阅读说明：本技术 抗cd47抗体及其应用 (anti-CD 47 antibodies and uses thereof ) 是由 王勇 赵立文 宋其峰 朱永强 张亚楠 韩璐薇 金亮 吴文明 于 2019-08-30 设计创作，主要内容包括：涉及抗体药物技术领域,尤其涉及一种抗CD47抗体或其抗原结合片段,包含抗CD47抗体或其抗原结合片段的药物组合物以及它们的应用。本发明的抗CD47抗体或其抗原结合片段具有显著的抗肿瘤活性,与人CD47蛋白的亲和力较高,可阻断SIRPa结合到细胞表面CD47的能力,且不具有显著的红细胞凝集活性,可在制备抗肿瘤的药物中应用。(Relates to the technical field of antibody medicines, in particular to an anti-CD 47 antibody or an antigen binding fragment thereof, a pharmaceutical composition containing the anti-CD 47 antibody or the antigen binding fragment thereof and application of the pharmaceutical composition. The anti-CD 47 antibody or the antigen binding fragment thereof has obvious anti-tumor activity, has higher affinity with human CD47 protein, can block the capacity of combining SIRPa to cell surface CD47, does not have obvious erythrocyte agglutination activity, and can be applied to the preparation of anti-tumor drugs.)

An anti-CD 47 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

(1) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 selected from the group consisting of:

(a1) amino acid sequences as shown in SEQ ID NO 1, 2 and 3;

(a2) amino acid sequences shown as SEQ ID NO 10, 2 and 11;

(a3) amino acid sequences as shown in SEQ ID NO 4, 5 and 6; and

(a4) amino acid sequences shown as SEQ ID NO. 7, 8 and 9;

(a5) a CDR having at least 85% sequence identity to the amino acid sequence set forth in (a1), (a2), (a3), or (a 4); and

(2) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 selected from the group consisting of:

(a6) amino acid sequences as shown in SEQ ID NO 12, 13 and 14;

(a7) amino acid sequences as shown in SEQ ID NO 15, 16 and 17; and

(a8) 18, 19 and 20;

(a9) a CDR having at least 85% sequence identity to the amino acid sequence set forth in (a6), (a7) or (a 8).

The anti-CD 47 antibody or antigen-binding fragment thereof of claim 1, having:

the HCDR1, HCDR2 and HCDR3 are heavy chain variable regions of SEQ ID NOs 1, 2 and 3 or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 are light chain variable regions of SEQ ID NOs 12, 13 and 14 or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 12, 13 and 14, respectively;

the HCDR1, HCDR2 and HCDR3 are heavy chain variable regions of SEQ ID NOs 10, 2 and 11, respectively, or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 10, 2 and 11, and the LCDR1, LCDR2 and LCDR3 are light chain variable regions of SEQ ID NOs 12, 13 and 14, respectively, or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 12, 13 and 14;

the HCDR1, HCDR2 and HCDR3 are heavy chain variable regions of SEQ ID NOs 4, 5 and 6, respectively, or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 4, 5 and 6, and the LCDR1, LCDR2 and LCDR3 are light chain variable regions of SEQ ID NOs 15, 16 and 17, respectively, or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 15, 16 and 17; or

The HCDR1, HCDR2 and HCDR3 are heavy chain variable regions of SEQ ID NOs 7, 8 and 9, respectively, or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 7, 8 and 9, and the LCDR1, LCDR2 and LCDR3 are light chain variable regions of SEQ ID NOs 18, 19 and 20, respectively, or CDRs having at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs 18, 19 and 20.

The anti-CD 47 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a murine monoclonal antibody, a human murine chimeric antibody, or a humanized antibody.

The anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein:

(1) the amino acid sequence of the heavy chain variable region is selected from:

(b1) the amino acid sequence shown as SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 27;

(b2) (b1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b1), and has the same or similar function with the amino acid sequence shown in (b 1); and

(b3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 1); and

(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:

(b4) amino acid sequences shown as SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 28;

(b5) (b4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (b4), and has the same or similar function with the amino acid sequence shown in (b 4); and

(b6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (b 4).

The CD47 antibody or antigen-binding fragment thereof of claim 4, wherein the antibody further comprises a heavy chain constant region of murine IgG1, IgG2, or variants thereof, and a light chain constant region of murine k chain or variants thereof.

The anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein

The amino acid sequence of the heavy chain variable region is SEQ ID NO 21, the amino acid sequence of the heavy chain variable region is obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO 21 and has the same function with the SEQ ID NO 21 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 21, the amino acid sequence of the light chain variable region is SEQ ID NO 24, the amino acid sequence of the light chain variable region is obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO 24 and has the same function with the SEQ ID NO 24 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 24;

the amino acid sequence of the heavy chain variable region is SEQ ID NO. 22, the amino acid sequence of the SEQ ID NO. 22 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO. 22 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO. 22, the amino acid sequence of the light chain variable region is SEQ ID NO. 25, the amino acid sequence of the SEQ ID NO. 25 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO. 25 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO. 25;

the amino acid sequence of the heavy chain variable region is SEQ ID NO:23, the amino acid sequence of the SEQ ID NO:23, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:23 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO:23, the amino acid sequence of the light chain variable region is SEQ ID NO:26, the amino acid sequence of the SEQ ID NO:26, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:26 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO: 26;

the amino acid sequence of the heavy chain variable region is SEQ ID NO:27, the amino acid sequence of the SEQ ID NO:27 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:27 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO:27, the amino acid sequence of the light chain variable region is SEQ ID NO:24, the amino acid sequence of the SEQ ID NO:24 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:24 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO: 24; or

The amino acid sequence of the heavy chain variable region is SEQ ID NO. 23, the amino acid sequence of the SEQ ID NO. 23 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO. 23 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO. 23, the amino acid sequence of the light chain variable region is SEQ ID NO. 28, and the amino acid sequence of the SEQ ID NO. 28 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO. 28 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO. 28.

The anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the anti-CD 47 antibody is a humanized antibody, wherein:

(1) the amino acid sequence of the heavy chain variable region is selected from:

(c1) the amino acid sequences shown as SEQ ID NO. 29, SEQ ID NO. 30 and SEQ ID NO. 31;

(c2) (c1) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (c1), and has the same or similar function with the amino acid sequence shown in (c 1); and

(c3) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (c 1); and

(2) the variable region of the light chain has an amino acid sequence selected from the group consisting of:

(c4) an amino acid sequence shown as SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34;

(c5) (c4) is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (c4), and has the same or similar function with the amino acid sequence shown in (c 4); and

(c6) an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in (c 4).

The anti-CD 47 antibody or antigen-binding fragment thereof of claim 7, wherein

The amino acid sequence of the heavy chain variable region is SEQ ID NO:29, the amino acid sequence of the SEQ ID NO:29 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:29 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO:29, the amino acid sequence of the light chain variable region is SEQ ID NO:32, the amino acid sequence of the SEQ ID NO:32 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO:32 or the amino acid sequence with at least 85 percent of sequence identity with the SEQ ID NO: 32;

the amino acid sequence of the heavy chain variable region is SEQ ID NO 30, the amino acid sequence of the SEQ ID NO 30, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 30 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 30, the amino acid sequence of the light chain variable region is SEQ ID NO 33, the amino acid sequence of the SEQ ID NO 33, which is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO 33 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO 33; or

The amino acid sequence of the heavy chain variable region is SEQ ID NO. 31, the amino acid sequence of the SEQ ID NO. 31 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO. 31 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO. 31, the amino acid sequence of the light chain variable region is SEQ ID NO. 34, and the amino acid sequence of the SEQ ID NO. 34 is obtained by substituting, deleting or adding one or more amino acids and has the same function with the SEQ ID NO. 34 or the amino acid sequence with at least 85% of sequence identity with the SEQ ID NO. 34.

An isolated nucleic acid encoding the anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-8.

The nucleic acid of claim 9, wherein:

(1) the nucleotide sequence for coding the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO 35, SEQ ID NO 36 or SEQ ID NO 37;

(2) the nucleotide sequence of the amino acid sequence of the light chain variable region is shown as SEQ ID NO 38, SEQ ID NO 39 or SEQ ID NO 40.

An expression vector comprising the nucleic acid of claim 9 or 10.

A host cell transformed with the expression vector of claim 11, said host cell being selected from the group consisting of prokaryotic and eukaryotic cells, preferably mammalian cells.

A method of making the anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-8, comprising the steps of expressing the antibody in the host cell of claim 12, and isolating the antibody from the host cell.

A pharmaceutical composition comprising the anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-8 and a pharmaceutically acceptable carrier.

Use of the anti-CD 47 antibody or antigen-binding fragment thereof of any one of claims 1-8 or the pharmaceutical composition of claim 14 in the manufacture of a medicament for inhibiting CD47 activity.

The use according to claim 15, wherein the medicament inhibiting CD47 activity is for the treatment of leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and/or melanoma.