 s. The experimental data were fitted with a 1:1 Langmuir model using Biacore T200 evaluation version 3.0 (GE) software to obtain an affinity value KD. The KD values of the mab15-c of the present invention are shown in Table 8.

Table 8 mab15-c Biacore Affinity

The above Biacore data indicates that the antibody of the present invention shows very good binding activity (both ELISA and Biacore are 1/10 nM grade), and surprisingly, its binding to specific antigen shows rapid binding (ka is 10 ⁵ ) and slow solution. Off (kd is 10 ^-4 ) characteristics. In particular, the kd is 10 ^{- 4} is 10 times slower than the usual 10 ^-3 . For example, another antibody (mabn) obtained in the screening process of the present invention, although its KD (0.78 nM) and the preferred antibody mab15-c KD (0.31 nM) of the present invention are in the same order of 1/10 nM, the dissociation speed is higher than mab15- c should be nearly an order of magnitude (kd is 10 ^-3 ). That is, the antibody and the human Tim-3 antigen rapidly bind and rapidly dissociate. Such antibodies are thus not preferred antibodies of the invention. These features of the preferred antibody mab15c of the present invention make this antibody molecule a great pharmaceutical advantage for therapeutic antibody drug development.

Example 8 Humanization of the antibody mab15 of the present invention

In the present invention, an anti-human TIM-3 antibody mab15 which binds to an unexpectedly preferable activity of the activity is obtained by immunizing a mouse, and the antibody is further humanized in this embodiment. Humanization is carried out as disclosed in many documents in the art.

Specifically, according to the antibody labeling system, the marker recognizes the CDR regions of the light and heavy chains of the antibody (see Example 6 above for details). The murine antibody sequence was compared with the human antibody germline database (v-base) to find a highly homologous human antibody light chain germline, including IGKV1D-39*01(F), IGKV1-12*01(F) , IGKV1-39*01(F), IGKV1-12*02(F), IGKV1-17*01(F), IGKV1-27*01(F), IGKV1-39*02(P), IGKV1-6* 01(F), IGKV1-NL1*01(F), IGKV1D-12*01(F), etc., preferably homology, and preferred frequency of germline, etc., preferably IGKV1-39*01(F) is a human source The germline light chain is used. The light chain J gene hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4, hJK3 and the like have high homology, and the sequence alignment is preferably hJK2.1. The human antibody germline having high homology with the heavy chain of the antibody of the present invention comprises IGHV3-7*01(F), IGHV3-7*02(F), IGHV3-7*03(F), IGHV3-48*01 ( F), IGHV3-48*02 (F), IGHV3-48*03 (F), IGHV3-21*01 (F), IGHV3-21*02 (F), IGHV3-21*03 (F), etc., preferably IGHV3-21*01(F). The antibody J gene of the present invention and the human antibody germline hJH1, hJH2, hJH3.1, hJH3.2, hJH4.1, hJH4.2, hJH4.3 and the like, preferably hJH4.1. The CDR region of the antibody mab15 of the present invention (see definition of the foregoing examples) was transplanted onto the selected humanized template and recombined with the IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the computer mimetic antibody, the residues which have an important interaction with the CDRs and the residues of the CDRs, and the residues which have an important influence on the conformation of VL and VH are subjected to back mutation and the CDR regions are Chemically labile amino acid residues are optimized to provide an optimized anti-TIM-3 humanized series of antibody molecules of the invention.

Humanized light chain variable region sequences of the invention:

L611:

L612:

L613:

L614:

L615:

L616

L617:

Humanized heavy chain variable region sequences of the invention:

H621:

H622:

H623:

H624:

There are different back mutations during humanization, and the back mutation can be 10 or more, preferably 0-10, such as the sequence of the light chain SEQ ID NO: 27-33. These arbitrary sequences are combined with a human antibody light chain constant region kappa chain or lambda chain, for example, with the light chain constant region sequence set forth in SEQ ID NO: 21 to give one of the light chain sequences of the antibody of the present invention. Similarly, the heavy chain used for humanization also has a different number of back mutations, and the number of back mutations can be 10 or more, preferably 0-10, as listed in SEQ ID NO: 34-37 of the heavy chain variable region sequence described above. of. These heavy chain variable region sequences containing different numbers of back mutations are recombined with the heavy chain constant region sequence selected from the above SEQ ID NOS: 22-24 to obtain one of the heavy chain sequences of the present invention. For example, some light and heavy chain sequences are listed, as shown in the following table.

Table 10 Humanized antibody sequences of the present invention (human kappa or lambda chain and hIgG4, hIgG1 as an example)

Partially humanized antibody sequence of the invention: humanized ab16 antibody amino acid sequence:

Light chain:

Heavy chain:

Humanized ab17 antibody amino acid sequence:

Light chain:

Heavy chain:

Humanized ab23 antibody amino acid sequence: light chain:

Heavy chain:

Humanized ab24 antibody amino acid sequence: light chain:

Heavy chain:

Humanized ab30 antibody amino acid sequence: light chain:

Heavy chain:

Humanized ab32 antibody amino acid sequence: light chain:

Heavy chain:

Humanized ab18 antibody amino acid sequence: light chain:

Heavy chain:

The recombinant antibody was cloned, expressed and purified by the method of Example 1, and the binding activity of these humanized antibodies and human TIM-3 was detected by the ELISA method described in Example 3, and the binding activity of these antibodies and hTim-3+ cells was examined. (Method of Example 2), and these antibodies activate the functional activity of human blood cell killer (NK) tumor cells (Example 4), and the results are shown in Table 11 below and Figure 2a (listing partial antibody and human Tim-3 antigen binding) Graph), Figure 2b (listed partial antibody and human Tim-3+ cell binding profile), and Figure 3 (antibody of the invention activates human blood cell killing tumor cell activity).

Table 11 Antibody and human Tim-3 ELISA binding activity of the present invention

本发明抗体Antibody of the invention	ELISA(nM)ELISA (nM)
mab15-cMab15-c	0.1830.183
ab16Ab16	0.1450.145
ab17Ab17	0.2120.212
ab23Ab23	0.2100.210
ab24Ab24	0.2430.243
ab30Ab30	0.2330.233
ab32Ab32	0.1840.184
ab18Ab18	0.1980.198

Table 11, the results of Figure 2a demonstrate that the humanized antibody has no loss of binding activity to human Tim-3 after humanization, and their EC50 is close to the murine antibody mab15-c.

In addition, the detection activity of the binding activity of the murine antibody of the present invention and the humanized antibody and human Tim-3+ cells is between 0.112 and 0.161 nM, and FIG. 2b lists the humanized, fully humanized antibody of the present invention. The binding activity of human Tim-3+ cells, wherein the ab16 has an EC50 of 0.151 nM; the ab32 has an EC50 of 0.112 nM.

In addition, the Biacore test results (method is the same as in Example 7), the kd of ab32 is 2.955E-4, and the characterization is the same as Table 8, which shows the characteristics of fast binding and slow dissociation.

In particular, the results in Figure 3 show that the fully humanized antibody ab32 of the present invention activates human blood cell killing of tumor cell antibody functional activity. In the experiment, 1 ug/ml, 10 ug/ml, 30 ug/ml of the control negative antibody (unrelated human IgG antibody) produced only a background value of 4.7% on average. The positive control antibody and ab32 activity values were subtracted from the background value of 4.7%, and the results are shown in Fig. 3. The results showed that the activity of the positive control antibody to activate human blood cells to kill tumor cells was 1.25%, 4.98%, and 7.9%, respectively, at 1 ug/ml, 10 ug/ml, and 30 ug/ml antibody concentration. The antibody ab32 of the present invention activated human blood cells to kill tumor cells. The activity was 3.08%, 13.1%, and 12.4%, respectively. That is to say, the activity of the antibody ab32 in the present invention was 2.5 times, 2.6 times and 1.6 times, respectively, of the positive control. Moreover, the activity of ab32-activated human blood cells to kill tumor cells has reached a peak at 10 ug/ml. The activity of the positive antibody was higher than 30 ug/ml.

These results indicate that the murine antibody mab15 and human Tim-3 found in the present invention have a good binding activity, and the binding speed is fast and the dissociation is slow. Suitable for the development of therapeutic antibody drugs, including for cancer therapy. After further humanization by the present invention, the humanized antibody obtained after optimization retains the binding activity of the murine antibody. In particular, the humanized antibody obtained by the present invention may have multiple back mutation sites, which are back-mutated into a murine antibody, and retain the same activity characteristics as the humanized antibody and mab15. Particularly unexpectedly, the humanized antibody (ab32, Table 11) is optimized as a fully humanized antibody, ie, the antibody is derived from the human antibody germline sequence except that the CDR sequence is derived from the murine antibody mab15 sequence, and The antibody retained the binding activity and binding characteristics of mab15, and the binding activity to human Tim-3 was better than that of the positive antibody (Table 7). It retains the characteristics of fast bonding speed and slow dissociation speed. More unexpectedly, the antibody activates human blood cells to kill tumor cell activity (NK kills tumor cell activity) more than twice as much as positive antibodies (10 ug/ml ab32 activity is even better than 30 ug/ml positive control activity). All of these unexpectedly significant advantages make the antibodies of the invention more suitable for the development of monoclonal antibody drugs for tumor therapy against the Tim3 target.

Example 9: Activity detection of anti-TIM-3 antibody of the present invention in mixed lymphocyte reaction (MLR assay)

The method of detecting INF-γ secretion by a mixed lymphocyte reaction (MLR assay) was used to evaluate the activity of the series of antibodies of the present invention on activating human blood cells. That is, the human blood cell PBMC isolated from the present invention (isolated from the peripheral blood of a healthy volunteer) was induced to obtain dendritic cells (DC), and T cells from different volunteers were stimulated.

Specifically, dendritic cells (DC) were cultured: on day 1 of the experiment, PBMC was inoculated into 6-well plates with 10% FBS RPMI 1640 medium, 2 ml per well, 2 x 10 ⁶ /ml, 37 ° C, 5%. After 2-4 hours of incubation in a CO2 incubator, the suspension cells were gently aspirated, and 2 ml of medium and 100 ng/ml GM-CSF (Peprotech, Cat#: 300-03) and 100 ng/ml IL-4 were added to the adherent cells ( Peprotech, Cat#: 200-04), after 2 days of incubation, 1 ml of fresh medium was added to each well. On day 5, 3 μl of 100 μg/ml TNF-α was added to each well to a final concentration of 100 ng/ml (TNF-α was purchased from Peprotech, Cat#: AF-300-01A), and culture was continued for 2 days to obtain dendrites. Cells (DC) were used in the following experiments.

DC stimulation of PBMC/T cells (MLR) assay: 10 ng/ml anti-CD3 antibody (Miltenyl Biotec, Cat#: 130-093-387), coated with 96-well cell culture plates at 100 ul/well, incubated at 37 °C 2 In the hour, wash it again with PBS. The DC cells on the 7th day of culture were collected, centrifuged, resuspended in 10% FBS RPMI 1640 medium, counted, formulated into 5× ^{10 4} cells/ml, and added to the above anti-CD3 coated 96 by 90 ul/well. - In the orifice plate. PBMC cells from different volunteers were taken, counted, and formulated into 5× ^{10 5} cells/ml, and added to the 96-well plate coated with the above-mentioned anti-CD3 and plated with DC cells at 90 ul/well. The sample to be tested was prepared in proportion to PBS, including a negative antibody, a control antibody, a PD-1 antibody (cloned according to the sequence disclosed by Opdivo, cloned according to Example 1 and purified by the expression), and added to the above 96-well plate at 20 ul/well. The concentration of the antibody to be tested in the 200 ul system is the desired concentration gradient. Control group = 90 ul PBMC cells + 90 ul DC + 20 ul PBS. After incubating the cell culture plate at 37 ° C for 6 days in a 5% CO ₂ incubator, the cell culture plate was taken out, centrifuged at 3000 rpm for 10 min, and 150 μl of the supernatant was taken out per well for detection of IFN-γ.

IFN-γ ELISA test, according to the kit (Shenzhen Xinbosheng Biotechnology Co., Ltd.; cat: EHC102g) instructions, the steps are as follows:

a. The extracted cell culture supernatant was diluted 25 times (different dilutions of different experiments) and added to the microplate (100 ul/well); the standard was diluted with the common dilution of the sample to a different concentration gradient: 1000 pg/ml 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, 100μl per well; blank wells plus standard dilutions.

b. Seal the reaction paper and seal the reaction well at 37 ° C for 90 minutes.

c. Wash the plate 5 times, each time for 3 minutes, add 100 μl biotin antibody working solution per well, add blank biotinylated antibody dilution solution, seal the reaction well with new sealing tape, and incubate for 60 minutes at 37 °C.

d. Wash the plate 5 times, each time for 3 minutes, add 100 μl enzyme-binding working solution to each well, add the enzyme to the blank well, and seal the reaction well with a new sealing tape. Incubate at 37 ° C for 30 minutes in the dark.

e. Wash the plate 5 times for 3 minutes each time, add 100 μl of chromogenic substrate TMB to each well, incubate at 36 ° C for 15 minutes in the dark.

f. Add stop solution, 100μl/well, mix and, within 3 minutes, read the OD450 by microplate reader.

g. Analysis of results: The IFN-γ value was calculated and evaluated as an increase percentage (%) in comparison with the blank control to evaluate the activity of the antibody of the present invention in the MLR assay. The results are shown in Table 12.

Table 12 Activity evaluation of anti-TIM-3 antibody of the present invention in mixed lymphocyte reaction (MLR assay)

待测样品/浓度Sample to be tested / concentration	0ug/ml0ug/ml	1ug/ml1ug/ml	5ug/ml5ug/ml
阴性+PD-1抗体Negative + PD-1 antibody	14.3％14.3%	15.8％15.8%	22.8％22.8%
ab6(对照)+PD-1抗体Ab6 (control) + PD-1 antibody	21.6％21.6%	28.6％28.6%	38.9％38.9%
ab32+PD-1抗体Ab32+PD-1 antibody	13.6％13.6%	18.9％18.9%	30.1％30.1%

The results in Table 12 indicate that the combination of negative antibody 0 ug/ml, 1 ug/ml, 5 ug/ml and PD-1 antibody (3 ug/ml) resulted in IFN-γ secreted by activated T cells between 14% and 23%. When antibody ab6 (as the control of this experiment, the antibody sequence sees patent application number: 2017103486994), or the ab32 antibody of the present invention 0 ug/ml, 1 ug/ml and PD-1 antibody (3 ug/ml), the IFN which activates T cell secretion is caused. - γ is between 13-29%, close to the level of negative antibody. However, when the ab6, ab32 antibody concentration was increased to 5 ug/ml, the IFN-γ secreted by T cells increased to 38.9% and 30.1%. That is, the antibody ab32 (5 ug/ml) and the PD-1 antibody (3 ug/ml) of the present invention can increase the secretion of IFN-γ by T cells, and the increase rate is (30.1-22.8)/22.8, that is, 32%.

Example 10 Binding activity of anti-TIM-3 antibody of the present invention and non-human Tim-3

The binding activity of the antibody of the present invention and non-human Tim-3 was examined by the method of Example 3. The non-human Tim-3 proteins used included macaque (Macaca) and marmoset Tim-3 proteins (see Table 1 for expression of each protein), and the results are shown in Figures 4a, 4b. The results showed that the antibody ab32 of the present invention and the macaque (Macaca) had weak binding, and the binding activity (EC50=11 nM) was nearly 100 times weaker than the positive control antibody (EC50=0.14 nM), and the antibody ab6 (see patent application number: 2017103486994) And the other antibody ab526 and macaque (Macaca) of the present invention have no binding at all, see Figure 4a. While ab32 and ab6 and marmoset Tim-3 protein have a good binding, and the binding activity is more than 4 times stronger than the positive control molecule (0.4nM), antibody ab526 and marmoset Tim-3 The protein is completely unbound, see Figure 4b. The binding activity of ab526 and human Tim-3 protein was very good, EC50=0.1 nM (experimental data of the present invention), close to the level of positive antibody and human Tim-3 binding.

These data indicate that the antibodies found in the present invention, including murine, humanized, fully humanized antibodies, and human Tim-3 have antigen binding sites that differ from both the positive control antibody and the ab6 antibody.

The data of the present invention shows that the antibody discovered by the invention has unique binding activity, fast binding and slow dissociation characteristics, humanized and fully humanized low immunogenicity, and activates the excellent activity of human blood cells to kill tumor cells. The PD-1 antibody alone and in combination can enhance PD-1 activated T cell (MLR) functional activity, etc., making it suitable for human Tim-3 target antibody drug development, and as an excellent drug candidate, tumors can be treated alone or in combination.

Claims

An antibody molecule or binding fragment thereof which specifically binds to a human T cell immunoglobulin domain and a mucin domain 3 (TIM-3), comprising at least one CDR region sequence selected from the group consisting of:

a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region ( VH) comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 8, a VHCDR2 amino acid sequence of SEQ ID NO: 9, and a VHCDR3 amino acid sequence of SEQ ID NO: 10;

b. VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 selected from SEQ ID NO: An amino acid sequence, the VHCDR2 amino acid sequence of SEQ ID NO: 12, and the VHCDR3 amino acid sequence of SEQ ID NO: 10;

c. VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 selected from SEQ ID NO: An amino acid sequence, the VHCDR2 amino acid sequence of SEQ ID NO: 14 and the VHCDR3 amino acid sequence of SEQ ID NO: 10;

d. VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 selected from SEQ ID NO: An amino acid sequence, the VHCDR2 amino acid sequence of SEQ ID NO: 9 and the VHCDR3 amino acid sequence of SEQ ID NO: 10;

e.VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 15, the VLCDR2 amino acid sequence of SEQ ID NO: 16 and the VLCDR3 amino acid sequence of SEQ ID NO: 17; and VH comprising a VHCDR1 selected from SEQ ID NO: Amino acid sequence, the VHCDR2 amino acid sequence of SEQ ID NO: 19, and the VHCDR3 amino acid sequence of SEQ ID NO: 20.
The TIM-3 antibody molecule of claim 1, or a binding fragment thereof, comprising at least one CDR region sequence selected from the group consisting of or a mutated sequence thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, SEQ ID a VLCDR2 amino acid sequence of NO: 6 and a VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 8, a VHCDR2 amino acid sequence of SEQ ID NO: 9, and SEQ ID NO: 10. The VHCDR3 amino acid sequence.
The TIM-3 antibody molecule of claim 1, or a binding fragment thereof, comprising at least one CDR region sequence selected from the group consisting of or a mutated sequence thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, SEQ ID a VLCDR2 amino acid sequence of NO: 6 and a VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 11, a VHCDR2 amino acid sequence of SEQ ID NO: 12, and SEQ ID NO: 10. The VHCDR3 amino acid sequence.
The TIM-3 antibody molecule of claim 1, or a binding fragment thereof, comprising at least one CDR region sequence selected from the group consisting of or a mutated sequence thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, SEQ ID a VLCDR2 amino acid sequence of NO: 6 and a VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 13, a VHCDR2 amino acid sequence of SEQ ID NO: 14, and SEQ ID NO: 10. The VHCDR3 amino acid sequence.
The TIM-3 antibody molecule of claim 1, or a binding fragment thereof, comprising at least one CDR region sequence selected from the group consisting of or a mutated sequence thereof, comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, SEQ ID a VLCDR2 amino acid sequence of NO: 6 and a VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 11, a VHCDR2 amino acid sequence of SEQ ID NO: 9, and SEQ ID NO: 10. The VHCDR3 amino acid sequence.
The TIM-3 antibody molecule of claim 1, or a binding fragment thereof, comprising at least one CDR region sequence selected from the group consisting of SEQ ID NO: 15 or a VLCDR2 amino acid sequence of NO: 16 and a VLCDR3 amino acid sequence of SEQ ID NO: 17; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 18, a VHCDR2 amino acid sequence of SEQ ID NO: 19, and SEQ ID NO: 20 The VHCDR3 amino acid sequence.
The TIM-3 antibody molecule or binding fragment thereof according to claims 1-6, wherein the TIM-3 antibody molecule or binding fragment thereof is a murine antibody molecule or a binding fragment thereof.
The TIM-3 antibody molecule of claim 7, or a binding fragment thereof, wherein the mouse antibody molecule or binding fragment thereof has a light chain variable region amino acid sequence of SEQ ID NO: 3 or a variant thereof.
The TIM-3 antibody molecule of claim 7, or a binding fragment thereof, wherein the murine antibody molecule or binding fragment thereof has a heavy chain variable region amino acid sequence which is a variant of SEQ ID NO: 4.
The TIM-3 antibody molecule of claim 7-9, or a binding fragment thereof, wherein the murine antibody light chain variable region amino acid sequence is SEQ ID NO: 3 or a variant thereof, and the heavy chain variable region amino acid The sequence is SEQ ID NO: 4 or a variant thereof.
The TIM-3 antibody molecule or binding fragment thereof according to any one of claims 1 to 6, wherein the TIM-3 antibody molecule or a binding fragment thereof is a variable region of a murine antibody molecule or a binding fragment thereof and a human antibody constant region, A chimeric antibody molecule comprising a human antibody heavy chain constant region IgG1, IgG2, IgG4, IgG4 and a light chain constant region kappa, lambda chain or the like, or a binding fragment thereof.
The TIM-3 antibody molecule of claim 11, or a binding fragment thereof, wherein the chimeric antibody molecule or binding fragment thereof has a light chain amino acid sequence of SEQ ID NO: 25 or a variant thereof.
The TIM-3 antibody molecule of claim 11, or a binding fragment thereof, wherein the chimeric antibody molecule or binding fragment thereof has a heavy chain amino acid sequence of SEQ ID NO: 26 or a variant thereof.
The TIM-3 antibody molecule of claim 11-12, or a binding fragment thereof, wherein the chimeric antibody molecule or binding fragment thereof has the light chain amino acid sequence of SEQ ID NO: 25 or a variant thereof, and the heavy chain amino acid sequence Is SEQ ID NO: 26 or a variant thereof.
The TIM-3 antibody molecule or binding fragment thereof according to claims 1-6, wherein the TIM-3 antibody molecule or binding fragment thereof is a humanized antibody molecule or a binding fragment thereof.
The TIM-3 antibody molecule of claim 15 or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof, the light chain variable region framework FR sequence is selected from the group consisting of a human germline light chain sequence, preferably IGKV1-39 *01. The FR sequence region preferably has a 0-10 amino acid back mutation.
The TIM-3 antibody molecule of claim 15-16, or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof has a light chain variable region sequence comprising a mutation selected from the group consisting of SEQ ID NO: 27-33 or a variant thereof Body sequence.
The TIM-3 antibody molecule of claim 15 or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof has a heavy chain variable region framework FR sequence selected from the group consisting of a human germline heavy chain sequence, preferably IGHV3-21 *01(F). The FR sequence region preferably has a 0-10 amino acid back mutation.
The TIM-3 antibody molecule or binding fragment thereof, according to claim 15, wherein the humanized antibody molecule or binding fragment thereof has a heavy chain variable region sequence comprising SEQ ID NO: 34-37 or a variant thereof. Body sequence.
The TIM-3 antibody molecule of claim 15-19, or a binding fragment thereof, wherein said humanized antibody comprises a light chain variable region selected from the group consisting of SEQ ID NO: 27-33 or a variant thereof A combination of heavy chain variable regions selected from the group consisting of SEQ ID NO: 34-37 or variants thereof.
The TIM-3 antibody molecule of claim 15-20, or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof light chain comprises a human kappa or lambda chain constant region or variant thereof.
The TIM-3 antibody molecule or binding fragment thereof, according to claim 15-21, wherein the humanized antibody molecule or binding fragment heavy chain thereof comprises a human antibody IgG1, IgG2, IgG4, IgG4 heavy chain constant region or Its variant.
The TIM-3 antibody molecule of claim 15-22, or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof has a light chain comprising SEQ ID NO: 38, SEQ ID NO: 40, or SEQ. ID NO: 43 or a full length light chain sequence with at least 85% sequence homology thereto.
The TIM-3 antibody molecule of claim 15-23, or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment heavy chain thereof comprises SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 42 or a full length heavy chain sequence with at least 85% sequence homology thereto.
The TIM-3 antibody molecule of claim 15-24, or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof is selected from the humanized light weight and claims of any one of claims 21, The humanized heavy chain combination of 22,24, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 38 and SEQ ID NO: 39; SEQ ID NO: 40 and SEQ ID NO: 39; SEQ ID NO: 38 and SEQ ID NO: 41; SEQ ID NO: 40 and SEQ ID NO: 41; SEQ ID NO: 38 and SEQ ID NO: 42; SEQ ID NO: 43 and SEQ ID NO: 42; Or SEQ ID NO: 43 and SEQ ID NO: 39.
The antibody molecule or binding fragment thereof according to claims 1-25, wherein the TIM-3 antibody molecule or binding fragment thereof comprises an antigen-binding fragment of a half antibody or a half antibody, preferably comprising Fab, F(ab")2 , Fv or single-chain Fv fragment (scFv).
The antibody molecule of claim 1-26, or a binding fragment thereof, wherein said TIM-3 antibody molecule or binding fragment thereof binds to a human blood cell PBMC antigen, enhancing the killing effect of PBMC on tumor cells; or wherein said TIM- 3 antibody molecules or binding fragments thereof can enhance or synergistically inhibit the growth of tumor cells by human blood T cells enhanced by PD-1 antibodies.
A DNA molecule encoding any of the antibody molecules of claim 1-27 or a binding fragment thereof.
An expression vector, which expresses any one of the DNA molecules of claim 28.
A method of producing an antibody comprising transforming a host cell, preferably a mammalian cell, more preferably a CHO cell, with any one of the antibody molecules of claim 1-27 or a binding fragment thereof, using an expression vector.
A pharmaceutical composition comprising the TIM-3 antibody molecule of any of claims 1-27, or a binding fragment thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer.
A method of treating cancer comprising administering to a subject in need thereof an antibody molecule of any of claims 1-27, or a binding fragment or pharmaceutical composition thereof, in an amount effective to treat cancer, preferably, the method comprising the present invention Any combination of the antibody molecules or binding fragments thereof, more preferably, the antibody molecule or binding fragment thereof of any of claims 1-27 and the antibody or antibody drug combination of PD-1.
The method for treating cancer according to claims 31-32, which comprises a solid tumor, a liquid tumor, preferably lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, bladder cancer, Metastatic lesions of esophageal cancer, neurological tumors, leukemia, etc. or cancer.