WO2023036041A1

WO2023036041A1 - Anti-4-1bb agonistic antibody and use thereof

Info

Publication number: WO2023036041A1
Application number: PCT/CN2022/116428
Authority: WO
Inventors: 孟祥雪; 史继亚; 介淼星; 任志衡; 李文佳
Original assignee: 广东东阳光药业有限公司
Priority date: 2021-09-09
Filing date: 2022-09-01
Publication date: 2023-03-16
Also published as: CN116253799B; CN116253799A

Abstract

An anti-4-1BB agonistic antibody and the use thereof, a humanized anti-4-1BB agonistic antibody or an antigen-binding portion thereof, and the use of the antibodies or antigen-binding portions thereof in the treatment of cancers.

Description

Agonistic antibody against 4-1BB and its application

technical field

The disclosure belongs to the field of biotechnology, and in particular relates to 4-1BB antibodies and applications thereof. More specifically, the present disclosure relates to murine antibodies capable of specifically recognizing human 4-1BB, humanized antibodies or antigen-binding fragments thereof, pharmaceutical compositions, detection kits and applications thereof.

Background technique

4-1BB (also known as CD137, TNFRSF9, etc.) is a member of the tumor necrosis factor receptor superfamily (TNFRS). 4-1BB is ubiquitously present in immune cell sublines such as activated natural killer (NK) and natural killer (NKT) cells, regulatory T cells, dendritic cells (DC), stimulated mast cells, differentiating In myeloid cells, monocytes, neutrophils and eosinophils (Wang, 2009, Immunological Reviews 229:192-215), it is involved in the regulation of cell proliferation, differentiation and programmed cell death.

Human 4-1BB is a protein with 255 amino acids. The protein contains a signal sequence at amino acid positions 1-17, followed by an extracellular domain of 169 amino acids, a transmembrane region of 27 amino acids, and an intracellular domain of 42 amino acids (Cheuk ATC et al., 2004, Cancer Gene Therapy, 11:215-226). 4-1BB is expressed on the cell surface as monomers and dimers, and readily trimerizes with ligands for signal transduction.

4-1BB has been reported to be a co-stimulatory receptor for activated T cells, and cross-linking of 4-1BB can enhance T cell proliferation, IL-2 secretion, survival, and cytolytic activity. 4-1BB can also induce proliferation of peripheral monocytes, enhance T cell apoptosis induced by TCR/CD3-triggered activation and regulated CD28 co-stimulation, thereby promoting Th1 cell responses. Its expression is induced by lymphocyte activation, and in addition to the 4-1BB ligand, the TNF receptor-associated factor (TRAF) adapter protein was found to bind to 4-1BB, resulting in the activation of NF-κB signaling. Specific agonistic antibodies against 4-1BB have been developed and found to enhance T cell function and promote antitumor activity.

Therefore, there remains a need to develop and improve specific agonistic antibodies against 4-1BB.

Contents of the invention

In order to solve one of the above-mentioned technical problems existing in the prior art, the present disclosure provides a therapeutically applicable and safe antibody specifically binding to 4-1BB or an antigen-binding portion thereof. Antibodies specific for 4-1BB of the present disclosure, or antigen-binding portions thereof, are agonistic antibodies against 4-1BB. Agonistic antibodies against 4-1BB show excellent antitumor effects. Accordingly, these agonistic antibodies, or antigen-binding portions thereof, are expected to be effective in the treatment of cancer or autoimmune diseases by modulating the immune response, for example, by modulating the activity of CD4 ⁺ cells, CD8 ⁺ cells, dendritic cells and/or natural killer cells .

To this end, the present disclosure relates to isolated antibodies, or antigen-binding portions thereof, that bind to 4-1BB and elicit cellular signaling mediated by 4-1BB.

According to one aspect of the present disclosure, there is provided an antibody or antigen-binding portion thereof that specifically binds 4-1BB or a fragment thereof, the antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region.

According to some embodiments of the present disclosure, the heavy chain variable region includes heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR1 includes amino acids selected from 1, 7, 13, 19, 25 or 31 sequence or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues, for example, an amino acid sequence in which 1 to 4 amino acid residues are replaced by different amino acid residues; the HCDR2 includes selected from such as SEQ ID NO : the amino acid sequence shown in 2, 8, 14, 20, 26 or 32 or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues, for example, wherein 1 to 10 amino acid residues are replaced by different amino acid residues The amino acid sequence of residue replacement; The HCDR3 comprises an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 3, 9, 15, 21, 27 or 33 or wherein one or more amino acid residues are substituted by different amino acid residues Amino acid sequence, for example, an amino acid sequence in which 1 to 2 amino acid residues are replaced by different amino acid residues.

According to some embodiments of the present disclosure, the light chain variable region includes light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 includes a sequence selected from the group consisting of SEQ ID NO: 4, 10, 16, 22, 28 or 34 The amino acid sequence shown or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues, for example, an amino acid sequence in which 1 to 10 amino acid residues are replaced by different amino acid residues; the LCDR2 includes selected From the amino acid sequence shown in SEQ ID NO: 5, 11, 17, 23, 29 or 35 or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues, for example, 1 to 6 amino acid residues thereof Amino acid sequence substituted by different amino acid residues; said LCDR3 comprises an amino acid sequence selected from amino acid sequences shown in SEQ ID NO: 6, 12, 18, 24, 30 or 36 or wherein one or more amino acid residues are substituted by different amino acids A residue-substituted amino acid sequence, for example, an amino acid sequence in which 1 to 8 amino acid residues are substituted with different amino acid residues.

According to some embodiments of the present disclosure, the antibody may be a bispecific antibody or a multispecific antibody. In some embodiments, an antibody or antigen binding portion of the disclosure is capable of binding a different epitope on 4-1BB, or binding an antigen or epitope other than 4-1BB.

According to some embodiments of the present disclosure, the heavy chain variable region comprises: comprising the amino acid sequence shown in SEQ ID NO: 1 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids HCDR1 of an amino acid sequence with residue substitution, including the amino acid sequence shown in SEQ ID NO: 2 or HCDR2 of an amino acid sequence in which one or more (for example, 1 to 10) amino acid residues are substituted by different amino acid residues , and HCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence in which one or more (for example, 1 to 2) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain variable region comprises: comprising the amino acid sequence shown in SEQ ID NO: 7 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids HCDR1 of an amino acid sequence substituted by residues, including an amino acid sequence as shown in SEQ ID NO: 8 or HCDR2 of an amino acid sequence in which one or more (for example, 1 to 10) amino acid residues are substituted by different amino acid residues , and HCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence in which one or more (for example, 1 to 2) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain variable region comprises: comprising the amino acid sequence shown in SEQ ID NO: 13 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids HCDR1 of an amino acid sequence substituted by residues, including the amino acid sequence shown in SEQ ID NO: 14 or HCDR2 of an amino acid sequence in which one or more (for example, 1 to 10) amino acid residues are substituted by different amino acid residues , and HCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence in which one or more (for example, 1 to 2) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain variable region comprises: comprising the amino acid sequence shown in SEQ ID NO: 19 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids HCDR1 of an amino acid sequence substituted by residues, including an amino acid sequence as shown in SEQ ID NO: 20 or HCDR2 of an amino acid sequence in which one or more (for example, 1 to 10) amino acid residues are substituted by different amino acid residues , and HCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence in which one or more (for example, 1 to 2) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain variable region comprises: comprising the amino acid sequence shown in SEQ ID NO: 25 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids HCDR1 of an amino acid sequence substituted by residues, including an amino acid sequence as shown in SEQ ID NO: 26 or HCDR2 of an amino acid sequence in which one or more (for example, 1 to 10) amino acid residues are substituted by different amino acid residues , and HCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence in which one or more (for example, 1 to 2) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain variable region comprises: comprising the amino acid sequence shown in SEQ ID NO: 31 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids HCDR1 of an amino acid sequence substituted by residues, including an amino acid sequence as shown in SEQ ID NO: 32 or HCDR2 of an amino acid sequence in which one or more (for example, 1 to 10) amino acid residues are substituted by different amino acid residues , and HCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence in which one or more (for example, 1 to 2) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the light chain variable region includes: comprising the amino acid sequence shown in SEQ ID NO: 4 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids LCDR1 of an amino acid sequence substituted by residues, including an amino acid sequence as shown in SEQ ID NO: 5 or LCDR2 of an amino acid sequence in which one or more (for example, 1 to 6) amino acid residues are substituted by different amino acid residues , comprising an amino acid sequence as shown in SEQ ID NO: 6 or an LCDR3 of an amino acid sequence in which one or more (for example, 1 to 8) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the light chain variable region includes: comprising the amino acid sequence shown in SEQ ID NO: 10 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids LCDR1 of an amino acid sequence substituted by residues, including LCDR2 of an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence in which one or more (for example, 1 to 6) amino acid residues are substituted by different amino acid residues , comprising the amino acid sequence shown in SEQ ID NO: 12 or the LCDR3 of an amino acid sequence in which one or more (for example, 1 to 8) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the light chain variable region includes: comprising the amino acid sequence shown in SEQ ID NO: 16 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids LCDR1 of an amino acid sequence substituted by residues, including an amino acid sequence as shown in SEQ ID NO: 17 or LCDR2 of an amino acid sequence wherein one or more (for example, 1 to 6) amino acid residues are substituted by different amino acid residues , comprising the amino acid sequence shown in SEQ ID NO: 18 or the LCDR3 of an amino acid sequence in which one or more (for example, 1 to 8) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the light chain variable region includes: comprising the amino acid sequence shown in SEQ ID NO: 22 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids LCDR1 of an amino acid sequence substituted by residues, including the amino acid sequence shown in SEQ ID NO: 23 or LCDR2 of an amino acid sequence in which one or more (for example, 1 to 6) amino acid residues are substituted by different amino acid residues , comprising an amino acid sequence as shown in SEQ ID NO: 24 or an LCDR3 of an amino acid sequence in which one or more (for example, 1 to 8) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the light chain variable region includes: comprising the amino acid sequence shown in SEQ ID NO: 28 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids LCDR1 of an amino acid sequence substituted by residues, including an amino acid sequence as shown in SEQ ID NO: 29 or LCDR2 of an amino acid sequence in which one or more (for example, 1 to 6) amino acid residues are substituted by different amino acid residues , comprising an amino acid sequence as shown in SEQ ID NO: 30 or an LCDR3 of an amino acid sequence in which one or more (for example, 1 to 8) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the light chain variable region includes: comprising the amino acid sequence shown in SEQ ID NO: 34 or wherein one or more (for example, 1 to 4) amino acid residues are different amino acids LCDR1 of an amino acid sequence substituted by residues, including LCDR2 of an amino acid sequence as shown in SEQ ID NO: 35 or an amino acid sequence in which one or more (for example, 1 to 6) amino acid residues are substituted by different amino acid residues , comprising an amino acid sequence as shown in SEQ ID NO: 36 or an LCDR3 of an amino acid sequence in which one or more (for example, 1 to 8) amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 37, 39, 41, 43, 45 or 47 or an amino acid wherein one or more amino acid residues are different Amino acid sequence of residue substitutions.

According to some embodiments of the present disclosure, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 38, 40, 42, 44, 46 or 48 or an amino acid wherein one or more amino acid residues are different Amino acid sequence of residue substitutions.

According to some embodiments of the present disclosure, the heavy chain of the above-mentioned antibody or its antigen-binding portion comprising the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues may be Variable region, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 38 or wherein one or more amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain of the above-mentioned antibody or its antigen-binding portion comprising the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues may be Variable region, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 40 or wherein one or more amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain of the above-mentioned antibody or its antigen-binding portion comprising the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues may be Variable region, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 42 or wherein one or more amino acid residues are replaced by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain of the above-mentioned antibody or its antigen-binding portion comprising the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues may be Variable region, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 44 or wherein one or more amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain of the above-mentioned antibody or its antigen-binding portion comprising the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues may be Variable region, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 46 or wherein one or more amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the heavy chain of the above-mentioned antibody or its antigen-binding portion comprising the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues may be Variable region, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 48 or wherein one or more amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the above-mentioned antibody comprises an amino acid sequence as shown in SEQ ID NO: 49 or a heavy chain of an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, and such as SEQ ID NO : The light chain of the amino acid sequence shown in 50 or the amino acid sequence wherein one or more amino acid residues are replaced by different amino acid residues.

According to some embodiments of the present disclosure, the above-mentioned antibody comprises an amino acid sequence as shown in SEQ ID NO: 51 or a heavy chain of an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, and such as SEQ ID NO : light chain of the amino acid sequence shown in 52 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the above-mentioned antibody comprises an amino acid sequence as shown in SEQ ID NO: 53 or a heavy chain of an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, and the amino acid sequence as shown in SEQ ID NO: The light chain of the amino acid sequence shown in :54 or the amino acid sequence wherein one or more amino acid residues are replaced by different amino acid residues.

According to some embodiments of the present disclosure, the above-mentioned antibody comprises an amino acid sequence as shown in SEQ ID NO: 55 or a heavy chain of an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, and the amino acid sequence as shown in SEQ ID NO: The light chain of the amino acid sequence shown in :56 or the amino acid sequence wherein one or more amino acid residues are replaced by different amino acid residues.

According to some embodiments of the present disclosure, the above-mentioned antibody comprises an amino acid sequence as shown in SEQ ID NO: 57 or a heavy chain of an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, and the amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in :58 or the light chain of the amino acid sequence wherein one or more amino acid residues are substituted by different amino acid residues.

According to some embodiments of the present disclosure, the above-mentioned antibody comprises an amino acid sequence as shown in SEQ ID NO: 59 or a heavy chain of an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, and the amino acid sequence as shown in SEQ ID NO: : light chain of the amino acid sequence shown in 60 or an amino acid sequence wherein one or more amino acid residues are substituted by different amino acid residues.

According to another aspect of the present disclosure, there is provided a humanized antibody or antigen-binding portion thereof derived from the above-mentioned antibody or antigen-binding portion thereof.

According to some embodiments of the present disclosure, the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region.

According to some embodiments of the present disclosure, the heavy chain variable region includes heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3; the HCDR1 has an amino acid sequence represented by SYWX ₁ X ₂ , wherein X ₁ is selected from isoleucine acid, methionine or valine, X ₂ is selected from asparagine, histidine or threonine; the HCDR2 has an amino acid sequence shown by X ₃ IYPSDTYTX ₄ YX ₅ QKFQX ₆ , wherein X ₃ is selected from From asparagine or isoleucine, _X4 is selected from asparagine, aspartic acid or serine, _X5 is selected from asparagine or alanine, _X6 is selected from aspartic acid or glycine; HCDR3 has the amino acid sequence shown in SEQ ID NO: 71 or an amino acid sequence in which one or more amino acid residues are replaced with different amino acid residues.

According to some embodiments of the present disclosure, the HCDR1 has the amino acid sequence shown in SEQ ID NO: 7, 61, 62 or 63; the HCDR2 has the amino acid sequence shown in SEQ ID NO: 64, 65, 66, 67, 68, 69 Or the amino acid sequence shown in 70; The HCDR3 has the amino acid sequence shown in SEQ ID NO:71.

According to some embodiments of the present disclosure, the above-mentioned heavy chain variable region also includes a framework region (FR); the framework region (H-FR) of the heavy chain variable region may include, comprising SEQ ID NO: 79 or 80 shown Amino acid sequence or H-FR1 of an amino acid sequence in which one or more (for example, 1 to 6) amino acid sequences are substituted, comprising the amino acid sequence shown in any one of SEQ ID NO: 81 to 84 or one or more of them H-FR2 of an amino acid sequence substituted by amino acid sequences (for example, 1 to 5), including the amino acid sequence shown in any one of SEQ ID NO: 85 to 89 or one or more of them (for example, 1 to 12 A) H-FR3 of an amino acid sequence whose amino acid sequence is substituted, and an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 90 or 91 or one or more (for example, 1 to 3) amino acid sequences are substituted The H-FR4.

According to some embodiments of the present disclosure, the heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO: 132-138 or an amino acid sequence in which one or more amino acid sequences are substituted.

According to some embodiments of the present disclosure, the light chain variable region includes light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; the LCDR1 has an amino acid sequence as shown in SEQ ID NO: 10 or one or more thereof (for example , 1 to 10) amino acid residues are replaced by different amino acid residues; the LCDR2 has an amino acid sequence as shown in SEQ ID NO: 11 or one or more (for example, 1 to 6) amino acids Amino acid sequence in which residues are replaced by different amino acid residues; the LCDR3 has the amino acid sequence shown in SEQ ID NO: 12 or one or more (for example, 1 to 8) amino acid residues are replaced by different amino acid residues Substituted amino acid sequences.

According to some embodiments of the present disclosure, the above-mentioned light chain variable region also includes a framework region (FR); the framework region (L-FR) of the light chain variable region may include, comprising SEQ ID NO: 92 or 93 shown in Amino acid sequence or L-FR1 in which one or more (for example, 1 to 5) amino acid sequences are substituted amino acid sequences; containing the amino acid sequence shown in any one of SEQ ID NO: 94 to 98 or one or L-FR2 of an amino acid sequence in which multiple (for example, 1 to 5) amino acid sequences are substituted; containing the amino acid sequence shown in any one of SEQ ID NO: 99 to 102 or one or more of them (for example, 1 to 5) 7) the L-FR3 of the amino acid sequence whose amino acid sequence is substituted; and the amino acid sequence containing the amino acid sequence shown in SEQ ID NO: 103 or 104 or one or more (for example, 1 to 3) amino acid sequences are substituted Sequence of L-FR4.

According to some embodiments of the present disclosure, the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO: 139-144 or an amino acid sequence in which one or more amino acid sequences are substituted.

According to some embodiments of the present disclosure, the antibody or antigen-binding portion thereof is a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 132-138, and having a sequence as shown in SEQ ID NO: 139 Any combination of the light chain variable regions of the amino acid sequences shown in any one of ~144.

According to some embodiments of the present disclosure, the antibody or antigen-binding portion thereof is a murine antibody, a human antibody, a primate antibody, preferably a murine antibody or a human antibody. According to some embodiments of the present disclosure, the humanized antibody comprises an amino acid sequence as shown in SEQ ID NO: 145 or a heavy chain constant region of an amino acid sequence in which one or more amino acids are substituted, and the amino acid sequence as shown in SEQ ID NO: 146 The light chain constant region of the amino acid sequence shown or an amino acid sequence in which one or more amino acids are substituted.

According to yet another aspect of the present disclosure, there is provided an antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region. According to some embodiments of the present disclosure, the heavy chain variable region includes heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3; the HCDR1 has the amino acid sequence shown by SYDIS or one or more of them (for example, 1 to 4 each) an amino acid sequence in which the amino acid residues are replaced by different amino acid residues; the HCDR2 has the amino acid sequence shown by VIWTGZ ₁ GTNYZ ₂ Z ₃ Z ₄ FZ ₅ S, wherein Z ₁ is selected from glycine or serine, and Z ₂ is selected from From asparagine or alanine, _Z3 is selected from serine or proline, _Z4 is selected from threonine or proline, _Z5 is selected from methionine or lysine; the HCDR3 has VDY The amino acid sequence shown or an amino acid sequence in which one or more (for example, 1 to 2) amino acid residues are replaced by different amino acid residues.

According to some embodiments of the present disclosure, the HCDR1 has an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence in which one or more amino acid sequences are substituted; the HCDR2 has an amino acid sequence such as SEQ ID NO: 72, 73 or The amino acid sequence shown in 74, or the amino acid sequence in which one or more amino acid sequences are substituted; the HCDR3 has the amino acid sequence shown in SEQ ID NO: 15, or the amino acid sequence in which one or more amino acid sequences are substituted .

According to some embodiments of the present disclosure, the heavy chain variable region further includes a framework region (FR); the framework region (H-FR) of the heavy chain variable region includes: any one of SEQ ID NO: 105-108 H-FR1 of the amino acid sequence shown or an amino acid sequence in which one or more amino acid sequences are substituted; H-FR1 containing the amino acid sequence shown in SEQ ID NO: 109 or 110 or an amino acid sequence in which one or more amino acid sequences are substituted H-FR2; H-FR3 containing the amino acid sequence shown in any one of SEQ ID NO: 111 to 116 or an amino acid sequence in which one or more amino acid sequences are substituted; and containing SEQ ID NO: 117 or 118 The amino acid sequence of or the H-FR4 of the amino acid sequence in which one or more amino acid sequences are substituted.

According to some embodiments of the present disclosure, the heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO: 147-152 or an amino acid sequence in which one or more amino acid sequences are substituted.

According to some embodiments of the present disclosure, the light chain variable region includes light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; the LCDR1 has the amino acid sequence shown by RSSQZ ₆ LVHSNTNTYLH, wherein Z ₆ is selected from serine or threonine acid; the LCDR2 has an amino acid sequence as shown in SEQ ID NO:77 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues; the LCDR3 has an amino acid sequence shown by SQZ ₇ THVPWT , wherein Z ₇ is selected from serine or threonine. According to some embodiments of the present disclosure, the LCDR1 has an amino acid sequence as shown in SEQ ID NO: 75 or 76, wherein one or more amino acid sequences are substituted amino acid sequences; the LCDR2 has an amino acid sequence as shown in SEQ ID NO: 77 The amino acid sequence shown, wherein one or more amino acid sequences are substituted amino acid sequences; the LCDR3 has an amino acid sequence as shown in SEQ ID NO: 18 or 78, wherein one or more amino acid sequences are substituted amino acid sequences.

According to some embodiments of the present disclosure, the above-mentioned light chain variable region also includes the following framework region (FR); the framework region (L-FR) of the light chain variable region includes any of SEQ ID NO: 119-122 The amino acid sequence shown in item or the L-FR1 of the amino acid sequence wherein one or more amino acid sequences are replaced; containing the amino acid sequence shown in any one of SEQ ID NO:123～126 or wherein one or more amino acid sequences are replaced L-FR2 containing a substituted amino acid sequence; L-FR3 containing an amino acid sequence shown in any one of SEQ ID NO: 127 to 129 or an amino acid sequence wherein one or more amino acid sequences are substituted; and containing SEQ ID NO: L-FR4 of the amino acid sequence represented by 130 or 131 or an amino acid sequence in which one or more amino acid sequences are substituted.

According to some embodiments of the present disclosure, the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO: 153-157 or an amino acid sequence in which one or more amino acid sequences are substituted.

According to some embodiments of the present disclosure, the antibody or its antigen-binding portion is a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 147-152, and having a sequence as shown in SEQ ID NO: 153 Any combination of the light chain variable regions of the amino acid sequences shown in any one of ~157.

According to some embodiments of the present disclosure, the heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NO:147～152, and the amino acid sequence shown in any one of SEQ ID NO:153～157 The light chain variable region is a humanized variable region sequence.

According to some embodiments of the present disclosure, the disclosed antibody or antigen-binding portion thereof is a murine antibody, a human antibody, a primate antibody, preferably a murine antibody or a human antibody. According to some embodiments of the present disclosure, the humanized antibody comprises an amino acid sequence as shown in SEQ ID NO: 158 or a heavy chain constant region of an amino acid sequence in which one or more amino acids are substituted, and the amino acid sequence as shown in SEQ ID NO: 159 The light chain constant region of the amino acid sequence shown or an amino acid sequence in which one or more amino acids are substituted.

According to some embodiments of the present disclosure, the disclosed antibody or antigen-binding portion thereof cross-reacts with at least one 4-1BB from the group consisting of cynomolgus monkey, mouse, rat, dog and/or human.

According to some embodiments of the present disclosure, the disclosed antibodies are selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE antibodies, preferably IgG1 or IgG4 antibodies.

According to yet another aspect of the present disclosure, there is provided a nucleic acid molecule encoding the above-mentioned antibody of the present disclosure or an antigen-binding portion thereof.

According to yet another aspect of the present disclosure, there is provided an expression vector, which includes the above-mentioned nucleic acid molecule.

According to yet another aspect of the present disclosure, a cell transfected with the above-mentioned expression vector of the present disclosure is provided.

According to yet another aspect of the present disclosure, a pharmaceutical composition is provided, the pharmaceutical composition comprising the above-mentioned antibody or its antigen-binding portion as disclosed herein, the above-mentioned nucleic acid molecule as disclosed herein, the above-mentioned expression vector as disclosed herein or The above cells as disclosed in the present disclosure, and a pharmaceutically acceptable carrier.

According to yet another aspect of the present disclosure, there is provided a method for inducing or enhancing cytokine production of immune cells in a subject, the method comprising administering to a subject in need an effective amount of the above-mentioned An antibody or an antigen-binding portion thereof, or the above-mentioned pharmaceutical composition of the present disclosure.

According to some embodiments of the present disclosure, the cytokine is IL-2, TNF-α, IL-13, IFN-γ or a combination thereof. The cytokines are produced in the tumor microenvironment.

According to yet another aspect of the present disclosure, there is provided a method of treating cancer, the method comprising administering to a subject in need an effective amount of the above-mentioned antibody of the present disclosure or an antigen-binding portion thereof, or the above-mentioned antibody of the present disclosure. pharmaceutical composition.

According to some embodiments of the present disclosure, the cancer is selected from melanoma, glioma, kidney cancer, breast cancer, blood cancer, and head and neck cancer. According to some embodiments of the present disclosure, the cancer is hematological cancer, such as B-cell lymphoma.

According to yet another aspect of the present disclosure, there is provided the above-mentioned antibody or its antigen-binding portion of the present disclosure, the above-mentioned nucleic acid molecule of the present disclosure, the above-mentioned expression vector of the present disclosure, the above-mentioned cells of the present disclosure, or the above-mentioned pharmaceutical composition of the present disclosure, in Application in the preparation of medicines for treating cancer.

According to some embodiments of the present disclosure, the cancer is selected from the group consisting of melanoma, glioma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, ovarian cancer, lung cancer, renal cell carcinoma, Nasopharyngeal cancer, kidney cancer, breast cancer, blood cancer and head and neck cancer.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present disclosure will become apparent from the following figures and detailed description of several embodiments, and also from the appended claims.

Description of drawings

In order to make the content of the present disclosure easier to understand, the present disclosure will be further described in detail below according to specific embodiments of the present disclosure and in conjunction with the accompanying drawings, wherein

Figure 1 shows the binding of antibodies according to some embodiments of the present disclosure to a CHO cell line highly expressing human 4-1BB.

Figure 2 shows the binding of antibodies according to some embodiments of the present disclosure to monkey-derived 4-1BB recombinant protein.

Figure 3 shows the binding of antibodies to activated T cells according to some embodiments of the present disclosure.

Figure 4 shows a T cell secretion IL2 indicator assay for agonist activity of antibodies according to some embodiments of the present disclosure.

FIG. 5 shows an indicator of T cell secretion of IFN-γ to measure the agonist activity of antibodies according to some embodiments of the present disclosure.

FIG. 6 shows the binding ability of humanized antibodies according to some embodiments of the present disclosure to a CHO cell line highly expressing human 4-1BB.

Figure 7 shows the anti-tumor activity of humanized antibodies according to some embodiments of the present disclosure.

Figure 8 shows the antitumor activity of humanized antibody HEC512119.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below in conjunction with embodiments. The specific embodiments described here are only used to explain the present disclosure, and are not intended to constitute any limitation to the present disclosure. Also, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concepts of the present disclosure.

definition

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the meaning commonly understood by one of ordinary skill in the art. It should be noted that the terms used herein should be interpreted to have a meaning consistent with the context of this specification, and not be interpreted in an idealized or overly rigid manner.

The expression "about" as used herein is understood by those of ordinary skill in the art, and varies within a certain range depending on the context in which it is used. If the use of this term is not understood by one of ordinary skill in the art depending on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

Numerical ranges as used herein, such as 1-10, are intended to encompass all values within the range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, carboxyl, amino and R groups, such as homoserine, norleucine, methionine sulfoxide, Methylsulfonium methionine. These analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics are compounds that differ structurally from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. As used herein, "polar amino acid" refers to an amino acid comprising a side chain that prefers to reside in an aqueous environment. In some embodiments, the polar amino acid is selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, and tyrosine. Polar amino acids can be positively charged, negatively charged, or neutrally charged. As used herein, "non-polar amino acid" is selected from the group consisting of alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan acid and valine.

"Substitution with one or more different amino acids" as used herein means that at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) is replaced by another different "replacement" amino acid residue . "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While inserts typically consist of insertions of 1 or 2 amino acid residues, larger "peptide insertions" can also be made, for example insertions of about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues may be naturally occurring or non-naturally occurring. "Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

Antibodies suitable for use in the present disclosure may comprise conservative amino acid substitutions at one or more amino acid residues, eg, at essential or non-essential amino acid residues. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (such as alanine, valine , leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, an essential or nonessential amino acid residue in an antibody is preferably replaced with another amino acid residue from the same side chain family. In certain embodiments, amino acid stretches may be replaced with structurally similar stretches that differ in the order and/or composition of side chain family members. Alternatively, in certain embodiments, mutations may be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be incorporated into the binding polypeptides of the invention and directed against these binding polypeptides The ability to bind to the desired target is screened.

As used herein, the term "anti-4-1BB agonistic antibody" or "specific agonistic antibody against 4-1BB" means that it specifically binds to 4-1BB and partially or completely promotes, induces, increases and/or activates Antibodies to 4-1BB biological activity, responses and/or downstream pathways mediated by 4-1BB signaling or other 4-1BB mediated functions. Tumor necrosis factor receptor (TNFR) signaling, particularly TNFRSF activation, requires receptor clustering and multimerization. 4-1BB is one of the TNFSF receptors known to require clustering to trigger downstream signaling. Formation of 2 or more trimers by cross-linking of 4-1BB has been reported to result in a stronger activated protein. In some embodiments, the anti-4-1BB agonistic antibody binds 4-1BB and induces multimerization of 4-1BB into 2 or more trimers.

The terms "specifically bind", "selectively bind", "selectively bind" and "specifically bind" as used herein refer to the binding of an antibody or antigen-binding portion thereof to an epitope on a predetermined antigen. Typically, when assayed by surface plasmon resonance (SPR) technique in a BIACORE 2000 instrument using recombinant human 4-1BB as the analyte and the antibody as the ligand, the antibody is present at about less than 10 ⁻⁶ M, such as about less than 10 ^-7 M, 10 ⁻⁸ M, 10 ⁻⁹ M or 10 ⁻¹⁰ M or even lower equilibrium dissociation constant (K _D ) binding, and binding to the predetermined antigen affinity is binding to other than the predetermined antigen or closely related At least twice the affinity of non-specific antigens (such as BSA, casein) outside the antigen.

The term "subject" as used herein includes any human or non-human animal. For example, the methods and compositions of the invention can be used to treat a subject with cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

As used herein, the terms "antibody fragment", "antigen-binding fragment", "antigen-binding portion" or similar terms refer to fragments of an antibody that retain binding to a target antigen (eg, 4-1BB) and have the same activity as the full-length antibody. Such fragments include, for example, single chain antibodies, single chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments or F(ab')2 fragments. A scFv fragment is a single polypeptide chain that includes the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, tribodies and diabodies are included within the definition of antibody and are suitable for use in the methods described herein. See eg Todorovska et al. (2001), J. Immunol. Methods, 248(1): 47-66; Hudson and Kortt, (1999), J. Immunol. Methods, 231(1): 177-189; Poljak, ( 1994), Structure, 2(12):1121-1123; Rondon and Marasco, (1997), Annu.Rev.Microbiol., 51:257-283, the respective disclosures of which are incorporated herein by reference in their entirety middle.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), where consideration is given to optimally aligning the two sequences The number of slots to introduce for alignment and the length of each slot. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and

length weight

1, 2, 3, 4, 5 or 6 determination. The percent identity between two nucleotide or amino acid sequences can also be calculated using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), using the PAM120 weight residue table, gap length penalty 12 and a gap penalty of 4, the algorithm has been incorporated into the ALIGN program (version 2.0). Alternatively, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970) ), using the Blossum 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and

length weights

1, 2, 3, 4, 5, or 6 determined, the algorithm has been incorporated into the GCG software package (available from http://www.gcg.com ) in the GAP program.

The term "tumor microenvironment" as used herein refers to the cellular environment or microenvironment in which tumors or neoplasms reside, including surrounding blood vessels and non-cancerous cells, including but not limited to immune cells, fibroblasts, myeloid-derived inflammatory cells and lymphocytes. Tumors and the surrounding microenvironment are closely related and constantly interact. Tumors can affect the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, and immune cells in the microenvironment can affect the growth and evolution of tumor cells.

Human 4-1BB is a transmembrane polypeptide with 255 amino acids (Accession No. NM_001561; NP_001552), which is a member of the phylogenetically conserved tumor necrosis factor receptor (TNFR) superfamily. 4-1BB and its ligands are involved in the regulation of many immune activities. The 4-1BB ligand crosslinks its receptor 4-1BB expressed on activated T cells and co-stimulates T cell activity. 4-1BB is an activation-induced co-stimulatory molecule. Recent studies have shown that the anticancer effect mediated by 4-1BB is mainly based on its ability to activate T cells, especially induce cytotoxic T lymphocyte (CTL) responses, and induce cytokine production, especially the induction of large amounts of IFNγ Ability.

The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "variable" as used herein means that certain segments of the variable domains generally differ in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β-sheet configuration, connected by three HVRs that form loops connecting and in some cases forming part of the β-sheet structure. The HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.

As used herein, the term "hypervariable region" or "HVR" refers to the region of an antibody variable domain region that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, native four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)", which have the highest sequence variability and/or are involved in antigen recognition. Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L26-L32(L1), L50-L52(L2), L91-L96(L3), H26-H32( H1), H52-H56 (H2) and H96-H101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) are located at amino acid residues L24-L34(L1), L50-L56(L2), L89-L97(L3), H31-H35( H1), H50-H65 (H2) and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). based on

Defining the rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L27-L32(L1), L50-L51(L2), L89-L97(L3), H26-H33(H1) , H51-H56(H2) and H93-H102(H3) (Honjo, T. and Alt, FW (1995) Immunoglobulin genes. Academic Press pp. 3-443). For comparison, in Table 1 are listed the corresponding amino acid residues comprising the CDRs defined in the references cited above. However, it is well known to those skilled in the art that the CDR of an antibody can be defined in various ways, such as the Kabat definition rule based on sequence variability, the Chothia definition rule based on the position of the structural loop region, and antibody humanization based on CDR grafting A reference tool for design (see J Mol Biol 273:927-48, 1997). It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing the above-mentioned existing ones as described by the present invention. Complementarity-determining regions defined in any one of the known schemes. Although the scope of protection claimed in the present disclosure is based on the sequence shown by the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.

Table 1. Amino acid numbering system for each CDR region (see http://bioinf.org.uk/abs/)

CDR\编号体系CDR\numbering system	KabatKabat	ChothiaChothia	IMGTIMGT
LCDR1LCDR1	24-3424-34	26-3226-32	27-3227-32
LCDR2LCDR2	50-5650-56	50-5250-52	50-5150-51
LCDR3LCDR3	89-9789-97	91-9691-96	89-9789-97

HCDR1HCDR1	31-3531-35	26-3226-32	26-3326-33
HCDR2HCDR2	50-6550-65	52-5652-56	51-5651-56
HCDR3HCDR3	95-10295-102	96-10196-101	93-10293-102

"Framework" or "FR" as used herein refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The "class" of an antibody as used herein refers to the type of constant domain or constant region that the heavy chain of the antibody has. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

The "humanized antibody" used herein comprises the amino acid residues from the non-human hypervariable region (HVR) and the constant regions of the heavy chain and light chain from the human antibody spliced to obtain the complete sequence of the humanized antibody. In certain embodiments, a humanized antibody comprises at least one, usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs Corresponds to FRs of human antibodies. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has been humanized.

As used herein, "cross-reactivity" refers to the ability of an antibody of the present disclosure to bind 4-1BB from a different species. For example, an antibody of the disclosure may bind human 4-1BB while also binding 4-1BB of another species (eg, mouse, rat, cynomolgus monkey, or dog). As used herein, cross-reactivity is measured by detecting specific reactivity with purified antigen in a binding assay (eg, SPR, ELISA), or with physiologically expressed cells that bind or otherwise functionally interact.

As used herein, "at least 95% sequence identity" with a sequence compared to that sequence means at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence.

The "expression vector" and "expression construct" used herein can be used interchangeably. When the above-mentioned isolated nucleic acid molecule is connected to the carrier, the nucleic acid sequence can be directly or indirectly connected to the regulatory elements on the carrier, as long as these regulatory elements It is sufficient that the element can regulate the translation and expression of the nucleic acid molecule. Of course, these regulatory elements may come directly from the vector itself, or be exogenous, that is, not from the vector itself. That is, a nucleic acid molecule is operably linked to a regulatory element. In this paper, "operably linked" refers to linking the exogenous gene to the carrier, so that the regulatory elements in the carrier, such as transcriptional regulatory sequences and translational regulatory sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function. Of course, the polynucleotides used to encode the heavy chain and light chain of the antibody can be independently inserted into different vectors, usually inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages and the like.

The "cell" or "recombinant cell" used herein may contain an expression vector. Expression vectors can be introduced into mammalian cells to obtain recombinant cells, which are then used to express the antibodies or antigen-binding portions provided in the present disclosure. The corresponding antibody can be obtained by culturing the recombinant cells. These usable mammalian cells are, for example, CHO cells and the like.

As used herein, "pharmaceutically acceptable carrier" may include any solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, the pharmaceutical composition may contain isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol) or sodium chloride, etc. Of course, pharmaceutically acceptable carriers may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to prolong the shelf life or potency of the antibody.

For example, an antibody of the disclosure, or an antigen-binding portion thereof, can be incorporated into a pharmaceutical composition suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, etc., including but not limited to liquid solutions (such as injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, Powders, liposomes and suppositories. Typical pharmaceutical compositions are in the form of solutions for injection or infusion. Antibodies of the disclosure, or antigen-binding portions thereof, can be administered by intravenous infusion, injection, intramuscular or subcutaneous injection.

As used herein, the term "cancer of the blood" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include B-cell lymphoma (B-cell blood cancer) and T-cell lymphoma. B-cell lymphomas include Hodgkin's lymphoma and most non-Hodgkin's lymphomas. Non-limiting examples of B-cell lymphoma include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma (MCL), Burkitt Lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, lymph node marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granuloma. Non-limiting examples of T cell lymphoma include extranodal T cell lymphoma, cutaneous T cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma. Hematological malignancies also include leukemias such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia. Hematologic malignancies also include myelomas such as, but not limited to, multiple myeloma and smoldering multiple myeloma. The term hematological malignancies encompasses other blood and/or B or T cell related cancers.

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. In the following description, descriptions of well-known technologies are omitted to avoid unnecessarily obscuring the concept of the present disclosure. Such techniques are described in a number of publications, such as "A Laboratory Guide to Molecular Cloning, Fourth Edition" (Cold Spring Harbor Laboratory Scientific Press).

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1. Process of Obtaining Antibody Sequence by Hybridoma

The hybridoma cell line that can produce the antibody combined with 4-1BB was screened by hybridoma technology and ELISA method. Briefly, human recombinant protein 4-1BB (UniProtKB-Q07011) was mixed with complete Freund's adjuvant or incomplete Freund's adjuvant to form an emulsion, and injected subcutaneously in Balb/c mice. After 2-4 times of immunization, the binding titer of mouse serum to human recombinant protein 4-1BB was determined by ELISA method. If the binding titer of the mouse serum reaches the standard, the spleen of the mouse can be obtained, and the spleen of the mouse can be fully ground to obtain a single cell suspension, which can be fused with myeloma cells to form hybridoma cells by PEG. After culturing for 7-14 days, the culture supernatant of the hybridoma cells is detected by ELISA method, and the positive cells meeting the standard are selected to continue culturing. The above-mentioned ELISA positive cell supernatant was obtained for the second time, and the FACs method was used to detect the binding of the antibody in the supernatant to the stably transfected CHO cell line highly expressing 4-1BB.

Cells showing positive binding were subcloned. By screening the hybridoma cells with a large sample size, 6 positive monoclonal hybridoma cell lines were obtained which could produce the molecular level combined with 4-1BB. The six positive monoclonal hybridoma cell lines were numbered HEC500, HEC501, HEC502, HEC503, HEC504 and HEC505, respectively. After the cells are harvested, the cellular mRNA is obtained and reverse transcribed into cDNA. Use commonly used antibody capture sequence primers to obtain the antibody sequence in the hybridoma cell line and sequence it. At the same time, the subcloned cells can be inoculated into the peritoneal cavity of the mouse at the number of 5×10 ⁵ -1×10 ⁶ cells. After 7 to 10 days, the ascites in the peritoneal cavity was collected and prepared by purification of Protein A medium to obtain mouse-derived monoantibody with a purity of more than 90%, which was used for further detection and evaluation. Antibody sequences are detailed in Table 2.

Table 2 below shows the CDR sequences of the six antibodies screened based on the Kabat definition rules.

Table 2. CDR sequences of antibodies

Example 2. Binding of the disclosed antibody to a CHO cell line stably transfected with human 4-1BB

In this example, the antibodies HEC500, HEC501, HEC502, HEC503, HEC504, and HEC505 screened in Example 1 were detected by flow cytometry, and they were highly expressed in human 4-1BB (UniProtKB-Q07011) stably transfected CHO cell lines binding ability. After the antibody was incubated with the cells, the FITC-labeled goat anti-mouse IgG Fc secondary antibody was used to bind, the isotype IgG antibody was used as a negative control, and the anti-4-1BB antibodies Urelumab (BMS Company) and Utomilumab (Pfizer Company) were used as positive control. The results are shown in Figure 1.

It can be seen from the results in Figure 1 that antibodies HEC500, HEC501, HEC502, HEC503, HEC504, and HEC505 can all effectively bind to proteins expressed on human 4-1BB membranes.

Example 3. ELISA binding of antibodies of the present disclosure to monkey-derived 4-1BB recombinant protein

In this example, ELISA was used to detect the binding ability of the antibody to the recombinant protein of cynomolgus monkey 4-1BB (XP_005544947.1).

Briefly, the recombinant 4-1BB protein was coated on a microtiter plate and left overnight at 4°C. After 3 washes, spin dry, seal at 37°C for 1 hour, wash 3 times, spin dry for later use. Incubate each antibody with the above spare microtiter plate at 4°C for 2 hours. After washing for 3 times, spin dry, and then bind with HRP-labeled goat anti-mouse IgG (Abcam: ab97023) secondary antibody. An isotype IgG antibody was used as a negative control, and Urelumab and Utomilumab antibodies were used as control samples. The results are shown in Figure 2.

It can be seen from Figure 2 that antibodies HEC500, HEC501, HEC502, HEC503, HEC504, and HEC505 can all effectively bind to monkey-derived 4-1BB protein. However, the binding ability of Urelumab and Utomilumab antibodies to monkey-derived 4-1BB protein was significantly weaker. Combining the results of Example 2 (Figure 1), it can be concluded that antibodies HEC500, HEC501, HEC502, HEC503, HEC504, and HEC505 can effectively bind to similar regions of monkey-derived and human-derived 4-1BB proteins. Antibodies that bind to these similar regions have the potential to become clinical drugs. Moreover, in the preclinical monkey toxicology experiment, it has advantages compared with Bristol-Myers Squibb (BMS) clinical antibody Urelumab and Pfizer clinical antibody Utomilumab.

Example 4. Binding of antibodies of the present disclosure to activated T cells isolated from human blood in vitro

T cells were isolated from human-derived peripheral blood mononuclear cell (PBMC) cell populations. T cells were stimulated in vitro for 48-72 hours using CD3 and CD28 antibody-coupled magnetic beads (Invitrogen-11161D). Antibodies HEC500, HEC501, HEC502, HEC503, HEC504, and HEC505 were incubated with T cells, respectively, and then PE-labeled goat anti-mouse IgG Fc secondary antibody (Jackson) was used to bind, and an isotype IgG antibody was used as a negative control. Urelumab and Utomilumab antibodies were used as control samples. Then, using a flow cytometer, the ability of the antibodies HEC500, HEC501, HEC502, HEC503, HEC504 and HEC505 to bind to the stimulated T cells was detected. The results are shown in FIG. 3 .

It can be seen from Figure 3 that the antibodies of the present disclosure can effectively bind to human activated T cells.

Example 5. Determination of Agonist Activity of Antibodies of the Disclosure Using IL2 Indicator of T Cell Activation

The Anti-CD3 antibody was pre-coated on the microtiter plate, and then the isolated human T cells were added, and the antibody diluted in a concentration gradient was added for incubation. Three days later, the expression level of IL2 in the supernatant was detected. Isotype IgG antibody (Isotype IgG) was used as a negative control, and Urelumab and Utomilumab antibodies were used as control samples. The results are shown in FIG. 4 .

It can be seen from FIG. 4 that the antibody of the present disclosure can activate T cells derived from the body in a concentration-dependent manner and secrete IL2 cytokines at a lower dose.

Example 6. Determination of Agonist Activity of Antibodies of the Disclosure Using T Cell Activation IFN-γ Indicator

The Anti-CD3 antibody was pre-coated on the microtiter plate, and then the isolated human T cells were added, and the antibody diluted in a concentration gradient was added for incubation. Three days later, the expression of IFN-γ in the supernatant was detected. An isotype IgG antibody was used as a negative control, and Urelumab and Utomilumab antibodies were used as control samples. The results are shown in FIG. 5 .

It can be seen from FIG. 5 that the antibody of the present disclosure can activate T cells derived from the body in a concentration-dependent manner and secrete IFN-γ cytokine at a lower dose.

Example 7. Preparation and expression detection of humanized antibody

In this example, humanized antibodies HEC501 and HEC502 were prepared according to conventional techniques in the art.

The variable region sequences of HEC501 and HEC502 and their humanized mutant sequences were obtained by whole gene synthesis. The HCDR sequences and LCDR sequences of the humanized antibodies obtained based on the HEC501 antibody are shown in Table 3. The HCDR sequences and LCDR sequences of the humanized antibodies obtained based on the HEC502 antibody are shown in Table 4. The variable region sequences of HEC501 and HEC502 were humanized to obtain the heavy chain variable region (HV) and light chain variable region (LV) shown in Table 5 and Table 6.

Table 3. CDR sequences of humanized antibodies obtained based on HEC501 antibody

Table 4. CDR sequences of humanized antibodies obtained based on HEC502 antibody

Table 5. Heavy chain variable region sequence and light chain variable region sequence after humanization of HEC501 antibody

Table 6. Heavy chain variable region sequence and light chain variable region sequence after humanization of HEC502 antibody

The constant region sequence required for the antibody comes from human IgG4 (EU: S228P), in which, the constant region of the heavy chain of the HEC501 humanized antibody is SEQ ID NO: 145; the constant region of the light chain of the HEC501 humanized antibody is SEQ ID NO: 146 The complete sequence of the humanized antibody can be obtained by directly splicing the constant region sequence with the humanized heavy chain variable region and light chain variable region shown in Table 5.

The constant region sequence required for the antibody comes from human IgG1, wherein the constant region of the heavy chain of the HEC502 humanized antibody is SEQ ID NO: 158; the constant region of the light chain of the HEC502 humanized antibody is SEQ ID NO: 159; the constant region sequence is the same as The complete sequence of the humanized antibody can be obtained by directly splicing the humanized heavy chain variable region and light chain variable region shown in Table 6.

The resulting construct was transiently transfected into HEK293 cells. Through the purification and preparation of Protein A medium, chimeric antibodies and humanized antibodies with a purity of more than 95% were obtained (wherein HEC51118 and HEC51225 are chimeric antibody chains). The expression level of the humanized antibody of the present disclosure was detected by conventional methods. Table 10 shows the detection results of the expression level of the HEC501 humanized antibody. Table 11 shows the detection results of the expression level of the HEC502 humanized antibody.

Table 10. Detection results of the expression level of HEC501 humanized antibody

Table 11. Detection results of the expression level of HEC502 humanized antibody

Example 8. ELISA binding of humanized antibody of the present disclosure to human 4-1BB protein and CHO cell line with high expression of 4-1BB.

In this example, a flow cytometer was used to detect the binding ability of the humanized antibody obtained in Example 7 to a stably transfected CHO cell line with high expression of human 4-1BB. After the antibody was incubated with the cells, the FITC-labeled goat anti-human IgG Fc secondary antibody was used to bind, and the isotype IgG antibody (Isotype IgG) was used as a negative control. The results are shown in Table 12 and Figure 6.

In addition, the binding ability of the humanized antibody obtained in Example 7 to the human 4-1BB (UniProtKB-Q07011) recombinant protein was tested using ELISA. The recombinant 4-1BB protein was coated on a microtiter plate and left overnight at 4°C. After 3 washes, spin dry, seal at 37°C for 1 hour, wash 3 times, spin dry for later use. Incubate the antibody with the above spare ELISA plate at 4°C for 2 hours, wash it three times and then spin dry. Subsequently, HRP-labeled goat anti-human IgG Fc secondary antibody was used for binding, and isotype IgG antibody (Isotype IgG) was used as a negative control. The results are shown in Table 13.

It can be seen from Tables 12 and 13 and FIG. 6 that the humanized antibodies of the present disclosure have a relatively high ability to bind human 4-1BB.

Table 12. Binding of HEC501 humanized antibody to CHO cell line with high expression of human 4-1BB

Table 13. Binding of HEC502 humanized antibody to CHO cell line with high expression of human 4-1BB

Example 9. Detecting the binding of the humanized antibody of the present disclosure to the CHO cell line with high expression of 41BB after heat treatment at 65°C for 5 minutes

Antibody samples at a concentration of 1 mg/ml were treated with different phosphate, citrate or acetate buffers at pH 5, 6, 7, 8 or 9 at 65°C for 5 minutes. Using flow cytometry, the binding ability of the antibody to the CHO cell line stably transfected with human 4-1BB was detected. And the activity retention rate (%) of the sample after heat treatment was calculated using the respective numbered samples which had not been heat-treated in pH 7.4 buffer solution as the denominator. The results are shown in Table 14.

Table 14. The heat resistance test results of HEC501 humanized antibody binding ability after heat treatment

It can be seen from Table 14 that the humanized antibodies of the present disclosure can still maintain a certain level of binding activity after heat treatment.

Embodiment 10. Biofilm light interference technique (Bio-Layer Interferometry, BLI) measures affinity

Immobilize the human 4-1BB protein on the sensor for 300s, then insert the sensor into the PBST solution to equilibrate the Baseline for 180s. Subsequently, the probe was immersed in the antibody solution for 600 s of binding, and then the probe was placed in PBST solution for 1200 s of dissociation. Finally, put the probe into the regeneration solution to complete the regeneration and preservation of the probe. The affinity of the antibody of the present disclosure to the human recombinant protein 4-1BB was tested using BLI. The results are shown in Table 7.

Table 7. Affinity of antibodies to human recombinant protein 4-1BB

编号serial number	K _D(M) K _D (M)	k _on(1/Ms) k _on (1/Ms)	k _dis(1/s) k _dis (1/s)
HEC502HEC502	8.13E-108.13E-10	2.23E+052.23E+05	1.81E-041.81E-04
HEC512119HEC512119	9.68E-109.68E-10	8.95E+058.95E+05	8.66E-048.66E-04

The results showed that there was no significant difference between the humanized antibody (HEC512119) and the unhumanized antibody (HEC502) in binding to human 4-1BB.

Example 11. Anti-tumor activity evaluation of the tested antibody in the tumor-bearing humanized mouse model of MC38 high-expression GPC3 stably transfected cell line

Use 4-1BB/4-1BBL double humanized C57BL/66 mice to inoculate MC38 tumor cell line with high expression of human GPC3 protein. When the average tumor volume reaches 100-150mm ³ , select the appropriate one according to the mouse body weight and tumor volume Mice were enrolled, Urelumab was used as a control, the test antibody HEC502 and Urelumab were intraperitoneally injected into the mice at a dose of 20mg/kg, the tumor size was measured regularly, and the growth or inhibition of the tumor was observed. The results are shown in Figure 7, showing that the tested antibody HEC502 can significantly inhibit the growth of GPC3-positive tumors, and the long-term inhibitory effect is better than Urelumab.

Example 12. Anti-tumor activity evaluation of the tested antibody in the tumor-bearing humanized mouse model of MC38 high expression GPC3 stably transfected cell line

Use 4-1BB/4-1BBL double humanized C57BL/66 mice to inoculate MC38 tumor cell line with high expression of human GPC3 protein. When the average tumor volume reaches 100-150mm ³ , select the appropriate one according to the mouse body weight and tumor volume The mice were enrolled, and the test antibody HEC512119 was injected intraperitoneally into the mice at doses of 20 mg/kg and 5 mg/kg, and the tumor size was measured regularly to observe the growth or inhibition of the tumor. The results are shown in Figure 8, showing that the tested antibody HEC512119 has the effect of significantly inhibiting the growth of GPC3-positive tumors.

The technical solution of the present disclosure is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

An antibody or antigen-binding portion thereof that specifically binds to 4-1BB or a fragment thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein,

The heavy chain variable region includes:

HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 2 or one or HCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, and an amino acid sequence comprising an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues HCDR3;

HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 8 or one or HCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, and an amino acid sequence comprising an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues HCDR3;

HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 14 or one or HCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, and an amino acid sequence comprising an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues HCDR3;

HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 20 or one or HCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, and an amino acid sequence comprising an amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues HCDR3;

HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 26 or one or HCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, and an amino acid sequence comprising an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues HCDR3; or

HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 32 or one or HCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, and an amino acid sequence comprising an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues HCDR3,

The light chain variable region includes:

LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 5 or one or LCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, including an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues LCDR3;

LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 11 or one or LCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, including an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues LCDR3;

LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 17 or one or LCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, including an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues LCDR3;

LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 23 or one or LCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, including an amino acid sequence as shown in SEQ ID NO: 24 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues LCDR3;

LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 29 or one or LCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, including an amino acid sequence as shown in SEQ ID NO: 30 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues LCDR3; or

LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, including the amino acid sequence shown in SEQ ID NO: 35 or one or LCDR2 of an amino acid sequence in which multiple amino acid residues are substituted by different amino acid residues, including an amino acid sequence as shown in SEQ ID NO: 36 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues LCDR3.
The antibody or antigen-binding portion thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, 39, 41, 43, 45 or 47 or one of them or an amino acid sequence in which multiple amino acid residues are replaced by different amino acid residues.
The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 38, 40, 42, 44, 46 or 48 or An amino acid sequence in which one or more amino acid residues are substituted with a different amino acid residue.
The antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the antibody heavy chain is selected from the amino acids shown in SEQ ID NO: 49, 51, 53, 55, 57 or 59 sequence or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues.
The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein the antibody light chain is selected from amino acids shown in SEQ ID NO: 50, 52, 54, 56, 58 or 60 sequence or an amino acid sequence in which one or more amino acid residues are replaced by different amino acid residues.
A humanized antibody or antigen-binding portion thereof derived from the antibody or antigen-binding portion thereof of any one of claims 1 to 5.
The humanized antibody or antigen-binding portion thereof according to claim 6, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region,

The heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein

The HCDR1 has the amino acid sequence shown by SYWX 1 X 2 ,

The HCDR2 has an amino acid sequence represented by X 3 IYPSDTYTX 4 YX 5 QKFQX 6 ,

The HCDR3 has an amino acid sequence as shown in SEQ ID NO: 71 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues;

The light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein

The LCDR1 has an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues,

The LCDR2 has an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues,

The LCDR3 has an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues,

in,

X is selected from isoleucine, methionine or valine,

X2 is selected from asparagine, histidine or threonine,

X is selected from asparagine or isoleucine,

X is selected from asparagine, aspartic acid or serine,

X is selected from asparagine or alanine,

X6 is selected from aspartic acid or glycine.
The antibody or antigen-binding portion thereof according to claim 7, wherein,

The HCDR1 has an amino acid sequence as shown in SEQ ID NO: 7, 61, 62 or 63,

The HCDR2 has an amino acid sequence as shown in SEQ ID NO: 64, 65, 66, 67, 68, 69 or 70,

The HCDR3 has an amino acid sequence as shown in SEQ ID NO:71;

The LCDR1 has an amino acid sequence as shown in SEQ ID NO: 10;

The LCDR2 has an amino acid sequence as shown in SEQ ID NO: 11;

The LCDR3 has an amino acid sequence as shown in SEQ ID NO:12.
The antibody or antigen-binding portion thereof according to claim 7 or 8, wherein the heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO: 132-138 or one or more thereof An amino acid sequence in which amino acid sequences are substituted.
The antibody or antigen-binding portion thereof according to any one of claims 7 to 9, wherein the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 139 to 144 or Amino acid sequences in which one or more amino acid sequences are substituted.
The humanized antibody or antigen-binding portion thereof according to claim 6, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region,

The heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein

The HCDR1 has an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues,

The HCDR2 has the amino acid sequence shown by VIWTGZ 1 GTNYZ 2 Z 3 Z 4 FZ 5 S, and

The HCDR3 has an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues,

The light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein

said LCDR1 has the amino acid sequence shown by RSSQZ 6 LVHSNTNTYLH,

The LCDR2 has an amino acid sequence as shown in SEQ ID NO: 77 or an amino acid sequence in which one or more amino acid residues are substituted by different amino acid residues, and

The LCDR3 has the amino acid sequence shown by SQZ 7 THVPWT,

in,

Z is selected from glycine or serine,

Z2 is selected from asparagine or alanine,

Z is selected from serine or proline,

Z is selected from threonine or proline,

Z is selected from methionine or lysine,

Z6 is selected from serine or threonine,

Z7 is selected from serine or threonine.
The antibody or antigen-binding portion thereof according to claim 11, wherein,

The HCDR1 has an amino acid sequence as shown in SEQ ID NO: 13,

The HCDR2 has an amino acid sequence as shown in SEQ ID NO:72, 73 or 74,

The HCDR3 has an amino acid sequence as shown in SEQ ID NO: 15,

The LCDR1 has an amino acid sequence as shown in SEQ ID NO: 75 or 76,

The LCDR2 has an amino acid sequence as shown in SEQ ID NO:77, and

The LCDR3 has an amino acid sequence as shown in SEQ ID NO: 18 or 78.
The antibody or antigen-binding portion thereof according to claim 11 or 12, wherein the heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO: 147-152 or one or more thereof An amino acid sequence in which amino acid sequences are substituted.
The antibody or antigen-binding portion thereof according to any one of claims 11 to 13, wherein the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 153 to 157 or Amino acid sequences in which one or more amino acid sequences are substituted.
A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the antibody or antigen-binding portion thereof according to any one of claims 1-14.
An expression vector, characterized in that the expression vector comprises the nucleic acid molecule according to claim 15.
A cell, characterized in that the cell is transfected with the expression vector according to claim 16.
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or antigen-binding portion thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the nucleic acid molecule according to claim 16 The expression vector or the cell according to claim 17, and a pharmaceutically acceptable carrier.
The antibody or antigen-binding portion thereof according to any one of claims 1 to 14, the nucleic acid molecule as claimed in claim 15, the expression vector as claimed in claim 16, the cell as claimed in claim 17, or the nucleic acid molecule as claimed in claim 16 The pharmaceutical composition of claim 18, the application in the preparation of the medicine for treating cancer,

Preferably, the cancer is selected from the group consisting of melanoma, glioma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, ovarian cancer, lung cancer, renal cell carcinoma, nasopharyngeal carcinoma, renal cancer, breast cancer, blood cancers, and head and neck cancers.