anti-PD-1 antibodies and uses thereof

文档序号：1574113 发布日期：2020-01-31 浏览：22次中文

阅读说明：本技术 抗pd-1抗体及其用途 (anti-PD-1 antibodies and uses thereof ) 是由黄承浩熊丹林超龙游敏罗文新张军夏宁邵于 2019-07-19 设计创作，主要内容包括：本发明涉及疾病治疗及免疫学领域,具体而言,本发明涉及抗PD-1的抗体或其抗原结合片段,编码它们的核酸分子,制备它们的方法,以及包含它们的药物组合物。本发明进一步涉及所述抗体(特别是人源化抗体)或其抗原结合片段在制备药物中的用途,所述药物用于提高免疫细胞活性、增强免疫应答,或者用于预防和/或治疗肿瘤或感染。(The present invention relates to the field of disease therapy and immunology, in particular, the present invention relates to antibodies against PD-1 or antigen binding fragments thereof, nucleic acid molecules encoding the same, methods of making the same, and pharmaceutical compositions comprising the same the present invention further relates to the use of said antibodies (particularly humanized antibodies) or antigen binding fragments thereof in the manufacture of a medicament for increasing immune cell activity, enhancing immune response, or for preventing and/or treating tumors or infections.)

1. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

(i) a VH CDR1 having the sequence of CDR1 contained in the VH set forth in SEQ ID NO:1, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., substitution of 1,2, 3 or 4 amino acids) compared to the sequence of CDR1 contained in said VH;

(ii) VH CDR2 having the sequence of CDR2 contained in the VH shown in SEQ ID NO:1, or a sequence having substitution, deletion or addition of or several amino acids (e.g., substitution of 1,2, 3, 4,5 or 6 amino acids) compared to the sequence of CDR2 contained in the VH, and

(iii) a VH CDR3 having the sequence of CDR3 contained in the VH set forth in SEQ ID NO:1, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution of 1,2, 3, 4,5, 6 or 7 amino acids) compared to the sequence of CDR3 contained in said VH;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) a VL CDR1 having the sequence of CDR1 contained in VL as shown in SEQ ID No. 2, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., substitution of 1,2, 3, 4,5, 6 or 7 amino acids) compared to the sequence of CDR1 contained in said VL;

(v) VL CDR2 having the sequence of CDR2 contained in VL as shown in SEQ ID NO:2, or a sequence having substitution, deletion or addition of or several amino acids (e.g., substitution of 1 or 2 amino acids) as compared with the sequence of CDR2 contained in said VL, and

(vi) a VL CDR3 having the sequence of CDR3 contained in VL as shown in SEQ ID No. 2, or a sequence having substitution, deletion or addition of or several amino acids (e.g., substitution of 1,2, 3, 4,5 or 6 amino acids) compared to the sequence of CDR3 contained in said VL;

preferably, the substitution recited in any one of (i) - (vi) under item is a conservative substitution;

preferably, the CDR1, CDR2 and CDR3 comprised in the heavy chain variable region (VH) and/or the CDR1, CDR2 and CDR3 comprised in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system;

preferably, the antibody or antigen-binding fragment thereof comprises:

(a) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1;

and/or the presence of a gas in the gas,

(b) 3 CDRs contained in the light chain variable region (VL) shown in SEQ ID NO: 2;

preferably, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system.

2. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) VH CDR1 consisting of SEQ ID NO:3, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution of 1,2, 3 or 4 amino acids) compared to SEQ ID NO:3,

(ii) VH CDR2 consisting of SEQ ID NO:4, or a sequence having a replacement, deletion or addition of or several amino acids (e.g., 1,2, 3, 4,5 or 6 amino acid substitutions) as compared to SEQ ID NO:4, and

(iii) VH CDR3 consisting of SEQ ID NO:5, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution of 1,2, 3, 4,5, 6 or 7 amino acids) as compared to SEQ ID NO: 5;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) VL CDR1 consisting of SEQ ID NO:6, or a sequence having a substitution, deletion or addition of or several amino acids compared to SEQ ID NO:6 (e.g. a substitution of 1,2, 3, 4,5, 6 or 7 amino acids),

(v) VL CDR2 consisting of SEQ ID NO:7, or a sequence having a replacement, deletion or addition of or several amino acids (e.g., a replacement of 1 or 2 amino acids) as compared to SEQ ID NO:7, and

(vi) VL CDR3 consisting of SEQ ID NO:8, or a sequence having a or several amino acid substitution, deletion or addition (e.g., 1,2, 3, 4,5 or 6 amino acid substitution) compared to SEQ ID NO: 8.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, which has or more of the following characteristics:

(i) VH CDR1 has a structure as X₁X₂AFX₃RFX₄(SEQ ID NO: 49);

(ii) VH CDR2 has a structure as X₅X₆GGX₇X₈X₉X₁₀(SEQ ID NO: 50);

(iii) VH CDR3 has, for example, ARHX₁₁X₁₂X₁₃TX₁₄AX₁₅X₁₆X₁₇(SEQ ID NO: 51);

(iv) VL CDR1 has a sequence as X₁₈X₁₉X₂₀X₂₁X₂₂YX₂₃YX₂₄F (SEQ ID NO:52)A sequence;

(v) VL CDR2 has a common reference position as RX₂₅A (SEQ ID NO: 53); and

(vi) VL CDR3 has a shape like QX₂₆X₂₇X₂₈X₂₉X₃₀PX₃₁(SEQ ID NO: 54);

wherein, X₁To X₃₁Independently of one another, are any amino acids.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein X₁Selected from G or

A，X₂Selected from F or A, X₃Selected from S or A, X₄Selected from D or A, X₅Selected from I or A, X₆To X₈Independently of one another, from G or A, X₉Selected from R or A, X₁₀Selected from T or A, X₁₁Selected from G or A, X₁₂Selected from T or A, X₁₃To X₁₄Independently of one another, from G or A, X₁₅Selected from M or A, X₁₆Selected from D or A, X₁₇Selected from Y or A, X₁₈Selected from K or A, X₁₉Selected from S or A, X₂₀Selected from V or A, X₂₁Selected from D or A, X₂₂Selected from N or A, X₂₃Selected from G or A, X₂₄Selected from S or A, X₂₅Selected from S or A, X₂₆Selected from Q or A, X₂₇Selected from S or A, X₂₈Selected from N or A, X₂₉Selected from E or A, X₃₀Selected from D or A, X₃₁Selected from T or A;

preferably, X₆Is G, X₇Is G, X₁₀Is T, X₁₁Is G, X₁₃Is G, X₁₄Is G, X₁₅Is M, X₂₃Is G, X₂₄Is S, X₂₆Is Q, X₃₁Is T.

5. The antibody or antigen-binding fragment thereof of claim 4, which has or more of the following characteristics:

(1)X₁to X₄Up to of which are A;

(2)X₅to X₁₀ToA plurality of are A;

(3)X₁₁to X₁₇Up to of which are A;

(4)X₁₈to X₂₄Up to of which are A;

(5)X₂₆to X₃₁Up to of which are A;

preferably, X₁To X₁₇Up to of which are A;

preferably, X₁₈To X₃₁Up to of which are A;

preferably, X₁To X₃₁Up to of which are a.

6. The antibody or antigen-binding fragment thereof of any of claims 1-5, comprising:

(1) VH CDR1 as shown in any of SEQ ID NOs 3, 55-58 and , VH CDR 2as shown in SEQ ID NO 4, VH CDR3 as shown in SEQ ID NO 5, VL CDR1 as shown in SEQ ID NO 6, VL CDR 2as shown in SEQ ID NO 7and VL CDR3 as shown in SEQ ID NO 8;

(2) VH CDR1 shown in SEQ ID NO. 3, VH CDR2 shown in any of SEQ ID NOs:4 and 59-61, VH CDR3 shown in SEQ ID NO.5, VL CDR1 shown in SEQ ID NO. 6, VL CDR2 shown in SEQ ID NO. 7, and VL CDR3 shown in SEQ ID NO. 8;

(3) VH CDR1 shown in SEQ ID NO. 3, VH CDR2 shown in SEQ ID NO.4, VH CDR3 shown in any of SEQ ID NOS:5 and 62-64, VL CDR1 shown in SEQ ID NO. 6, VL CDR2 shown in SEQ ID NO. 7, and VL CDR3 shown in SEQ ID NO. 8;

(4) VH CDR1 shown as SEQ ID NO. 3, VH CDR2 shown as SEQ ID NO.4, VH CDR3 shown as SEQ ID NO.5, VL CDR1 shown as any one of SEQ ID NOs 6 and 65-69 to , VL CDR2 shown as SEQ ID NO. 7and VL CDR3 shown as SEQ ID NO. 8;

(5) VH CDR1 shown in SEQ ID NO 3; VH CDR2 shown in SEQ ID NO. 4; and, a VH CDR3 shown in SEQ ID NO. 5; VL CDR1 as shown in SEQ ID NO. 6; VL CDR 2as set forth in SEQ ID NO. 7 or 70; and, VL CDR3 as set forth in SEQ ID NO. 8; or

(6) VH CDR1 shown in SEQ ID NO. 3, VH CDR2 shown in SEQ ID NO.4, VH CDR3 shown in SEQ ID NO.5, VL CDR1 shown in SEQ ID NO. 6, VL CDR2 shown in SEQ ID NO. 7, and VL CDR3 shown in any of SEQ ID NOs:71-74 and .

7. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

(i) a VH CDR1 having the sequence of CDR1 contained in the VH set forth in SEQ ID NO:9, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR1 contained in said VH;

(ii) VH CDR2 having the sequence of CDR2 contained in the VH shown in SEQ ID NO:9 or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR2 contained in the VH, and

(iii) a VH CDR3 having the sequence of CDR3 contained in the VH set forth in SEQ ID NO:9, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VH;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) a VL CDR1 having the sequence of CDR1 contained in VL as shown in SEQ ID No. 10, or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR1 contained in said VL;

(v) VL CDR2 having the sequence of CDR2 contained in VL as shown in SEQ ID NO:10 or a sequence having substitution, deletion or addition of or several amino acids (e.g. substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR2 contained in said VL, and

(vi) a VL CDR3 having the sequence of CDR3 contained in VL as shown in SEQ ID No. 10, or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VL;

preferably, the substitution recited in any one of (i) - (vi) under item is a conservative substitution;

preferably, the antibody or antigen-binding fragment thereof comprises:

(a) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 9;

and/or the presence of a gas in the gas,

(b) 10 in the light chain variable region (VL) containing 3 CDR;

8. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) VH CDR1 consisting of SEQ ID NO:11, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:11,

(ii) VH CDR2 consisting of SEQ ID NO:12, or a sequence having a replacement, deletion or addition of or several amino acids (e.g., a replacement, deletion or addition of 1,2 or 3 amino acids) as compared to SEQ ID NO:12, and

(iii) VH CDR3 consisting of SEQ ID NO:13, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO: 13;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) VL CDR1 consisting of SEQ ID NO:14, or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:14,

(v) VL CDR2 consisting of SEQ ID NO:15, or a sequence having a replacement, deletion or addition of or several amino acids (e.g. a replacement, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:15, and

(vi) VL CDR3 consisting of SEQ ID NO:16, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO: 16;

preferably, the substitution recited in any one of (i) - (vi) under item is a conservative substitution;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown in SEQ ID NO. 11; VH CDR2 shown in SEQ ID NO. 12; and, VH CDR3 shown in SEQ ID NO 13; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 14; VL CDR 2as shown in SEQ ID NO. 15; and, VL CDR3 as shown in SEQ ID NO 16.

9. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

(i) a VH CDR1 having the sequence of CDR1 contained in the VH set forth in SEQ ID NO:17, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR1 contained in said VH;

(ii) VH CDR2 having the sequence of CDR2 contained in the VH shown in SEQ ID NO:17 or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR2 contained in the VH, and

(iii) a VH CDR3 having the sequence of CDR3 contained in the VH set forth in SEQ ID NO:17, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VH;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) a VL CDR1 having the sequence of CDR1 contained in VL as set forth in SEQ ID NO:18, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) as compared to the sequence of CDR1 contained in said VL;

(v) a VL CDR2 having the sequence of CDR2 contained in VL as shown in SEQ ID NO:18 or a sequence having substitution, deletion or addition of or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) as compared with the sequence of CDR2 contained in said VL, and

(vi) a VL CDR3 having the sequence of CDR3 contained in VL as set forth in SEQ ID NO:18, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) as compared to the sequence of CDR3 contained in said VL;

preferably, the substitution recited in any one of (i) - (vi) under item is a conservative substitution;

preferably, the antibody or antigen-binding fragment thereof comprises:

(a) the 3 CDRs contained in the variable region of the heavy chain (VH) shown in SEQ ID NO: 17;

and/or the presence of a gas in the gas,

(b) 18 in the light chain variable region (VL) as shown in SEQ ID NO 3 CDR;

10. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) VH CDR1 consisting of SEQ ID NO:19, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:19,

(ii) VH CDR2 consisting of SEQ ID NO:20, or a sequence having a replacement, deletion or addition of or several amino acids (e.g., a replacement, deletion or addition of 1,2 or 3 amino acids) as compared to SEQ ID NO:20, and

(iii) VH CDR3 consisting of SEQ ID NO:21, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO: 21;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) VL CDR1 consisting of SEQ ID NO:22, or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:22,

(v) VL CDR2 consisting of SEQ ID NO:23, or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:23, and

(vi) VL CDR3 consisting of SEQ ID NO:24, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO: 24;

preferably, the substitution recited in any one of (i) - (vi) under item is a conservative substitution;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown in SEQ ID NO. 19; VH CDR2 shown as SEQ ID NO: 20; and, VH CDR3 shown in SEQ ID NO: 21; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 22; VL CDR 2as shown in SEQ ID NO. 23; and, VL CDR3 as shown in SEQ ID NO. 24.

11. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

(i) a VH CDR1 having the sequence of CDR1 contained in the VH set forth in SEQ ID NO:25, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR1 contained in said VH;

(ii) VH CDR2 having the sequence of CDR2 contained in the VH shown as SEQ ID NO:25, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR2 contained in the VH, and

(iii) a VH CDR3 having the sequence of CDR3 contained in the VH set forth in SEQ ID NO:25, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VH;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) a VL CDR1 having the sequence of CDR1 contained in VL as shown in SEQ ID No. 26, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR1 contained in said VL;

(v) VL CDR2 having the sequence of CDR2 contained in VL as shown in SEQ ID NO:26 or a sequence having substitution, deletion or addition of or several amino acids (e.g. substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR2 contained in said VL, and

(vi) a VL CDR3 having the sequence of CDR3 contained in VL as shown in SEQ ID No. 26, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VL;

preferably, the substitution recited in any one of (i) - (vi) under item is a conservative substitution;

preferably, the antibody or antigen-binding fragment thereof comprises:

(a) the 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 25;

and/or the presence of a gas in the gas,

(b) 26 in the light chain variable region (VL) containing 3 CDRs;

12. An antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, said antibody or antigen-binding fragment thereof comprising:

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) VH CDR1 consisting of SEQ ID NO:27, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:27,

(ii) VH CDR2 consisting of SEQ ID NO:28, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) as compared to SEQ ID NO:28, and

(iii) VH CDR3 consisting of SEQ ID NO:29, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO: 29;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) VL CDR1 consisting of SEQ ID NO:30, or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:30,

(v) VL CDR2 consisting of SEQ ID NO:31, or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO:31, and

(vi) VL CDR3 consisting of SEQ ID NO:32, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2 or 3 amino acids) compared to SEQ ID NO: 32;

preferably, the substitution recited in any one of (i) - (vi) under item is a conservative substitution;

preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown as SEQ ID NO: 27; VH CDR2 shown as SEQ ID NO 28; and, VH CDR3 shown in SEQ ID NO. 29; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 30; VL CDR 2as shown in SEQ ID NO. 31; and, VL CDR3 as shown in SEQ ID NO: 32.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-12 to , wherein the antibody or antigen-binding fragment thereof is humanized;

preferably, the antibody or antigen binding fragment thereof is humanized to a degree of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98%;

preferably, the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin (e.g., a framework region comprised in an amino acid sequence encoded by a human germline antibody gene), the framework region optionally comprising or more (e.g., 1,2, 3, 4,5, 6, 7,8, 9, or 10) back mutations from human residues to murine residues;

preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region comprised in the amino acid sequence encoded by the human heavy chain germline gene and/or a light chain framework region comprised in the amino acid sequence encoded by the human light chain germline gene.

14. The antibody or antigen-binding fragment thereof of of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof further comprises steps comprising heavy chain framework regions comprised in the amino acid sequence encoded by IGVH3-23 a 04 and light chain framework regions comprised in the amino acid sequence encoded by IGKV3-11 a 01, said heavy chain framework regions and/or light chain framework regions optionally comprising or more (e.g., 1,2, 3, 4,5, 6, 7,8, 9, or 10) back mutations from human residues to murine residues;

preferably, the antibody or antigen-binding fragment thereof is humanized to a degree of at least 95%.

15. The antibody or antigen-binding fragment thereof of claim 7 or 8, wherein the antibody or antigen-binding fragment thereof further comprises steps of including a heavy chain framework region in the amino acid sequence encoded by IGVH1-2 x 02 and a light chain framework region in the amino acid sequence encoded by IGKV1-16 x 01, the heavy chain framework region and/or the light chain framework region optionally comprising or more (e.g., 1,2, 3, 4,5, 6, 7,8, 9, or 10) back mutations from human residues to murine residues;

preferably, the antibody or antigen-binding fragment thereof is humanized to a degree of at least 84%.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-6, 13-14 to , wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) 1, 33, 35 or ;

(ii) a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2, 3, 4 or 5 amino acids) as compared to the sequence represented by any one of SEQ ID NOs:1, 33, 35 and , or

(iii) A sequence -identical to the sequence set forth in any one of SEQ ID NOs:1, 33, 35 and , at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) the sequence shown in any items of SEQ ID NOs:2, 34 and 36;

(v) a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2, 3, 4 or 5 amino acids) as compared to the sequence represented by any one of of SEQ ID NOs:2, 34, 36, or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence represented by any one of of SEQ ID NOs:2, 34, 36;

preferably the substitutions described in (ii) or (v) are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof comprises:

(1) VH having a sequence shown as SEQ ID NO. 1and VL having a sequence shown as SEQ ID NO. 2;

(2) VH having a sequence shown as SEQ ID NO. 33 and VL having a sequence shown as SEQ ID NO. 34; or

(3) VH having a sequence shown as SEQ ID NO. 35 and VL having a sequence shown as SEQ ID NO. 36.

17. The antibody or antigen-binding fragment thereof of any one of claims 7-8, 13, 15 to , wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) 9, 37, 39 or ;

(ii) a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2, 3, 4 or 5 amino acids) as compared with the sequence represented by any one of SEQ ID NOs: 9, 37, 39 and , or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence presented in any of SEQ ID NOs 9, 37, 39;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) 10, 38, 40 or ;

(v) a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution, deletion or addition of 1,2, 3, 4 or 5 amino acids) as compared with the sequence represented by any one of SEQ ID NOs: 10, 38, 40 and , or

(vi) A sequence -identical to the sequence set forth in any one of SEQ ID NOs 10, 38, 40, , at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;

preferably the substitutions described in (ii) or (v) are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof comprises:

(1) VH having a sequence shown as SEQ ID NO.9 and VL having a sequence shown as SEQ ID NO. 10;

(2) VH having a sequence shown as SEQ ID NO. 37 and VL having a sequence shown as SEQ ID NO. 38; or

(3) VH having the sequence shown as SEQ ID NO. 39 and VL having the sequence shown as SEQ ID NO. 40.

18. The antibody or antigen-binding fragment thereof of any one of claims 9-10, 13 to , wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 17;

(ii) a sequence having or several amino acid substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to the sequence shown in SEQ ID NO:17, or

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO:18, or a sequence shown in seq id no;

(v) a sequence having or several amino acid substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared to the sequence shown in SEQ ID NO:18, or

preferably the substitutions described in (ii) or (v) are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO. 17 and VL having a sequence shown as SEQ ID NO. 18.

19. The antibody or antigen-binding fragment thereof of any of claims 11-13, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 25;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 26;

preferably the substitutions described in (ii) or (v) are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO. 25 and VL having the sequence shown as SEQ ID NO. 26.

20. The antibody or antigen-binding fragment thereof of of any one of claims 1-19, wherein the antigen-binding fragment is selected from the group consisting of scFv, Fab ', (Fab')₂Fv fragments and diabodies (diabodies); and/or, the antibody is a chimeric antibody or a humanized antibody.

21. The antibody or antigen-binding fragment thereof of any one of claims 1-20, , wherein the antibody is an IgG antibody (e.g., an IgG1, IgG2, IgG3, or IgG4 antibody).

22. The antibody or antigen-binding fragment thereof of any one of claims 1 to 21, , wherein the antibody or antigen-binding fragment thereof carries a label, preferably wherein the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

23. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-22 to , or a heavy chain variable region and/or a light chain variable region thereof.

24. A vector comprising the nucleic acid molecule of claim 23; preferably, the vector is a cloning vector or an expression vector.

25. A host cell comprising the nucleic acid molecule of claim 23 or the vector of claim 24.

26. A method of making the antibody or antigen-binding fragment thereof of any of claims 1-22, comprising culturing the host cell of claim 25 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

27. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22 to and a therapeutic agent linked to the antibody or antigen-binding fragment thereof;

preferably, the therapeutic agent is selected from a cytotoxin or a radioisotope.

28. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22 to or the conjugate of claim 27, and a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent;

preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an additional immune checkpoint inhibitor, an oncolytic virus, a chemotherapeutic agent, an anti-angiogenic drug, an anti-metabolite drug, a tumor-targeting drug or an immune stimulant;

preferably, the additional pharmaceutically active agent is a drug for the treatment of infections, such as an antiviral, antifungal, antibacterial or immunostimulating agent;

preferably, the antibody or antigen-binding fragment thereof or the conjugate and the additional pharmaceutically active agent are provided as separate components or as components of a composition with .

29. An immunogenic composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-22 to , and an immunogen;

preferably, the antibody or antigen-binding fragment thereof is used as an adjuvant;

preferably, the immunogen is selected from the group consisting of a tumor-associated antigen (e.g., a protein, polypeptide, or carbohydrate molecule), a tumor cell, a dendritic cell primed with the antigen, and any combination thereof;

preferably, the immunogen is selected from the group consisting of an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen, an inactivated or attenuated pathogen, a dendritic cell primed by the antigen, and any combination thereof;

preferably, the antibody or antigen-binding fragment thereof is provided as a separate component from the immunogen or as a component of a composition with .

30. Use of the antibody or antigen-binding fragment thereof of any one of of claims 1-22, or the conjugate of claim 27, or the pharmaceutical composition of claim 28, in the preparation of a medicament for:

(1) increasing immune cell activity in vitro or in a subject (e.g., a human);

(2) enhancing an immune response in a subject (e.g., a human);

(3) preventing and/or treating a tumor in a subject (e.g., a human); or

(4) Preventing and/or treating infection in a subject (e.g., a human);

preferably, the immune cell is a T cell;

preferably, the immune response is a T cell mediated immune response;

preferably, the tumor is a tumor with microsatellite high instability (MSI-H) and/or mismatch repair deficiency (dMMR);

preferably, the tumor is selected from melanoma (e.g., metastatic malignant melanoma), breast cancer, kidney cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, and hematological malignancies (e.g., lymphoma, leukemia);

preferably, the infection is selected from the group consisting of viral infection, bacterial infection, fungal infection and parasitic infection;

preferably, the subject is a mammal, such as a human.

31. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-22 to in the preparation of an immunogenic composition for enhancing an immune response in a subject, wherein the immunogenic composition comprises the antibody or antigen-binding fragment thereof and an immunogen;

preferably, the infection is selected from the group consisting of viral infection, bacterial infection, fungal infection and parasitic infection;

preferably, the subject is a mammal, such as a human.

Technical Field

The present invention relates to the field of disease therapy and immunology, in particular, the present invention relates to antibodies against PD-1 or antigen binding fragments thereof, nucleic acid molecules encoding the same, methods of making the same, and pharmaceutical compositions comprising the same the present invention further relates to the use of said antibodies (particularly humanized antibodies) or antigen binding fragments thereof in the manufacture of a medicament for increasing immune cell activity, enhancing immune response, or for preventing and/or treating tumors or infections.

Background

The activation process of T cells requires two signals is that the antigenic peptide bound by MHC presented by APCs of antigen presenting cells is cross-linked with cell receptors on the surface of T cells to generate stimulating signal which is antigen specific, and the second stimulating signal provided by the binding of APCs surface molecules (such as B7 family) with corresponding molecules on the surface of T cells (such as CD28) mainly comprises costimulatory factors for activating T cells and costimulatory factors for regulating the non-overstimulation of T cells, and is non-antigen specific, but is necessary for the antigen-specific activation of T cells, and determines whether the antigen-stimulated T cells enter proliferation and differentiation processes called effector T cells or enter inhibition state or apoptosis.

Programmed death molecule (Programmed death gene 1, PD-1; also known as CD279), which is an immunosuppressive molecule in members of the CD28 family, plays an important role in maintaining the immune tolerance of the organism and functions to protect the organism from the attack of the autoimmune system, the PD-1 gene is cloned by Ishida et al in 1992 from the T cell hybridoma 2B4.11 in the apoptotic stage of mice by subtractive hybridization techniques (Ishida Y, Agata Y, et al expressed expression of PD-1, antibody chamber of the immunoglobulin gene transmembrane [ J ]. EMBO J,1992,11(11): 3887. 3895). PD-1 consists of 288 amino acids, the molecular weight is 50-55kDa, the genus I type Pdptransmembrane protein, the member of the CD28 family, whose gene is located on the mouse cell surface region containing tyrosine kinase No. 1, cDNA-12, the chromosome 14, cDNA-1, the cDNA-11, the cDNA sequence of the immunoglobulin gene, the cDNA sequence of the cDNA receptor (III) is found in the human cell receptor gene, the immunoglobulin gene containing tyrosine motif, the immunoglobulin gene of the immunoglobulin gene, the cDNA sequence of the cDNA (I type Pdpastoma [ J. (III) and the cDNA sequence of the cDNA, the cDNA sequence of the cDNA, wherein the cDNA sequence of the cDNA, the.

PD-1 is expressed primarily on the surface of activated T cells, B cells, natural killer T cells, monocytes and dendritic cells. The resting T cells do not express PD-1 on their surface, but are activated to induce expression. PD-1 expression on the cell surface can be detected within 24h after T cell activation and can exert a functional effect within a few hours after binding to its ligand (Riley JL. PD-1signaling in primary T cells [ J ]. Immunol Rev,2009,229(1): 114-) -125). PD-1 has two ligands, PD-L1 (also known as B7-H1, CD274) and PD-L2 (also known as B7-DC, CD273), which are found in 1999 and 2001 respectively, belong to B7family I transmembrane proteins, and the extracellular domain has IgV and IgC domains. PD-L1 is expressed encoded by the gene CD274, 290 amino acids in total, which maps to mouse chromosome 19 and human chromosome 9. PD-L2is encoded and expressed by gene Pdcd1lg2, which has 247 amino acids, and the gene is located on the same chromosome as the gene encoding PD-L1.

PD-L1 is expressed in both hematopoietic and non-hematopoietic cells. PD-L1 is constitutively expressed at high levels in T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells and myeloid mast cells, and continues to upregulate expression upon cell activation. In some nonhematopoietic cells, mainly solid tumors such as kidney, ovarian cancer and non-small cell lung cancer, etc., PD-L1 also exhibits high expression level due to the abnormality of tumor signaling pathway or the induction of tumor cells by external signals. It has also been found that type I and type II interferons also cause immune cells to upregulate the expression of PD-L1 (Francisco L M, Sage P.T., et al. the PD-1pathway in toll and autoimmunity [ J ]. Immunol Rev,2010,236: 219-242). Compared with PD-L1, the expression profile of PD-L2is relatively narrow, and usually is not expressed on the surface of resting cells, mainly expressed in myeloid DC cells, macrophage myeloid-derived mast cells and part of B cell lines, and is limited in expression in peripheral tissues and other cell types. Studies have also found that the effect of PD-L2 in inhibiting T cell activity is lower than that of PD-L1(Latchman Y, et al. PD-L2is as attached ligand for PD-1and inhibitors T cell activation [ J ]. Nat Immunol,2001,2(3): 261-268).

The molecular mechanism of PD-1 to inhibit T cell activity is primarily through the PI3K/Akt signaling pathway. After binding of PD-1and PD-L1/PD-L2 on the surface of T cells, tyrosine in ITIM and ITSM in the membrane domain of PD-1 is phosphorylated, dephosphorylating enzymes SHP-2 and SHP-2 dephosphorylating TCR complex containing SH2 homologous domain are recruited, phosphorylation of CD3 zeta, ZAP70 and PKC theta is inhibited, and phosphorylation of PI3K is inhibited, so that phosphorylation of Akt is inhibited, down-regulation expression of cytokines IL-2 and IFN-gamma is caused, activity, proliferation and cytokine expression of T cells are inhibited (Chemnitz J M, Parry R V, et al, SHP-1and SHP-2 associated with expression of cytokine-based expressed receptor type of modulated death 1 uplink immune man T cell stimulation, butyl receptor expression J2, 954J 954, 954 prediction promoter J2, 954J).

In addition to the regulation and maintenance of autoimmune tolerance, PD-1/PD-L1 signals induce high expression of PD-L1 on the cell surface and cause T cell apoptosis or failure in patients with chronic infectious diseases, such as Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) and other infectious patients, and in tumor patients, the high expression of PD-L1 can enhance the growth and metastasis of tumors, and thus can be used as an indicator of prognosis of tumor patients (Ceraz S, Nowak E C, et al. B7family cancer regulation in immune regulation and disease [ J ]]Trends Immunol,2013,34(11): 556-563). It was found that various tumor cells highly express PD-L1, such as melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, lymphoma, gastric cancer, liver cancer, breast cancer, pancreatic cancer, esophageal cancer, bladder cancer, liver cancer and the like (Chen L, Han, X.anti-PD-1/PD-L1therapy of human cancer: past, present, and future [ J]J Clin Invest,2015,125(9): 3384-. In addition, tumor site invasive CD8⁺PD-1 is also highly expressed on the surface of the T cell, and the PD-1 interacts with PD-L1 on the surface of the tumor cell, so that the activation and the proliferation of the T cell are inhibited, the killing effect of the T cell on the tumor cell is lost, and the immune escape is caused. Therefore, the signal path of PD-1/PD-L1 is blocked in a targeted manner, and the killing of T cells to infectious disease pathogens and tumor cells can be enhanced. Currently, several anti-PD-1 and PD-L1 monoclonal antibody drugs have been approved by the FDA for cancer therapy with good clinical therapeutic efficacy (Sunshine J, Taube JM. PD-1/PD-L1inhibitors [ J)]Curr Opin Pharmacol,2015,23: 32-38). By 2 months 2018, Pembrolizumab and Nivolumab have been approved by the U.S. FDA for clinical treatment of nearly ten malignancies, including non-small cell lung cancer, head and neck cancer, melanoma, bladder cancer, kidney cancer, hodgkin's lymphoma, urothelial cancer, and gastric cancer.

In general, the existing PD-1 antibodies have the defects of toxic and side effects, obviously limited treatment dose in partial population and the like, which greatly weakens the clinical treatment effect and universality of the PD-1 antibodies and causes safety hidden trouble (Michot J M, Bigenwald C, et. Immune-related adjuvant vectors with animal checkpoint: a comprehensive review [ J ]. Eur J Cancer,2016,54: 139-48).

Therefore, the development of anti-PD-1 antibodies with higher specificity, lower toxic and side effects and better clinical efficacy is urgent and necessary, which provides more medication options for patients with cancer, infection and other diseases.

Disclosure of Invention

In the present application, the inventors first developed a murine antibody with superior properties, which is capable of specifically recognizing/binding to human PD-1, blocking the binding of PD-1 to its ligand PD-L1, and is capable of enhancing immune cell activity in vitro/in vivo, stimulating immune response. Therefore, the murine antibody has potential for use in the prevention and/or treatment of tumors or infections.

On the basis, the inventor carries out a great deal of creative work, and carries out intensive research and modification on the murine antibody, thereby developing a humanized antibody of the murine antibody: the humanized antibody of the present invention has not only a very high degree of humanization but also substantially the same biological function as the murine antibody.

Thus, the antibodies (particularly humanized antibodies) of the invention are highly advantageous, not only retaining the functions and properties of the parent murine antibody, thereby having potential for the prevention and treatment of tumors or infections; but also has a high degree of humanisation so that it can be safely administered to human subjects without eliciting an immunogenic response. The antibody of the invention has great clinical value.

Antibodies of the invention

Thus, in aspects, the invention provides antibodies or antigen-binding fragments thereof capable of specifically binding to PD-1, the antibodies or antigen-binding fragments thereof comprising:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

(i) a VH CDR1 having the sequence of CDR1 contained in VH as shown in any of SEQ ID NOs:1, 9, 17, 25, , or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR1 contained in said VH;

(ii) VH CDR2 having the sequence of CDR2 contained in the VH shown in any of items of SEQ ID NOs:1, 9, 17, 25, or a sequence having substitution, deletion or addition of or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) as compared with the sequence of CDR2 contained in the VH, and

(iii) a VH CDR3 having the sequence of CDR3 contained in VH as shown in any of SEQ ID NOs:1, 9, 17, 25, , or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VH;

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(iv) a VL CDR1 having the sequence of CDR1 contained in VL as shown in any of SEQ ID NOs:2, 10, 18, 26, , or a sequence having a substitution, deletion, or addition of or several amino acids (e.g., a substitution, deletion, or addition of 1,2, or 3 amino acids) as compared to the sequence of CDR1 contained in said VL;

(v) a VL CDR2 having the sequence of CDR2 contained in VL as shown in any of items of SEQ ID NOs:2, 10, 18, 26 or a sequence having substitution, deletion or addition of or several amino acids (e.g. substitution, deletion or addition of 1,2 or 3 amino acids) as compared to the sequence of CDR2 contained in said VL, and

(vi) a VL CDR3 having the sequence of CDR3 contained in VL as shown in any of SEQ ID NOs:2, 10, 18, 26, , or a sequence having a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VL.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain exemplary embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat numbering system.

In some embodiments of , the antibody or antigen-binding fragment thereof of the invention comprises:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(vi) VL CDR3 having the sequence of CDR3 contained in VL as shown in SEQ ID NO:2 or a sequence with a substitution, deletion or addition of or several amino acids (e.g. a substitution of 1,2, 3, 4,5 or 6 amino acids) compared to the sequence of CDR3 contained in said VL.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system. In certain exemplary embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1;

and/or the presence of a gas in the gas,

(b) the variable region of the light chain (VL) as shown in SEQ ID NO 2 contains 3 CDRs.

In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering system. In certain exemplary embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the Kabat numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and/or, 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO: 2;

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are determined by the Kabat numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a) the following 3 heavy chain variable region (VH) CDRs:

(i) VH CDR1 consisting of SEQ ID NO:3, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution of 1,2, 3 or 4 amino acids) compared to SEQ ID NO:3,

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(v) VL CDR2 consisting of SEQ ID NO:7, or a sequence having a replacement, deletion or addition of or several amino acids (e.g., a replacement of 1 or 2 amino acids) as compared to SEQ ID NO:7, and

(vi) VL CDR3 consisting of SEQ ID NO:8, or a sequence having a substitution, deletion or addition of or several amino acids (e.g., a substitution of 1,2, 3, 4,5 or 6 amino acids) as compared to SEQ ID NO: 8;

wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof has or more of the following characteristics:

(i) VH CDR1 has a structure as X₁X₂AFX₃RFX₄(SEQ ID NO: 49);

(ii) VH CDR2 has a structure as X₅X₆GGX₇X₈X₉X₁₀(SEQ ID NO: 50);

(iii) VH CDR3 has, for example, ARHX₁₁X₁₂X₁₃TX₁₄AX₁₅X₁₆X₁₇(SEQ ID NO: 51);

(iv) VL CDR1 has a sequence as X₁₈X₁₉X₂₀X₂₁X₂₂YX₂₃YX₂₄F (SEQ ID NO: 52);

(v) VL CDR2 has a common reference position as RX₂₅A (SEQ ID NO: 53); and

(vi) VL CDR3 has a shape like QX₂₆X₂₇X₂₈X₂₉X₃₀PX₃₁(SEQ ID NO: 54);

wherein, X₁To X₃₁Independently of one another, are any amino acids.

In certain preferred embodiments, X₁To X₃₁Independently of one another, from alanine (A), arginine (R), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), histidine (H), isoleucine (I), glycine (G), aspartic acid (A), (B), (C), (N), leucine (L), lysine (K), methionine (M), phenylalanine (F), coenzyme (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

In certain preferred embodiments, X₁Selected from G or A, X₂Selected from F or A, X₃Selected from S or A, X₄Selected from D or A, X₅Selected from I or A, X₆To X₈Independently of one another, from G or A, X₉Selected from R or A, X₁₀Selected from T or A, X₁₁Selected from G or A, X₁₂Selected from T or A, X₁₃To X₁₄Independently of one another, from G or A, X₁₅Selected from M or A, X₁₆Selected from D or A, X₁₇Selected from Y or A, X₁₈Selected from K or A, X₁₉Selected from S or A, X₂₀Selected from V or A, X₂₁Selected from D or A, X₂₂Selected from N or A, X₂₃Selected from G or A, X₂₄Selected from S or A, X₂₅Selected from S or A, X₂₆Selected from Q or A, X₂₇Selected from S or A, X₂₈Selected from N or A, X₂₉Selected from E or A, X₃₀Selected from D or A, X₃₁Is selected from T or A.

In certain preferred embodiments, X₆Is G, X₇Is G, X₁₀Is T, X₁₁Is G, X₁₃Is G, X₁₄Is G, X₁₅Is M, X₂₃Is G, X₂₄Is S, X₂₆Is Q, X₃₁Is T.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof has or more of the following characteristics:

(1)X₁to X₄Up to of which are A;

(2)X₅to X₁₀Up to of which are A;

(3)X₁₁to X₁₇Up to of which are A;

(4)X₁₈to X₂₄Up to of which are A;

(5)X₂₆to X₃₁Up to of which are a.

In some preferredIn an embodiment, X₁To X₁₇Up to of which are a.

In certain preferred embodiments, X₁₈To X₃₁Up to of which are a.

In certain preferred embodiments, X₁To X₃₁Up to of which are a.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(3) VH CDR1 shown in SEQ ID NO. 3, VH CDR2 shown in SEQ ID NO.4, VH CDR3 shown in any of SEQ ID NOs:5 and 62-64, VL CDR1 shown in SEQ ID NO. 6, VL CDR2 shown in SEQ ID NO. 7, and VL CDR3 shown in SEQ ID NO. 8;

(4) VH CDR1 shown in SEQ ID NO. 3, VH CDR2 shown in SEQ ID NO.4, VH CDR3 shown in SEQ ID NO.5, VL CDR1 shown in any of SEQ ID NOs 6 and 65-69, VL CDR2 shown in SEQ ID NO. 7, and VL CDR3 shown in SEQ ID NO. 8;

(5) VH CDR1 shown in SEQ ID NO 3; VH CDR2 shown in SEQ ID NO. 4; and, VH CDR3 shown as SEQ ID NO. 5; VL CDR1 as shown in SEQ ID NO. 6; a VLCDR 2as set forth in SEQ ID NO. 7 or 70; and, VL CDR3 as set forth in SEQ ID NO. 8; or

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: VH CDR1 shown in SEQ ID NO. 3; VH CDR2 shown in SEQ ID NO. 4; and, VH CDR3 shown in SEQ ID NO. 5; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 6; VL CDR 2as shown in SEQ ID NO. 7; and, VL CDR3 as shown in SEQ ID NO. 8.

In still other embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(vi) VL CDR3 having the sequence of CDR3 contained in VL as shown in SEQ ID NO 10 or a sequence with a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VL.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 9;

and/or the presence of a gas in the gas,

(b) the variable region of the light chain (VL) as shown in SEQ ID NO 10 contains 3 CDRs.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 9; and/or, 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO: 10;

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are determined by the Kabat numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a) the following 3 heavy chain variable region (VH) CDRs:

and/or

(b) The following 3 light chain variable region (VL) CDRs:

wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: VH CDR1 shown in SEQ ID NO. 11; VH CDR2 shown in SEQ ID NO 12; and, VHCDR3 as set forth in SEQ ID NO. 13; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 14; VL CDR 2as shown in SEQ ID NO. 15; and, VL CDR3 as shown in SEQ ID NO 16.

In still other embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(vi) VL CDR3 having the sequence of CDR3 contained in VL as shown in SEQ ID NO:18 or a sequence with a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VL.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) the 3 CDRs contained in the variable region of the heavy chain (VH) shown in SEQ ID NO: 17;

and/or the presence of a gas in the gas,

(b) the variable region of the light chain (VL) shown in SEQ ID NO:18 contains 3 CDRs.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: the 3 CDRs contained in the variable region of the heavy chain (VH) shown in SEQ ID NO: 17; and/or, 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO: 18;

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are determined by the Kabat numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a) the following 3 heavy chain variable region (VH) CDRs:

and/or

(b) The following 3 light chain variable region (VL) CDRs:

wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: VH CDR1 shown in SEQ ID NO. 19; VH CDR2 shown as SEQ ID NO: 20; and, VHCDR3 as set forth in SEQ ID NO. 21; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 22; VL CDR 2as shown in SEQ ID NO. 23; and, VL CDR3 as shown in SEQ ID NO. 24.

In still other embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

(a) the following 3 heavy chain variable region (VH) Complementarity Determining Regions (CDRs):

and/or

(b) The following 3 light chain variable region (VL) CDRs:

(vi) VL CDR3 having the sequence of CDR3 contained in VL as shown in SEQ ID NO:26 or a sequence with a substitution, deletion or addition of or several amino acids (e.g. a substitution, deletion or addition of 1,2 or 3 amino acids) compared to the sequence of CDR3 contained in said VL.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) the 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 25;

and/or the presence of a gas in the gas,

(b) the variable region of the light chain (VL) as shown in SEQ ID NO 26 contains 3 CDRs.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: the 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 25; and/or, 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO: 26;

wherein the 3 CDRs contained in the heavy chain variable region (VH) and the 3 CDRs contained in the light chain variable region (VL) are determined by the Kabat numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a) the following 3 heavy chain variable region (VH) CDRs:

and/or

(b) The following 3 light chain variable region (VL) CDRs:

wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

In certain preferred embodiments, any of the substitutions (i) - (vi) described in items is a conservative substitution.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: VH CDR1 shown in SEQ ID NO. 27; VH CDR2 shown as SEQ ID NO 28; and, VHCDR3 as set forth in SEQ ID NO. 29; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 30; VL CDR 2as shown in SEQ ID NO. 31; and, VL CDR3 as shown in SEQ ID NO: 32.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention further comprises a Framework Region (FR) derived from a mammalian (e.g., murine or human) immunoglobulin.

In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) Framework Region (FR) derived from a murine immunoglobulin, and/or the VL of said antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a murine immunoglobulin.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are humanized. In certain preferred embodiments, the antibodies or antigen binding fragments thereof of the invention are humanized to a degree of at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) Framework Region (FR) derived from a human immunoglobulin, and/or the VL of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a human immunoglobulin in such embodiments, the heavy chain variable region FR and/or the light chain variable region FR of the antibody or antigen-binding fragment thereof of the present invention may comprise or more non-human (e.g., murine) amino acid residues, e.g., the heavy chain framework region FR and/or the light chain framework region FR may comprise or more amino acid back mutations in which there is a corresponding murine amino acid residue.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain framework region of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1,2, 3, 4, or 5 amino acids) in comparison to the sequence from which it is derived; and/or

(b) A light chain framework region of a human immunoglobulin or a variant thereof, which variant has conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1,2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise framework regions of a human immunoglobulin, e.g., framework regions comprised in an amino acid sequence encoded by a human germline antibody gene. In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region comprised in the amino acid sequence encoded by the human heavy chain germline gene and/or a light chain framework region comprised in the amino acid sequence encoded by the human light chain germline gene.

In certain preferred embodiments, the framework regions (heavy chain framework regions and/or light chain framework regions) comprise or more amino acid residues that are back-mutated to the corresponding murine residue or conservative amino acid substitution of the corresponding murine residue (such mutations are referred to as back mutations).

Thus, in certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a framework region of a human immunoglobulin (e.g., a framework region comprised in an amino acid sequence encoded by a human germline antibody gene), which optionally comprises or more back mutations from human residues to murine residues.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof comprise heavy chain framework regions comprised in the amino acid sequence encoded by IGVH3-23 x 04, and light chain framework regions comprised in the amino acid sequence encoded by IGKV3-11 x 01, the heavy chain framework regions and/or light chain framework regions optionally comprising or more back mutations from residues of human origin to residues of murine origin.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof comprise heavy chain framework regions comprised in the amino acid sequence encoded by IGVH1-2 x 02 and light chain framework regions comprised in the amino acid sequence encoded by IGKV1-16 x 01, the heavy chain framework regions and/or light chain framework regions optionally comprising or more back mutations from residues of human origin to residues of murine origin.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) 1, 9, 17, 25, 33, 35, 37, 39 or of SEQ ID NOs;

and/or

(b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) the sequence shown in any of SEQ ID NOs:2, 10, 18, 26, 34, 36, 38 and 40;

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to to the sequence represented by any of of SEQ ID NOs:2, 10, 18, 26, 34, 36, 38, 40.

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 1;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 2;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 9;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 10;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 17;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO:18, or a sequence shown in seq id no;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 25;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 26;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 33;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 34;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 35;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 36;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 37;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 38;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 39;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 40;

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(1) VH having a sequence shown as SEQ ID NO. 1and VL having a sequence shown as SEQ ID NO. 2;

(2) VH having a sequence shown as SEQ ID NO.9 and VL having a sequence shown as SEQ ID NO. 10;

(3) VH having a sequence shown as SEQ ID NO. 17 and VL having a sequence shown as SEQ ID NO. 18;

(4) VH having a sequence shown as SEQ ID NO. 25 and VL having a sequence shown as SEQ ID NO. 26;

(5) VH having a sequence shown as SEQ ID NO. 33 and VL having a sequence shown as SEQ ID NO. 34;

(6) VH having a sequence shown as SEQ ID NO. 35 and VL having a sequence shown as SEQ ID NO. 36;

(7) VH having a sequence shown as SEQ ID NO. 37 and VL having a sequence shown as SEQ ID NO. 38; or

(8) VH having the sequence shown as SEQ ID NO. 39 and VL having the sequence shown as SEQ ID NO. 40.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin or a variant thereof having a substitution, deletion, or addition of or more amino acids compared to the sequence from which it is derived.

In certain preferred embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having a substitution, deletion or addition of or more amino acids (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., a substitution, deletion or addition of 1,2, 3, 4 or 5 amino acids) compared to the sequence from which it is derived, and/or,

the light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1,2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

In certain preferred embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain preferred embodiments, the heavy chain constant region is a murine IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain preferred embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain embodiments, preferably the heavy chain constant region is a human IgG1 or IgG4 heavy chain constant region.

In certain preferred embodiments, the light chain constant region is a kappa light chain constant region. In certain preferred embodiments, the light chain constant region is a murine kappa light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.

In certain preferred embodiments, the antibodies of the invention are chimeric or humanized antibodies. In certain preferred embodiments, the antigen binding fragment of the invention is selected from the group consisting of scFv, Fab ', (Fab')₂Fv fragments, diabodies (diabodies).

The antibody or antigen-binding fragment thereof of the present invention has high specificity and high affinity for PD-1 (particularly human PD-1). In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention can be present at about 1 × 10^-10K of M or less_DBinding to PD-1 (particularly human PD-1); preferably at about 5 × 10^-11K of M or less_DBinds to PD-1 (especially human PD-1), or, alternatively, at about 1X 10^-11K of M or less_DBinding to PD-1 (in particular human PD-1).

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention have at least of the following biological functions:

(1) specifically recognizes/binds to PD-1 (particularly human PD-1);

(2) blocking the binding of PD-1 to PD-L1, or inhibiting and/or blocking intracellular signaling mediated by PD-1 binding to PD-L1;

(3) increasing immune cell (particularly T cell, e.g., antigen-specific T cell) activity in vitro and in vivo in a subject;

(4) increasing the level of secretion, proliferative activity, expression of an activation marker (e.g., CD25, CD69, etc.), and/or cell killing activity of an effector cytokine (e.g., IL-2, IFN- γ, etc.) by an immune cell (particularly a T cell, e.g., an antigen-specific T cell) in vitro and in vivo in a subject;

(5) enhancing immune responses (particularly T cell-mediated immune responses) in vitro and in a subject;

(6) preventing and/or treating a tumor (e.g., a tumor with microsatellite height instability (MSI-H) or mismatch repair deficiency (dMMR)) or an infection in a subject.

In the present invention, an antibody or antigen-binding fragment thereof of the present invention may include variants that differ from the antibody or antigen-binding fragment thereof from which it is derived only by conservative substitutions of or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids) amino acid residues, or that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the antibody or antigen-binding fragment thereof from which it is derived, and that substantially retain the above-described biological functions of the antibody or antigen-binding fragment thereof from which it is derived.

Derivatized antibodies

An antibody or antigen-binding fragment thereof of the invention can be derivatized, e.g., attached to another molecules (e.g., another polypeptides or proteins). generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment thereof does not adversely affect its binding to PD-1, particularly human PD-1. thus, an antibody or antigen-binding fragment thereof of the invention is also intended to include such derivatized forms. for example, an antibody or antigen-binding fragment thereof of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent linking, or otherwise) to or more other molecular groups, e.g., another antibodies (e.g., to form a bispecific antibody), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides (e.g., avidin or polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment to another molecules.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are labeled with a detectable label, such as an enzyme, radionuclide, fluorescent dye, luminescent material (e.g., chemiluminescent material), or biotin.³H、¹²⁵I、³⁵S、¹⁴C or³²P), fluorescent dyes (e.g., Fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), Phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine ester compounds), magnetic beads (e.g.,

) And measureThermal labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above patents which teach the use of such labels include, but are not limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference.) detectable labels as described above can be detected by methods known in the art.

In another aspects, the invention also provides conjugates comprising an antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

In certain preferred embodiments, the conjugate is an antibody-drug conjugate (ADC). In certain preferred embodiments, the therapeutic agent is selected from a cytotoxin or a radioisotope. Non-limiting examples of suitable therapeutic agents in the present invention include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is conjugated to the therapeutic agent, optionally through a linker. In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are conjugated to the therapeutic agent via a cleavable linker (e.g., a peptide linker, disulfide, or hydrazone linker).

Preparation of antibodies

The antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cells are then cultured under specific conditions and the antibodies of the invention are expressed.

Antigen-binding fragments of the invention may be obtained by hydrolysis of the whole antibody molecule (see Morimoto et al, J.Biochem.Biophys.methods 24:107-117(1992) and Brennan et al, Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol.11:548-557 (1999); Little et al, Immunol.today,21:364-370 (2000)). For example, Fab' fragments can be obtained directly from the host cell; fab 'fragments can be chemically coupled to form F (ab')₂Fragments (Carter et al, Bio/Technology,10: 163-. In addition, Fv, Fab or F (ab')₂The fragments may also be isolated directly from the culture medium of the recombinant host cell. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

Thus, in another aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region and/or a light chain variable region thereof.

In another aspects, the invention provides vectors (e.g., cloning vectors or expression vectors) comprising an isolated nucleic acid molecule of the invention.

In another aspect, the invention provides host cells comprising the isolated nucleic acid molecules of the invention or the vectors of the invention, such host cells include, but are not limited to, prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

In another aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

Methods of treatment and pharmaceutical compositions

The antibodies or antigen-binding fragments thereof of the present invention are useful for inhibiting and/or blocking intracellular signaling mediated by PD-1 binding to PD-L1, increasing immune cell activity, enhancing immune response, and for preventing and/or treating tumors or infections, in vitro or in a subject, or as an immunological adjuvant.

Thus, in another aspects, the invention provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the invention or a conjugate of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

In certain preferred embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as an additional immune checkpoint inhibitor, an oncolytic virus, a chemotherapeutic agent, an anti-angiogenic drug, an anti-metabolite drug, a tumor-targeting drug, an immunostimulant, and the like.

In certain preferred embodiments, the additional pharmaceutically active agent is a drug for treating an infection, such as an antiviral agent, an antifungal agent, an antibacterial agent, an immunostimulant, and the like.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof or conjugate of the invention and the additional pharmaceutically active agent are provided in the pharmaceutical composition as separate components or as components of a composition with .

In certain exemplary embodiments, the medicament comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.

In another aspect, the invention relates to the use of an antibody, or antigen-binding fragment or conjugate thereof, of the invention in the preparation of a medicament for:

(1) increasing immune cell (e.g., T cell) activity in vitro or in a subject (e.g., a human);

(2) enhancing an immune response (e.g., a T cell-mediated immune response) in a subject (e.g., a human);

(3) treating a tumor in a subject (e.g., a human); or

(4) Treating an infection in a subject (e.g., a human).

In certain preferred embodiments, the tumor is a tumor having microsatellite height instability (MSI-H) and/or mismatch repair deficiency (dMMR).

In another aspects, the invention provides immunogenic compositions comprising an antibody or antigen-binding fragment thereof of the invention, and an immunogen.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention act as adjuvants.

In certain preferred embodiments, the immunogen is selected from the group consisting of a tumor-associated antigen (e.g., a protein, polypeptide, or carbohydrate molecule), a tumor cell, a dendritic cell primed with the antigen, and any combination thereof.

In certain preferred embodiments, the immunogen is selected from the group consisting of an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen (e.g., a virus), an inactivated or attenuated pathogen, a dendritic cell primed by the antigen, and any combination thereof.

In certain preferred embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier and/or excipient. In certain preferred embodiments, the immunogenic composition comprises a stabilizer.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are provided as separate components from the immunogen or as components of a composition with in the immunogenic composition.

In another aspects, the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention as an adjuvant, or in the preparation of an immunogenic composition for enhancing an immune response (e.g., a T cell-mediated immune response) in a subject, wherein the immunogenic composition comprises an antibody or antigen-binding fragment thereof of the invention and an immunogen.

In another aspects, the invention provides methods of increasing the activity of an immune cell (e.g., a T cell) in vitro, the method comprising the step of contacting the immune cell with an antibody or antigen-binding fragment thereof of the invention.

In certain preferred embodiments, any suitable indicator can be used to measure the activity of the immune cells. Non-limiting examples of such suitable indicators include: in the presence of an antibody or antigen-binding fragment thereof of the invention, an immune cell (e.g., a T cell) has an increased level of cytokine (e.g., IL-2, IFN- γ, etc.) secretion, proliferative activity, and/or expression of an activation marker (e.g., CD25, CD69, etc.).

In certain preferred embodiments, the methods are used to treat tumors. In such embodiments, the immune cells obtained by the above methods can be adoptively transferred into a subject to treat a tumor. Activation in vitro in the presence of an antibody or antigen-binding fragment thereof of the invention is expected to increase the activity of adoptively transferred immune cells, thereby facilitating tumor killing of these adoptively transferred immune cells in a subject. In certain preferred embodiments, the immune cell is a tumor-infiltrating lymphocyte (e.g., a tumor-infiltrating T cell).

In certain exemplary embodiments, the additional pharmaceutically active agent is selected from, for example, IL-2, IL-3, IL-12, IL-15, IL-18, IFN- γ, IL-10, TGF- β, GM-CSF, and any combination thereof.

In certain preferred embodiments, the immune cell is a T cell, such as a cytotoxic T Cell (CTL), an antigen-specific T cell, or a tumor-infiltrating T cell (TIL-T). In certain exemplary embodiments, the immune cell is a tumor-infiltrating lymphocyte (e.g., a tumor-infiltrating T cell).

In another aspects, the invention provides methods of increasing immune cell activity and/or enhancing an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of an antibody (or antigen-binding fragment thereof) of the invention, a conjugate of the invention, a pharmaceutical composition of the invention, or an immunogenic composition of the invention.

In certain preferred embodiments, the immune response is a T cell-mediated immune response.

In certain preferred embodiments, the methods are used for the prevention and/or treatment of tumors. In such embodiments, the subject has a tumor. In certain preferred embodiments, the tumor is a tumor having microsatellite height instability (MSI-H) and/or mismatch repair deficiency (dMMR).

In certain preferred embodiments, the methods are used to prevent and/or treat infection. In such embodiments, the subject has an infection. In certain preferred embodiments, the infection is a viral infection, such as a chronic viral infection.

In another aspects, the invention provides methods for preventing and/or treating a tumor in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of an antibody (or antigen-binding fragment thereof) of the invention, a conjugate of the invention, a pharmaceutical composition of the invention, or an immunogenic composition of the invention.

In certain preferred embodiments, the antibodies (or antigen-binding fragments thereof) or conjugates of the invention are used in combination with another drug having anti-tumor activity. Such additional agents having anti-tumor activity may be administered prior to, concurrently with, or subsequent to the administration of the antibody (or antigen-binding fragment thereof) or conjugate.

In certain preferred embodiments, the antibodies (or antigen-binding fragments thereof), conjugates, pharmaceutical compositions, or immunogenic compositions of the invention are administered in combination with additional therapies. Such additional therapy may be any therapy known for tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy, or palliative therapy. Such additional therapies may be administered prior to, concurrently with, or subsequent to the administration of the antibody (or antigen-binding fragment thereof), conjugate, pharmaceutical composition, or immunogenic composition of the invention.

In another aspects, the invention provides methods for preventing and/or treating infection in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of an antibody (or antigen-binding fragment thereof) of the invention, a conjugate of the invention, a pharmaceutical composition of the invention, or an immunogenic composition of the invention.

In certain preferred embodiments, the antibodies (or antigen-binding fragments thereof) or conjugates of the invention are used in combination with another agent for treating infection. Such additional drugs for treating infection may be administered prior to, concurrently with, or subsequent to administration of the antibody (or antigen-binding fragment thereof) or conjugate.

In certain preferred embodiments, the antibodies (or antigen-binding fragments thereof) of the present invention are used in combination with a vaccine. In certain preferred embodiments, the vaccine can be an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen, an inactivated or attenuated pathogen, a dendritic cell primed by the antigen, or any combination thereof. In certain preferred embodiments, the pathogen may be a virus (e.g., hepatitis a, b, c virus, human immunodeficiency virus, human papilloma virus, or herpes virus), a fungus, a bacterium, or a parasite.

In the present invention, non-limiting examples of the tumor include melanoma (e.g., metastatic malignant melanoma), breast cancer, kidney cancer (e.g., clear cell cancer), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell cancer, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, and hematological malignancies (e.g., lymphoma, leukemia), and the like.

It has been previously reported that Microsatellite Instability (MSI) is tumor phenotypes associated with inactivating changes in the DNA Mismatch Repair (MMR) gene, which are present in up to 24 types of tumors MSI is generally expressed as any change in the length of a Microsatellite in tumor tissue due to insertion or deletion of repeat units compared to normal tissue.

According to the classification criteria for MSI detection established by the National Cancer Institute (NCI) (Boland CR, equivalent. cancer Res.1998Nov 15; 58(22):5248-57.), BAT26, BAT25, D2S123, D5S346, D17S250 for 5 microsatellite standard sites, as compared to normal tissue, are judged to be microsatellite highly unstable (MSI-H), i.e., mismatch repair function defective (dMMR), if tumor tissue contains 2 or more sites of instability; if less than 2 or no bit instabilities, then microsatellite low instability (MSI-L) or microsatellite stability (MSS), i.e., complete mismatch repair function (pMMR), is determined.

Methods for detecting microsatellite height instability (MSI-H) or mismatch repair deficiency (dMMR) are known to those skilled in the art, and MSI-H can be determined by detecting length change of the above 5 standard microsatellite loci in tumor tissue, for example, by PCR or secondary sequencing, and when 2 or more loci are unstable, expression of MLH1, MSH2, MSH6 and PSM2 in tumor tissue can be detected by Immunohistochemistry (ICH), and if all four proteins are expressed positively, pMMR is determined, and if proteins are expressed negatively, dMMR is determined.

Thus, in certain preferred embodiments, the tumor according to the invention is a tumor with microsatellite height instability (MSI-H) and/or mismatch repair deficiency (dMMR). In certain preferred embodiments, the tumor is selected from the group consisting of colon adenocarcinoma, esophageal carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, uterine body and endometrial carcinoma, and the like.

In the present invention, the infection refers to any infection caused by any pathogenic microorganism such as virus, bacteria, fungi, parasite, etc. Non-limiting examples of such viruses include hepatitis A, B, C, human immunodeficiency virus, human papilloma virus or herpes virus, and the like; non-limiting examples of such bacteria include chlamydia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, and the like; non-limiting examples of such fungi include Trichophyton, Epidermophyton, Microsporum, Candida albicans, Cryptococcus neoformans, and the like; non-limiting examples of such parasites include plasmodium, schistosoma, leishmania donovani, filarial worms, hookworms, and the like.

The antibody or antigen-binding fragment thereof of the present invention, the conjugate of the present invention, the pharmaceutical composition of the present invention, or the immunogenic composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, and the like, the preferred dosage forms depend on the intended mode of administration and therapeutic use.

Furthermore, the antibody or antigen-binding fragment thereof of the invention or the conjugate of the invention may be present in a pharmaceutical or immunogenic composition in unit dosage form for ease of administration.

The antibodies or antigen binding fragments thereof, conjugates, pharmaceutical compositions or immunogenic compositions of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes.

A "prophylactically effective amount" refers to an amount sufficient to prevent, or delay the onset of disease, a "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent disease and its complications in a patient already suffering from disease.

In the present invention, the dosage regimen may be adjusted to achieve the optimal desired response (e.g., therapeutic or prophylactic response.) for example, a single administration may be given, multiple administrations may be given over periods of time, or the dosage may be reduced or increased proportionally to the exigencies of the therapeutic situation.

A typical non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof of the invention is 0.01-100 mg/kg, e.g., 0.1-100 mg/kg, 0.1-50 mg/kg, or 1-50 mg/kg. it is noted that the dosage may vary depending on the type and severity of the condition to be treated, furthermore, one skilled in the art understands that for any specific patient, the particular dosage regimen will be adjusted over time according to the patient's needs and the professional assessment of the physician, and the dosage ranges given herein are for illustrative purposes only and are not limiting as to the use or scope of a pharmaceutical or immunogenic composition of the invention.

In the present invention, the subject may be a mammal, such as a human.

Detection method and kit

The antibodies or antigen-binding fragments thereof of the present invention are capable of specifically binding to PD-1and thus can be used to detect the presence or level of PD-1 in a sample.

Thus, in another aspects, the invention provides kits comprising an antibody or antigen-binding fragment thereof of the invention in certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention bears a detectable label in preferred embodiments the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the invention.

In the present invention, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means.³H、¹²⁵I、³⁵S、¹⁴C or³²P), fluorescent dyes (e.g., Fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), Phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine ester compounds), magnetic beads (e.g.,

) For example, radioactive labels can be detected using photographic film or scintillation calculators, fluorescent labels can be detected using photodetectors to detect emitted light, enzyme labels are generally detected by providing a substrate for the enzyme and detecting the light emitted to the substrate by the enzyme, and enzyme labels are generally detected by providing a substrate for the enzyme and detecting the light by the enzyme to the substrateThe reaction products resulting from the action of the substance are detected, and the calorimetric markers are detected by simply visualizing the colored marker. In certain embodiments, a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

In another aspects, the invention provides a method of detecting the presence or level of PD-1 in a sample, comprising the step of using an antibody or antigen-binding fragment thereof of the invention in preferred embodiments, the antibody or antigen-binding fragment thereof of the invention also carries a detectable label in another preferred embodiments, the method further comprises using a reagent carrying a detectable label to detect the antibody or antigen-binding fragment thereof of the invention.

In another aspects, there is provided use of an antibody or antigen-binding fragment thereof of the invention in the preparation of a kit for detecting the presence or level of PD-1 in a sample.

Definition of terms

In the present invention, unless otherwise defined, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art, and the procedures of cell culture, biochemistry, nucleic acid chemistry, immunology laboratories and the like used herein are all conventional procedures used widely in in the corresponding field.

As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having Light Chains (LC) and Heavy Chains (HC), the antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains, the heavy chains can be classified as mu, delta, gamma, α, or epsilon, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and ige, respectivelyWithin chains and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, the heavy chain also comprises a "D" region of about 3 or more amino acids, each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH 3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of domains CL.the constant domains are not directly involved in antibody binding to antigen, but exhibit multiple effector functions, such as mediating binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the component of the classical complement system (C1 q). the VH and VL regions can also be subdivided into regions of high denaturation (termed Complementarity Determining Regions (CDRs)) between which there are relatively heavy regions termed Framework Regions (FRs)_HAnd V_LBy the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antigen-binding sites, respectively. The distribution of amino acids in each region or domain may follow Kabat, Sequences of Proteins of Immunological Interest (national institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia&Lesk (1987) J.mol.biol.196: 901-917; chothia et al (1989) Nature 342: 878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues may be defined according to various numbering systems known in the art, for example as defined in the Kabat numbering system (Kabat et al, Sequences of Proteins of immunological interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md.,1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol.biol.196: 901-917; Chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, Dev.Complex.Immunol.27: 55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al, Dev. company. Immunol.27:55-77,2003).

In the present invention, the CDRs contained in the antibodies of the present invention or antigen binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia, or IMGT numbering system. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat numbering system.

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch.7(Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes₂Fd, Fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies), linear antibodies (linear antibodies), nanobodies (technology from Domanitis), domain antibodies (technology from Ablynx), probody, and polypeptides comprising at least portions of an antibody sufficient to confer specific antigen binding capability to the polypeptideDescribed in Holliger et al, 2005; nat Biotechnol,23: 1126-.

As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains", wherein "full-length heavy chain" refers to a polypeptide chain consisting of, in the N-terminal to C-terminal direction, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain, and, when the full-length antibody is of IgE isotype, optionally further comprises a heavy chain constant region CH4 domain, preferably "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction, "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.

As used herein, the term "Fd fragment" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al, Nature 341: 544546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F (ab')₂Fragment "means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reductively linked F (ab')₂The fragment obtained after disulfide bonding of the two heavy chain fragments in the fragment consists of intact Fd fragments of the light and heavy chains (consisting of VH and CH1 domains).

As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. the Fv fragment is generally considered to be the smallest antibody fragment that can form the complete antigen binding site. generally considers that six CDRs confer antigen binding specificity on an antibody, however, even variable regions (e.g., the Fd fragment, which contains only three CDRs specific for an antigen) are capable of recognizing and binding antigen, although their affinity may be lower than the complete binding site.

As used herein, the term "Fc fragment" means an antibody fragment formed by disulfide bonding of the second and third constant regions of the th heavy chain of an antibody to the second and third constant regions of the second heavy chain.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (linker) (see, e.g., Bird et al, Science 242:423-₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS)₄Linker of (1), although variants thereof may also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but that a linker is used that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger P. et al, Proc. Natl. Acad. Sci. USA 90: 6444-.

As used herein, the term "probody" has the meaning commonly understood by those of skill in the art, which refers to masked antibodies that remain inert in healthy tissue but are specifically activated in the disease environment (e.g., via protease cleavage by proteases that are either enriched or characteristic of the disease environment.) detailed teachings thereof can be found, for example, in Desnoyers et al, sci.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or competes with the full length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of antibodies are specifically screened for specificity in the same manner as for intact antibodies.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

As used herein, the terms "monoclonal antibody," "mAb" have the same meaning and are used interchangeably to refer to antibodies or fragments of antibodies from a highly homologous population of antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously.

Monoclonal antibodies of the invention can be prepared by a variety of techniques, such as hybridoma techniques (see, e.g., Kohler et al, Nature,256:495,1975), recombinant DNA techniques (see, e.g., U.S. Pat. No.4,816,567), or phage antibody library techniques (see, e.g., Clackson et al, Nature 352: 624-.

Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G. Subsequently or alternatively, the specific antigen (the target molecule recognized by the antibody) or an epitope thereof may be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography. Purification of immunoglobulins can be found, for example, in D.Wilkinson (The Scientist, public shared by The Scientist, Inc., Philadelphia Pa., Vol.14, No.8(Apr.17,2000), pp.25-28).

As used herein, the term "Chimeric antibody" refers to an antibody whose portion of the light or/and heavy chain is derived from antibodies (which may be derived from a certain specific species or belong to a certain specific antibody class or subclass) and another portion of the light or/and heavy chain is derived from another antibodies (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which nevertheless retains binding activity to an antigen of interest (u.s.p4,816,567to harvesting et al.; Morrison et al, proc.natl.acad.sci.usa,81:68516855(1984)) for example, the term "Chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., murine antibody) and the heavy and light chain variable regions of the antibody are derived from a second antibody (e.g., human antibody).

As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity, the ability to enhance an immune response, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having a desired property (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and/or ability to enhance an immune response).

Humanized antibodies are particularly advantageous because they retain both the desirable properties of non-human donor antibodies (e.g., murine antibodies) and are effective in reducing the immunogenicity of non-human donor antibodies (e.g., murine antibodies) in human subjects. However, due to matching issues between the CDRs of the donor antibody and the FRs of the acceptor antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and/or ability to enhance an immune response) are generally lower than non-human donor antibodies (e.g., murine antibodies).

In the present invention, in order for the humanized antibody to retain as much as possible the properties of the donor antibody (including, for example, antigen specificity, affinity, reactivity, ability to enhance the activity of immune cells and/or ability to enhance the immune response), the Framework Regions (FRs) in the humanized antibody of the present invention may comprise both the amino acid residues of the recipient antibody of human origin and the amino acid residues of the corresponding donor antibody of non-human origin.

In the present application, the desired properties of the antibodies of the invention include (1) specific recognition/binding of PD-1 (especially human PD-1), (2) the ability to block the binding of PD-1 to PD-L1, (3) the ability to inhibit and/or block intracellular signaling mediated by PD-1 binding to PD-L1, (4) the ability to increase the activity of immune cells (especially T cells, such as antigen-specific T cells), (5) the ability to enhance immune responses (especially T cell-mediated immune responses), (6) the ability to prevent and/or treat tumors, (7) the ability to prevent and/or treat infections.

The chimeric antibody or humanized antibody of the present invention can be prepared based on the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chains can be obtained from a murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

For making chimeric antibodies, the murine immunoglobulin variable region may be linked to a Human immunoglobulin constant region using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to Cabilly et al.) for example, DNA encoding VH may be operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene the sequence of the Human heavy chain constant region gene is known in the art (see, e.g., Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Humanservices, NIH Publication No. 91-42), DNA fragments comprising these regions may be amplified by standard PCR the heavy chain constant region may be 1, IgG2, IgG3, IgG4, IgA, IgM or IgD constant regions, but typically IgG2 or 4 is preferably amplified, e.g., the constant region may be linked to a Human immunoglobulin constant region, light chain constant region, e.g., Fab, light chain constant region is preferably obtained by standard PCR, light chain constant region (see, light chain) Sequences of Human immunoglobulin constant region, light chain, see, light chain constant region, preferably DNA of Fab, SEQ ID No. 3.

To make humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see, e.g., U.S. Pat. No.5,225,539 to Winter; U.S. Pat. No.5,530,101 to Queen et al; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, K.C., editor, in Antibody Engineering: methods and Protocols, volume 248, Humana Press, N.J., 2004). Alternatively, transgenic animals can also be used which do not produce endogenous immunoglobulins after immunization andand is capable of generating a repertoire of fully human antibodies. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production, and then transfer of a human germline immunoglobulin gene array into the germline mutant mice results in the mice producing human antibodies upon encountering antigen stimulation (see, e.g., Jakobovits et al, 1993, Proc. Natl. Acad. Sci. USA 90: 2551; Jakobovits et al, 1993, Nature 362: 255-. Non-limiting examples of such transgenic animals include, HuMAb mice (Metarex, Inc.) that contain a human immunoglobulin gene minilocus (minioci) encoding unrearranged human heavy (μ and γ) and kappa light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and kappa chain loci (see, e.g., Lonberg et al (1994) Nature 368(6474): 856-859); or "KM mice carrying human heavy chain transgenes and human light chain transchromosomes^TM"(see patent application WO 02/43478). Other methods of humanizing antibodies include phage display techniques (Hoogenboom et al, 1991, J.mol.biol.227: 381; Marks et al, J.mol.biol.1991, 222: 581 597; Vaughan et al, 1996, Nature Biotech 14: 309).

As used herein, the term "degree of humanization" is an index used to evaluate the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be calculated, for example, by: the degree of humanization is (number of amino acids in the FR region-number of non-human amino acids remaining in the FR region)/number of amino acids in the FR region × 100%.

As used herein, the term "germline antibody gene (germline antibody gene)" or "germline antibody gene segment (germline antibody gene segment)" refers to immunoglobulin-encoding sequences present in the genome of an organism that have not undergone a maturation process that can lead to genetic rearrangements and mutations that express specific immunoglobulins. In the present invention, the expression "heavy chain germline gene" means the germline antibody gene or gene segment encoding the immunoglobulin heavy chain, which includes the V gene (variable), the D gene (diversity), the J gene (conjugation), and the C gene (constant); similarly, the expression "light chain germline gene" refers to germline antibody genes or gene segments encoding immunoglobulin light chains, which include the V gene (variable), the J gene (junction), and the C gene (constant). In the present invention, the amino acid sequence encoded by the germline antibody gene or the germline antibody gene segment is also referred to as "germline sequence". Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from specialized databases (e.g., IMGT, unsmig, NCBI, or VBASE 2).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction may be the equilibrium dissociation constant (K) of the interaction_D) And (4) showing. In the present invention, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody has less than about 10^-9M, e.g. less than about 10^-9M、10^-10M、10^-11M or 10^-12M or less affinity (K)_D) Binding the antigen. Specific binding properties between two molecules can be determined using methods well known in the art, for example in a BIACORE instrument using Surface Plasmon Resonance (SPR).

As used herein, the term "vector" refers to nucleic acid delivery vehicles into which a polynucleotide may be inserted when the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector, the vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements it carries are expressed in the host cell.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

As used herein, the term "homo " is used to refer to the match of sequences between two polypeptides or between two nucleic acids when a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine or a position in each of two polypeptides is occupied by lysine), then each molecule is at that position in the same 567. the "homo " between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions being compared x 100. for example, if there are 6 matches in 10 of the two sequences, then the two sequences have 60% homo . for example, DNA sequences CTGACT and CAGGTT share 50% homo (3 of the total 6 positions) typically, when the two sequences are aligned to produce maximum homo . such comparisons can be made using alignment programs such as the alignment machines, e.g. the alignment can be carried out using the alignment algorithm, e.g. the alignment algorithm, the alignment of the homology 3. 7. the homology, 12. the alignment algorithm (see 7. the alignment algorithm) and the alignment algorithm (see the alignment algorithm, the alignment of homology, 12. 7. the alignment of the sequences can be determined using the alignment algorithm, 12. the alignment of homology, 12. 7. the alignment of the sequences, 12. 3. 7. 3. the alignment of the sequences (see [ 12. 3. 7. 3. the alignment of.

As used herein, the term "conservative substitutions" means amino acid substitutions that do not adversely affect or alter the intended properties of the Protein/polypeptide comprising the amino acid sequence.conservative substitutions may be introduced, for example, by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.conservative substitutions include substitutions in which amino acid residues are replaced with amino acid residues having similar side chains, for example, substitutions with residues that are physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.. families of amino acid residues having similar side chains have been defined in the art. these families include those having basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β branching side chains (e.g., threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, phenylalanine, valine, phenylalanine, tyrosine, tryptophan, tyrosine, tryptophan, tyrosine, tryptophan.

The twenty conventional amino acids referred to herein are written following conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)) which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19the d. pennsylvania: machine Publishing Company,1995), and include, but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.

As used herein, the term "prevention" refers to a method performed in order to prevent or delay the onset of a disease or disorder or symptom (e.g., tumor or infection) in a subject. As used herein, the term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., no longer worsening) of the state of the disease, delay or slowing of disease progression, amelioration or palliation of the state of the disease, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to prolonging survival as compared to expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, e.g., a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor or an infection, or is at risk for having a disease as described above.

As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect.A prophylactically effective amount, e.g., a tumor or infection, refers to an amount sufficient to prevent, arrest, or delay the onset of a disease, e.g., a tumor or infection, a therapeutically effective amount, e.g., an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.

As used herein, the term "immune cell" includes cells having hematopoietic origin and functioning in an immune response, such as lymphocytes, e.g., B cells and T cells; a natural killer cell; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes. In certain preferred embodiments, the immune cell is a T cell, such as a cytotoxic T Cell (CTL), an antigen-specific T cell, or a tumor-infiltrating T cell (TIL-T).

As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen presenting cells, phagocytes, or granulocytes) and soluble macromolecules produced by the immune cells or liver (including antibodies, cytokines, and complement) that results in the selective damage, destruction, or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation from the human body. In certain preferred embodiments, the immune response is a T cell-mediated immune response that results when the T cell-specific antigen stimulates the T cell. Non-limiting examples of responses produced by T cells upon antigen-specific stimulation include T cell proliferation and cytokine (e.g., IL-2) production.

Advantageous effects of the invention

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

(1) the antibody of the invention can not only specifically recognize/bind PD-1and block the binding of PD-1and PD-L1, but also enhance the activity of immunocytes in vitro/in vivo and stimulate immune response. Thus, the antibodies of the invention have potential for use in the prevention and/or treatment of tumors or infections.

(2) The antibodies (particularly humanized antibodies) of the invention not only retain the functions and properties of the parent murine antibody, thereby having potential for the prevention and treatment of tumors or infections; but also has a high degree of humanisation so that it can be safely administered to human subjects without eliciting an immunogenic response. It is particularly surprising that the antibodies of the invention have significantly improved blocking activity and the ability to induce T cell activation compared to commercial anti-PD-1 antibodies. Thus, the antibodies of the invention (particularly humanized antibodies) are of significant clinical value.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1A schematically shows the principle of ELISA for detecting the binding activity between an antibody and PD-1/His.

FIG. 1B schematically shows the principle of FACS method for detecting the binding activity between an antibody and human PD-1 expressed on the cell surface.

FIG. 1C schematically shows the principle of ELISA to detect the ability of antibodies to block the binding of human PD-1/His to PD-L1/Fc.

FIG. 2 shows the results of detection of the binding activity of the murine antibodies (16F2, 10D6, 48G 7and 17D5) to human PD-1and murine PD-1 by ELISA in example 1.

FIG. 3 shows the results of the detection of the binding activity of murine antibodies (16F2, 10D6, 48G 7and 17D5) to cell surface-expressed human PD-1 by FACS method in example 2.

FIG. 4 shows the results of the detection of the ability of murine antibodies (16F2, 10D6, 48G 7and 17D5) to block the binding between human PD-1/His and PD-L1/Biotin by the CLEIA method in example 2. Control refers to a positive Control group in which PBS was used instead of anti-PD-1 monoclonal antibody.

FIG. 5 shows the inhibition rate curves of murine antibodies (16F2, 10D6, 48G 7and 17D5) on binding between human PD-1/His and PD-L1/Biotin in example 2. Control refers to a positive Control group in which PBS was used instead of anti-PD-1 monoclonal antibody.

FIG. 6 shows the results of measuring the level of IL-2 secretion induced by murine antibodies (16F2, 10D6, 48G 7and 17D5) in example 4, wherein control refers to a negative control group to which an anti-PD-1 antibody was not added.

FIG. 7 shows the results of detection of the antigen-binding activity of the chimeric antibodies 17D5-cAb and 48G7-cAb by ELISA in example 5.

FIG. 8 shows the results of the detection of the binding activity of humanized antibodies 17D5H1, 17D5H2, 48G7H1, 48G7H2 by the CLEIA method in example 6. Wherein PEM is Pembrolizumab, NIV is Nivolumab.

FIG. 9 shows the results of the detection of the blocking activity of humanized antibodies 17D5H1, 17D5H2, 48G7H1, 48G7H2 by the CLEIA method in example 6. Wherein PEM is Pembrolizumab, NIV is Nivolumab.

FIG. 10 shows the results of measurement of IL-2 secretion levels induced by humanized antibodies 17D5H1, 17D5H2, 48G7H1, 48G7H2 in example 6. Wherein PEM is Pembrolizumab, NIV is Nivolumab, and control is negative control group without anti-PD-1 antibody.

Sequence information

Information on the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

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