Anti-CD47 monoclonal antibody and use thereof

Abstract

To provide antibody medicaments with high affinity for CD47, higher efficacy and fewer toxic side effects for treating tumors.SOLUTION: The present invention relates to an anti-CD47 monoclonal antibody and use thereof, wherein the antibody is secreted by the hybridoma cell line deposited under CCTCC NO: C2018135.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of autoimmune disease treatment and molecular immunology, particularly to anti-CD47 antibodies, pharmaceutical compositions containing same, and uses thereof. More particularly, the present invention relates to anti-CD47 monoclonal antibodies.

[0002] background CD47, also known as integrin-associated protein (IAP), is a five-span transmembrane protein with a molecular weight of approximately 50 kDa and belongs to the immunoglobulin superfamily. Its extracellular N-terminus is an IgV domain, which binds to αvβ3 (CD51 / CD61) and αIIbβ3 (CD41 / CD61) integrins. CD47 is involved in various physiological functions, such as cell transplantation, T cell and dendritic cell (DC) activation, and axon formation.

[0003] CD47 is expressed on all cell types, including erythrocytes, and is highly expressed on tumor cells. CD47 has two ligands: signal-regulatory protein-α (SIRPα) and thrombospondin-1 (TSP1). SIRPα, an immunoglobulin domain-containing receptor transmembrane glycoprotein, belongs to the SIRP family and is primarily expressed on macrophages and neurons. In the CD47-SIRPα pathway, CD47 binds to SIRPα, phosphorylating its immunoreceptor tyrosine-based inhibitory motif (ITIM), and then recruits SHP-1 protein into the cell, triggering a series of cascade reactions that inhibit macrophage phagocytosis (Matozaki T, Murata Y, Okazawa H, et al. Functions and molecular mechanisms of the CD47-SIRPα signaling pathway. Trends in cell biology, 2009, 19(2): 72-80). However, normal red blood cells are not phagocytosed due to an inhibitory signal generated by CD47 binding to SIPRα on the cell membrane of macrophages (Oldenborg PA, Zheleznyak A, Fang YF, et al. Role of CD47 as a marker of self on red blood cells. Science, 2000, 288(5473): 2051-2054.). TSP1, a homotrimer composed of three peptide chains, is involved in cell proliferation, apoptosis, adhesion, migration, angiogenesis, and other processes through interactions with other cell surface receptors, matrix components, and growth factors (Jiang P, Lagenaur CF, Narayanan V. Integrin-associated Protein Is a Ligand for the P84 Neural Adhesion Molecule. Journal of Biological Chemistry, 1999, 274:559-62).

[0004] Macrophages are derived from monocytes, which in turn originate from precursor cells in the bone marrow. Their main function is to phagocytose cellular debris and pathogens in the form of fixed or free cells, and activate lymphocytes or other immune cells to respond to pathogens. Currently, research has shown that tumor cells have mechanisms to escape macrophage phagocytosis. During tumor cell proliferation, specific proteins such as calreticulin are formed on the surface, exposing identical tumor cells so that they can be phagocytosed by attracted macrophages. However, because the CD47-SIRPα pathway activates inhibition of macrophage phagocytosis (CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis), tumor cells with high CD47 expression are mistaken for normal cells by macrophages with SIRPα and thus escape macrophage phagocytosis (Jaiswal S, Jamieson CHM, Pang WW, et al. Cell, 2009, 138(3) 271-285).

[0005] Current research suggests that anti-CD47 antibodies kill tumor cells primarily through two mechanisms: 1. By binding to CD47, anti-CD47 antibodies block the CD47-SIRPα pathway, allowing macrophages to phagocytose tumors. 2. Anti-CD47 antibodies exert their tumor-killing effect through DC cells and CD8+ T cells. DC cells phagocytose tumor cells through the synergistic effect of anti-CD47 antibodies and phagocytosis-promoting molecules such as calreticulin, presenting tumor-associated antigens to CD8+ T cells, thereby enabling CD8+ T cells to specifically kill tumors (CD47 blockade as another immune checkpoint therapy for cancer. Vonderheide R H. Nature Medicine, 2015, 21(10):1122). These two mechanisms suggest that anti-CD47 antibodies are highly likely to have the ability to activate both nonspecific and specific immune responses.

[0006] Currently, anti-CD47 monoclonal antibody drugs have promising applications in various fields and are effective in treating tumors. These drugs can be used to treat a variety of tumors. The anti-CD47 monoclonal antibody drug Hu5F9-G4 effectively inhibits the growth and metastasis of hematological malignancies and solid tumors in preclinical studies (Abstract PR13: The anti-CD47 antibody Hu5F9-G4 is a novel immune checkpoint inhibitor with synergistic efficacy in combination with clinically active cancer targeting antibodies [J] Chao MP, McKenna KM, Cha A, et al., 2016).

[0007] Therefore, the development of antibody drugs with high affinity for CD47, higher efficacy and fewer toxic side effects for treating tumors is of great importance.

[0008] overview We used a mammalian cell expression system to express recombinant human CD47 as an antigen for immunizing mice, and then fused mouse spleen cells with myeloma cells to obtain hybridoma cells. By screening a large number of samples, we obtained the following hybridoma cell lines:

[0009] The inventors have found that: The hybridoma cell line LTO12 can secrete a monoclonal antibody (designated 6F7) that can specifically bind to CD47, and the monoclonal antibody can compete with the receptor SIRPαECD-hFc-biotin for binding to CD47, effectively blocking the binding of SIRPα to CD47 and further promoting the phagocytosis of tumor cells by macrophages. Furthermore, the present inventors prepared humanized monoclonal antibodies 6F7 (designated 6F7H1L1, 6F7H2L2, and 6F7H3L3).

[0010] The present invention is described in detail below. One aspect of the present invention relates to an antibody or antigen-binding fragment thereof, wherein the antibody comprises the following CDR sequences contained in the heavy chain variable region and the light chain variable region: (1) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO: 4; or (2) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 12, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO: 14; or (3) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 16, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO: 18; or (4) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 20, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO: 22; and a CDR sequence selected from Preferably, the antibody comprises: an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 5, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 6, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 7, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence which has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, Furthermore, the antibody LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; and The LCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 10, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

[0011] In one embodiment of the invention, the antibody (1) the amino acid sequence set forth in SEQ ID NO: 2, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 2; or a heavy chain variable region comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO:2; and the amino acid sequence set forth in SEQ ID NO: 4, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 4, or a light chain variable region comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO:4; (2) the amino acid sequence set forth in SEQ ID NO: 12, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 12; or a heavy chain variable region comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 12; and the amino acid sequence set forth in SEQ ID NO: 14, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 14; or a light chain variable region comprising, or consisting of, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 14; (3) the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 16; or a heavy chain variable region comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 18, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 18; a light chain variable region comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 18; and (4) the amino acid sequence set forth in SEQ ID NO: 20, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 20; or a heavy chain variable region comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 20; and the amino acid sequence set forth in SEQ ID NO: 22, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 22; or a light chain variable region comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 22; Includes. The amino acid sequences of the CDR regions of the antibodies in (1) to (12) above are analyzed using technical means well known to those skilled in the art, for example, through the VBASE2 database.

[0012] Antibodies 6F7, 6F7H1L1, 6F7H2L2 and 6F7H3L3 disclosed herein share the same HCDRs 1-3 and LCDRs 1-3. The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GYTFTSYW (SEQ ID NO: 5), HCDR2: IDPSDSET (SEQ ID NO: 6), and HCDR3: as follows: ARLYRWYFDV (SEQ ID NO: 7); The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1:EIVGTY (SEQ ID NO: 8), LCDR2:GAS (SEQ ID NO: 9), and LCDR3: As shown in GQSYNFPYT (SEQ ID NO: 10).

[0013] In some embodiments, the antibodies disclosed herein are Amino acid sequence of 6F7H1L1(G1M) heavy chain (SEQ ID NO: 59): Amino acid sequence of 6F7H1L1(G1M) light chain (SEQ ID NO: 60): The amino acid sequence of 6F7H2L2(G1M) heavy chain (SEQ ID NO: 61): Amino acid sequence of 6F7H2L2(G1M) light chain (SEQ ID NO: 62): The amino acid sequence of 6F7H3L3(G1M) heavy chain (SEQ ID NO: 63): Amino acid sequence of 6F7H3L3(G1M) light chain (SEQ ID NO: 64): Amino acid sequence of 6F7H1L1 (hG4) heavy chain (SEQ ID NO: 65): Amino acid sequence of 6F7H1L1 (hG4) light chain (SEQ ID NO: 66): Amino acid sequence of 6F7H2L2 (hG4) heavy chain (SEQ ID NO: 67): Amino acid sequence of 6F7H2L2 (hG4) light chain (SEQ ID NO: 68): Amino acid sequence of 6F7H3L3 (hG4) heavy chain (SEQ ID NO: 69): Amino acid sequence of 6F7H3L3 (hG4) light chain (SEQ ID NO: 70) 6.

[0014] In one embodiment of the present invention, the antibody (preferably 6F7 antibody) further comprises FRs in the heavy chain variable region, preferably FRs comprising FR-H1, FR-H2, FR-H3 and FR-H4 (wherein the FR-H1 has an amino acid sequence set forth in SEQ ID NO: 23, a sequence which is at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or more of the amino acid sequence set forth in SEQ ID NO: 23). FR-H2 comprises or consists of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 24; 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 25; said FR-H3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, or consisting of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 25, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 25;The FR-H4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 26, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 26;

[0015] In one embodiment of the present invention, the antibody (preferably 6F7 antibody) further comprises FRs in the light chain variable region, preferably FRs comprising FR-L1, FR-L2, FR-L3 and FR-L4 (wherein the FR-L1 is the amino acid sequence set forth in SEQ ID NO: 27, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 27, or one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid residues compared to the amino acid sequence set forth in SEQ ID NO: 27). the FR-L2 comprises or consists of an amino acid sequence having conservative amino acid mutations (preferably substitutions, insertions or deletions); the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 28, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 28;the FR-L3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 29, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 29; The FR-L4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 30, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 30);

[0016] In one embodiment of the present invention, the antibody (preferably 6F7H1L1 antibody) further comprises FRs in the heavy chain variable region, preferably FRs comprising FR-H1, FR-H2, FR-H3 and FR-H4 (wherein the FR-H1 has an amino acid sequence set forth in SEQ ID NO: 31, a sequence which is at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 1 FR-H2 comprises or consists of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 32; said FR-H2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 32, an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 32 , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 32; said FR-H3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 33. comprising or consisting of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 33, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 33;The FR-H4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 34, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 34);

[0017] In one embodiment of the present invention, the antibody (preferably 6F7H1L1 antibody) further comprises FRs in the light chain variable region, preferably FRs comprising FR-L1, FR-L2, FR-L3 and FR-L4 (wherein the FR-L1 has an amino acid sequence set forth in SEQ ID NO: 35, a sequence which is at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% of the sequence set forth in SEQ ID NO: 35). the FR-L2 comprises or consists of an amino acid sequence having 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 35; the FR-L2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 36, an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, the FR-L3 comprises or consists of an amino acid sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 36; the FR-L3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 37, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 37; or consisting of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 37, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 37;The FR-L4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 38, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 38);

[0018] In one embodiment of the present invention, the antibody (preferably the 6F7H2L2 antibody) further comprises FRs in the heavy chain variable region, preferably FRs comprising FR-H1, FR-H2, FR-H3 and FR-H4 (wherein the FR-H1 has an amino acid sequence set forth in SEQ ID NO: 39, a sequence which is at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% of the sequence set forth in SEQ ID NO: 39). the FR-H2 comprises or consists of an amino acid sequence having 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 39; the FR-H2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 40, an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, the FR-H3 comprises or consists of an amino acid sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 40; the FR-H3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 41, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 41; or consisting of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 41, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 41;The FR-H4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 42, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 42);

[0019] In one embodiment of the present invention, the antibody (preferably 6F7H2L2 antibody) further comprises FRs in the light chain variable region, preferably FRs comprising FR-L1, FR-L2, FR-L3 and FR-L4 (wherein the FR-L1 has an amino acid sequence set forth in SEQ ID NO: 43, a sequence at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 44, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 44; said FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 44, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 44; said FR-L3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 45 or comprising or consisting of an amino acid sequence having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 45;The FR-L4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 46, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 46);

[0020] In one embodiment of the present invention, the antibody (preferably the 6F7H3L3 antibody) further comprises FRs in the heavy chain variable region, preferably FRs comprising FR-H1, FR-H2, FR-H3 and FR-H4 (wherein the FR-H1 has an amino acid sequence set forth in SEQ ID NO: 47, a sequence which is at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% of the sequence set forth in SEQ ID NO: 47). the FR-H2 comprises or consists of an amino acid sequence having 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 47; the FR-H2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 48, an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, the FR-H3 comprises or consists of an amino acid sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 49; the FR-H3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 49, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 49; or consisting of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 49, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 49;The FR-H4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 50, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 50);

[0021] In one embodiment of the present invention, the antibody (preferably 6F7H3L3 antibody) further comprises FRs in the light chain variable region, preferably FRs comprising FR-L1, FR-L2, FR-L3 and FR-L4 (wherein the FR-L1 has an amino acid sequence set forth in SEQ ID NO: 51, a sequence similar to the sequence set forth in SEQ ID NO: 51, at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 11109, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, the FR-L2 comprises or consists of an amino acid sequence having 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 51; the FR-L2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 52, an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, the FR-L3 comprises or consists of an amino acid sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 52; the FR-L3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 53, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 53; or consisting of an amino acid sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 53;The FR-L4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 54, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 54);

[0022] One aspect of the present invention relates to an isolated polypeptide comprising the sequence set forth in SEQ ID NOs: 5, 6 and 7, wherein said polypeptide specifically binds to human CD47 as part of an anti-human CD47 antibody, said antibody further comprising the sequence set forth in SEQ ID NOs: 8, 9 and 10.

[0023] One aspect of the present invention relates to an isolated polypeptide comprising the sequence set forth in SEQ ID NOs: 8, 9 and 10, wherein said polypeptide specifically binds to human CD47 as part of an anti-human CD47 antibody, said antibody further comprising the sequence set forth in SEQ ID NOs: 5, 6 and 7.

[0024] One aspect of the present invention relates to an isolated polypeptide comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 2, 12, 16 and 20, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is an antibody As part of a human CD47 antibody, the antibody specifically binds to human CD47, said antibody further comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 4, 14, 18 and 22, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, said sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence); or One aspect of the present invention relates to an isolated polypeptide comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 4, 14, 18 and 22, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is an isolated polypeptide comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 4, 14, 18 and 22, and the monoclonal antibody specifically binds to human CD47, as part of the method of the present invention, and further comprises a sequence selected from the sequences set forth in SEQ ID NOs: 2, 12, 16 and 20, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the foregoing sequences, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the foregoing sequences.

[0025] In one embodiment of the invention, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), bivalent antibodies, and domain antibodies.

[0026] In one embodiment of the invention, the antibody is a humanized antibody, a chimeric antibody or a multispecific antibody (eg, a bispecific antibody).

[0027] In one embodiment of the invention, the antibody is -5 Less than m, e.g., about 10 -6 M, 10 -7M, 10 -8 M, 10 -9 Less than M or 10 -10 It binds to human CD47 protein with a KD of no more than M. Preferably, the KD is measured by a Fortebio molecular interaction analyzer.

[0028] In one embodiment of the invention, the antibody binds to human CD47 protein with an EC50 of less than about 100 nM, e.g., less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. Specifically, the EC50 is measured by indirect ELISA.

[0029] In one embodiment of the invention, the antibody comprises a constant region, and the constant region is derived from a species other than mouse, such as a human antibody, preferably human IgG, more preferably IgG1 or IgG4.

[0030] In one embodiment of the invention, the constant region of the antibody is humanized, for example, the heavy chain constant region is an Igγ-1 chain C region, more preferably an Igγ-1 chain C region based on GenBank Accession No. P01857 (SEQ ID NO: 58), or an Igγ-4 chain C region, more preferably an Igγ-4 chain C region based on GenBank Accession No. P01861.1 (SEQ ID NO: 56); and the light chain constant region is an Igκ chain C region, more preferably an Igκ chain C region based on GenBank Accession No. P01834 (SEQ ID NO: 57). The antibodies disclosed herein use the following constant regions based on the variable regions of 6F7H1L1, 6F7H2L2, and 6F7H3L3: the heavy chain constant region is an Igγ-1 chain C region based on Accession No. P01857 (SEQ ID NO: 58) or an Igγ-4 chain C region based on Accession No. P01861.1 (SEQ ID NO: 56); and the light chain constant region is an Igκ chain C region based on Accession No. P01834 (SEQ ID NO: 57). Another aspect of the present invention relates to isolated polynucleotides encoding polypeptides comprising the sequences set forth in SEQ ID NOs: 5, 6, and 7, wherein the polypeptide specifically binds to human CD47 as part of an anti-human CD47 antibody, and the antibody further comprises the sequences set forth in SEQ ID NOs: 8, 9, and 10.

[0031] One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising the sequence set forth in SEQ ID NOs: 8, 9 and 10, wherein said polypeptide specifically binds to human CD47 as part of an anti-human CD47 antibody, said antibody further comprising the sequence set forth in SEQ ID NOs: 5, 6 and 7.

[0032] One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 2, 12, 16 and 20, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polynucleotide the peptide specifically binds to human CD47 as part of an anti-human CD47 antibody, said antibody further comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 4, 14, 18 and 22, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; or One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 4, 14, 18 and 22, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polynucleotide The peptide specifically binds to human CD47 as part of an anti-human CD47 antibody, the antibody further comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 2, 12, 16 and 20, a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

[0033] In particular, the polynucleotide molecule comprises or consists of the sequence set forth in SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:15 or SEQ ID NO:19, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity thereto.

[0034] Specifically, the polynucleotide molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:17 or SEQ ID NO:21, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity thereto.

[0035] Yet another aspect of the present invention relates to a vector comprising any one of the polynucleotide molecules disclosed herein as described above.

[0036] Yet another aspect of the present invention relates to a host cell comprising any one of the polynucleotide molecules disclosed herein or the vectors disclosed herein, as described above.

[0037] Yet another aspect of the present invention relates to a method comprising preparing any one of the antibodies or antigen-binding fragments thereof disclosed herein as described above, culturing under suitable conditions a host cell disclosed herein, and isolating the antibody or antigen-binding fragment thereof from the cell culture.

[0038] One embodiment of the present invention further provides an antibody conjugate comprising an anti-human CD47 antibody or antigen-binding fragment thereof and a conjugate moiety attached thereto, wherein the conjugate moiety is a purification tag (e.g., a His tag), a cytotoxic agent, or a detectable label. Preferably, the conjugate moiety is a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

[0039] One embodiment of the present invention further provides a multispecific antibody, preferably a bispecific antibody, comprising an anti-human CD47 antibody or antigen-binding fragment thereof and an antibody or antigen-binding fragment against another antigen and / or another antigen epitope.

[0040] One aspect of the present invention further provides a fusion protein comprising any one of the anti-human CD47 antibodies or antigen-binding fragments thereof disclosed herein as described above.

[0041] One aspect of the present invention further provides a kit comprising any one of the antibodies or antigen-binding fragments thereof disclosed herein, or comprising an antibody conjugate, multispecific antibody, or fusion protein disclosed herein as described above.

[0042] Preferably, the kit further comprises a second antibody that specifically identifies the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label such as a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

[0043] One aspect of the present invention further provides a hybridoma cell line selected from the hybridoma cell line LT012 hybridoma cell line under CCTCC number 2018135, and a monoclonal antibody produced by said hybridoma cell line.

[0044] Yet another aspect of the present invention relates to the use of any one of the antibodies or antigen-binding fragments thereof, antibody conjugates, multispecific antibodies or fusion proteins disclosed herein as described above in detecting the presence or level of human CD47 in a sample or in the preparation of a kit for detecting the presence or level of human CD47 in a sample.

[0045] Yet another aspect of the present invention relates to a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof disclosed herein or the antibody conjugates, multispecific antibodies or fusion proteins disclosed herein as described above, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0046] Yet another aspect of the present invention is a method for producing a semiconductor device comprising: an agent that blocks the binding of human CD47 to human SIRPα; an agent that blocks the activity of human CD47 or downregulates the level of human CD47; or Agents that block cellular responses mediated by binding of human SIRPα to CD47 In adjusting The present invention relates to the use of any one of the antibodies or antigen-binding fragments thereof disclosed herein, or the antibody conjugates, multispecific antibodies or fusion proteins disclosed herein, as described above.

[0047] One aspect of the invention relates to the use of an antibody or antigen-binding fragment thereof as described above or any one of the antibody conjugates, multispecific antibodies or fusion proteins disclosed herein in the treatment of a tumor or in the preparation of a medicament for the treatment of a tumor.

[0048] Yet another aspect of the present invention relates to an in vivo or in vitro method comprising administering a cell comprising an antibody or antigen-binding fragment thereof, an antibody conjugate, multispecific antibody or fusion protein disclosed herein, or administering to a subject in need thereof an effective amount of any one of the above-described antibodies or antigen-binding fragments thereof, or antibody conjugates, multispecific antibodies or fusion proteins disclosed herein, said method comprising: Methods for blocking the binding of CD47 to human SIRPα Methods for blocking the activity of or downregulating the levels of human CD47, and Methods for blocking cellular responses mediated by binding of human SIRP.ALPHA. to CD47 is selected from.

[0049] In one embodiment of the invention, the in vitro method is for non-therapeutic and / or non-diagnostic purposes.

[0050] Yet another aspect of the present invention relates to the use of an antibody or antigen-binding fragment thereof as disclosed herein, or any one of the antibody conjugates, multispecific antibodies or fusion proteins as disclosed herein as above, in the prevention and / or treatment and / or adjuvant treatment and / or diagnosis of an associated tumor, or in the preparation of a medicament for the prevention and / or treatment and / or adjuvant treatment and / or diagnosis of an associated tumor.

[0051] In one embodiment of the invention, the tumor is preferably a CD47-expressing tumor, preferably a cancer, such as a hematological malignancy or a solid tumor, more preferably a lymphoma, colon cancer or breast cancer, more preferably a non-Hodgkin's lymphoma, even more preferably a B-cell lymphoma cell.

[0052] In one embodiment of the invention, the medicament is in a form suitable for injection, preferably suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.

[0053] In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry, and immunology used in the present invention are routine procedures widely used in the corresponding fields. In addition, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0054] As used herein, the term "antigen-binding region" refers to a protein or a portion of a protein that specifically binds to a given antigen. For example, a portion of an antibody that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antibody for said antigen is called an "antigen-binding region." The antigen-binding region generally contains one or more "complementarity-determining regions" (CDRs). Certain antigen-binding regions further contain one or more "framework" regions (FRs). CDRs are amino acid sequences that contribute to antigen-binding specificity and affinity.

[0055] As used herein, the term "antibody" refers to any isotype or antigen-binding fragment thereof that can compete with an intact antibody for specific binding to a target antigen and includes, for example, chimeric, humanized, fully humanized, and bispecific antibodies or antigen-binding fragments thereof. Such "antibodies" are antigen-binding proteins. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as antibodies naturally occurring in camelids, which may contain only heavy chains. An antibody or antigen-binding fragment thereof may be derived from only a single source or may be "chimeric," i.e., different portions of the antibody are derived from two different sources, as described further below. Antibodies or antigen-binding fragments thereof may be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise specified, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, and fragments thereof.

[0056] As used herein, the term "antigen-binding fragment" (or abbreviated "fragment") of an "antibody" or "immunoglobulin chain" (heavy or light chain) includes a portion of an antibody (whether obtained or synthetic) that lacks at least some of the amino acids present in the full-length antibody, but is capable of specifically binding to its antigen. Such fragments are biologically active because they specifically bind to the target antigen and can compete with other antibodies or antigen-binding fragments thereof for specific binding to a given epitope. In one aspect, such a fragment retains at least one CDR present in a full-length light or heavy chain of the antibody, and in some embodiments, such a fragment comprises a single heavy and / or light chain or portion thereof. Such biologically active fragments can be produced by recombinant DNA techniques or by, for example, enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fab / c, dAb, Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including, but not limited to, human, mouse, rat, camelid, and rabbit. It is further contemplated that functional portions of the antibodies disclosed herein, such as one or more CDRs, can be covalently linked to a second protein or small molecule to generate therapeutic agents directed to specific targets within the body, thereby possessing bifunctional therapeutic properties, such as fusion proteins, or having extended serum half-lives.

[0057] As used herein, the terms "full antibody chain," "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having a heavy chain in the Fc region, as defined herein.

[0058] The term "light chain" includes full-length light chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. L and constant region domain C LThe light chain variable region domain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0059] The term "heavy chain" includes full-length heavy chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. H and three constant region domains CH1, CH2 and CH3. H The domain is at the amino terminus of the polypeptide and H The CH3 domain is at the carboxyl terminus, with the CH3 domain being closest to the carboxyl terminus of the polypeptide. The heavy chain may be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0060] As used herein, the term "Fab fragment" refers to a fragment containing one light chain, C H1 and one heavy chain variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0061] As used herein, the term Fc region refers to the C region of an antibody. H1 Domain and C H2 The two heavy chain fragments comprise two heavy chain fragments containing the C domain, the two heavy chain fragments being connected by two or more disulfide bonds and the C H3 The domains are held together by hydrophobic interactions.

[0062] As used herein, the term "Fab' fragment" refers to a portion of one light chain and one heavy chain (the V Fab' fragments) such that an interchain disulfide bond can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule. H Domain, C H1 domain, and the C H1 Domain and C H2 domain).

[0063] As used herein, the term "F(ab')2" fragment refers to the C fragment in which an interchain disulfide bond is formed between the two heavy chains. H1 Domain and C H2 It contains two light chains and two heavy chains, including part of the constant region between the light and heavy chains. Thus, an F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0064] As used herein, the term "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0065] As used herein, the term "Fd" fragment refers to a V H Domain and C H1 It refers to an antibody fragment consisting of domains (Ward et al., Nature, 341:544-546 (1989)).

[0066] As used herein, the term "dAb" fragment refers to a V H It consists of domains (Ward et al., Nature 341:544-546 (1989)).

[0067] As used herein, the term "Fab'-SH" is the designation herein for Fab' in which one or more cysteine residues of the constant domains contain a free thiol group.

[0068] As used herein, the term "Fab / c" fragment refers to an intermediate formed by pepsin digestion of immunoglobulins, which combines the advantages of the Fab and Fc regions, i.e., high in vivo diffusibility and low metabolic clearance, while retaining high affinity (Liu Jianjun, Chinese Journal of Cellular and Molecular Immunology, 1989(4):29-29).

[0069] As used herein, the term "single-chain antibody" refers to an Fv molecule in which the heavy and light chain variable regions are linked by a flexible linker to form a single polypeptide chain (which forms the antigen-binding region) (see, e.g., Bird et al., Science, 242:423-426 (1988), and Huston et al., Proc. Natl. Acad. Sci. USA, 90:5879-5883 (1988)). Single-chain antibodies are described in detail in International Patent Publication No. 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference.

[0070] As used herein, the term "domain antibody" refers to an immunologically functional immunoglobulin fragment comprising only the variable region of the heavy chain or the light chain, including multivalent domain antibodies or bivalent domain antibodies. In some cases, two or more V H The domains are covalently linked by peptide linkers to form multivalent domain antibodies (particularly bivalent domain antibodies). H The regions may target the same or different antigens.

[0071] As used herein, the term "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. The bivalent antibody may be bispecific.

[0072] As used herein, the term "multispecific antigen-binding protein" or "multispecific antibody" is an antigen-binding protein or antibody that targets multiple antigens or epitopes.

[0073] As used herein, the terms "bispecific," "dual-specific," or "bifunctional" antigen-binding protein or antibody refer to a hybrid antigen-binding protein or antibody, respectively, having two different antigen-binding sites. Bispecific antibodies are multispecific antigen-binding proteins or antibodies and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes present on the same or different protein targets.

[0074] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or antibody fragment derived from a population of highly homologous antibodies, i.e., derived from a population of identical antibody molecules except for naturally occurring mutations that may occur spontaneously. The monoclonal antibody is highly specific to a single epitope on an antigen. Compared to the monoclonal antibody, the polyclonal antibody generally comprises at least two or more different antibodies that generally recognize different epitopes on the antigen. Monoclonal antibodies can generally be obtained using hybridoma technology, first described by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. Pat. No. 4,816,567).

[0075] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by the CDR regions of a non-human antibody (donor antibody), where the donor antibody may be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody can also be replaced by the corresponding amino acid residues of a non-human antibody or by amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For further details on humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21:397-402 (2000).

[0076] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant." The epitope or antigenic determinant generally consists of a chemically active surface grouping of molecules such as amino acids, carbohydrates, or sugar side chains, and usually has specific three-dimensional structural and charge characteristics. For example, the epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, which can be "linear" or "conformational." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are located linearly along the primary amino acid sequence of the protein, whereas in a conformational epitope, the interaction sites are located across amino acid residues of the protein that are separated from each other.

[0077] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms are also used to refer to amino acid polymers in which one or more amino acid residues are analogs or mimetics of naturally occurring amino acids, as well as naturally occurring amino acid polymers. The terms can also include amino acid polymers that have been modified, for example, by the addition of sugar residues to form glycoproteins, or phosphorylated amino acid polymers. Polypeptides and proteins can be produced by naturally occurring cells and non-recombinant cells, or they can be produced by genetically engineered or recombinant cells, and include molecules with the amino acid sequence of a naturally occurring protein or molecules with deletions, insertions, and / or substitutions of one or more amino acids of the naturally occurring sequence.

[0078] In some embodiments, the terms "polypeptide" and "protein" specifically include antibodies such as anti-human CD47 antibodies (also referred to as CD47 antibodies), CD47 binding proteins, or variants thereof, e.g., antibodies or sequences having one or more amino acid deletions, insertions, and / or substitutions.

[0079] The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length protein. Such fragments may also contain modified amino acids compared to the full-length protein. In certain embodiments, such fragments are about 5 to 500 amino acids in length. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies containing the binding domain. In the case of human p40 antibodies, useful fragments include, but are not limited to, CDR regions, heavy or light chain variable domains, portions of antibody chains, and variable domains containing exactly two CDRs.

[0080] A "derivative" of a polypeptide is a polypeptide (e.g., an antigen-binding protein or antibody) that is chemically modified in a way other than by insertion, deletion, or substitution, e.g., by conjugation with another chemical moiety (e.g., a PEG-conjugated polypeptide).

[0081] As used herein, the term "isolated" refers to something obtained by artificial means from a natural state. When a particular "isolated" substance or component exists in nature, it may be altered in its natural environment, isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally exists in a particular living animal, and the same polynucleotide or polypeptide, isolated in such a natural state and in a highly purified form, is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0082] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors well known to those skilled in the art include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). The vector may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, the vector may further contain a replication origin.

[0083] As used herein, the term "host cell" refers to a cell that can be introduced with a vector, including, but not limited to, prokaryotic cells such as E. coli or B. subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0084] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or is specific for an antigen) is one in which the antibody binds to an antigen within about 10 -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, less than or equal to 10 -10 Affinity (K D ) to bind to an antigen.

[0085] As used herein, the term "K D " refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. Among several parameters measured by molecular binding kinetics, K D The value is the dissociation equilibrium constant. In antibody drug research, the KD value is a parameter that characterizes the strength of the affinity effect between the antibody of interest and the target antigen molecule, and is expressed by the formula: KD = k dis / k on A smaller equilibrium dissociation constant indicates stronger antibody-antigen binding and higher affinity between the antibody and the antigen. k on (association rate constant) is the rate of formation of the antigen-antibody complex, and is the smaller of k on indicates a faster binding rate of the antibody to the antigen. dis(dissociation rate constant) is the rate at which an antibody dissociates from an antigen-antibody complex, and is the smaller of k dis indicates that the antibody dissociates from the antigen more slowly and that the binding between the antibody and the antigen is stronger. Generally, antibodies have a binding affinity of about 10, as measured, for example, by a BIACORE surface plasmon resonance (SPR) instrument or a Fortebio molecular interaction analyzer. -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 Less than M or 10 -10 The dissociation equilibrium constant (K D ) and binds to an antigen (e.g., L1 protein).

[0086] As used herein, the terms "monoclonal antibody" and "McAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "PcAb" have the same meaning and can be used interchangeably; and the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Additionally, amino acids are generally represented herein by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0087] As used herein, the terms "hybridoma" and "hybridoma cell line" can be used interchangeably, and references to the terms "hybridoma" and "hybridoma cell line" also include subclones and progeny of hybridomas.

[0088] As used herein, the terms "percent sequence identity" and "percent sequence homology" are used interchangeably.

[0089] As used herein, the terms "similarity," "sequence similarity," and "identity" refer to the correlation between the sequences of two or more protein or polypeptide molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical amino acid residues in the compared molecules and can be calculated based on the size of the smallest molecule being compared. For such calculations, gaps in the alignment, if any, must be addressed by a particular mathematical model or computer program (i.e., "algorithm"). When used in reference to polypeptides, the term "substantial identity" means that two peptide sequences have at least 70%, 75%, or 80% sequence identity, at least 90% or 95% sequence identity, or at least 97%, 98%, or 99% sequence identity when optimally aligned, e.g., using the programs GAP or BESTFIT, using default gap weights provided by the programs. In some cases, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain R group possessing similar chemical properties (e.g., charge or hydrophilicity). Generally, conservative amino acid substitutions substantially preserve the function and properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be increased to correct for the conservative nature of the substitutions. Methods for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol., 243:307-31 (1994). Examples of groups of amino acids with side chains possessing similar chemical properties include: 1) aliphatic hydroxyl side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.For example, conservative amino acids substitution groups are: valine-leucine-isoleucine-glycine-alanine, phenylalanine-tyrosine, threonine-serine, lysine-arginine, glutamic-aspartic, and asparagine-glutamine.

[0090] Optionally, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science, 256:1443-45 (1992), which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0091] Polypeptide sequence identity is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to different substitutions, deletions, and other modifications (including conservative amino acid substitutions). For example, GCG, including programs such as "Gap" and "Bestfit" (using default parameters specified by the program), can be used to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different species) or between a wild-type protein and its mutant protein. See, e.g., GCG Version 6.1 (University of Wisconsin, WI). Polypeptide sequences can also be compared using FASTA with default or recommended parameters. See GCG version 6.10 FASTA (e.g., FASTA2 and FASTA3), which provides alignment and percent sequence identity for regions of optimal overlap between challenge and query sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol., 132:185-219 (2000)). Another preferred algorithm for use when comparing sequences to databases containing large sequences from different organisms is the computer program BLAST, particularly blasp or blasn (using the default parameters provided by the program). See, e.g., Altschul et al., Mol. Biol., 215:403-410 (1990); Altschul et al., Nucleic Acids Res., 25:3389-402 (1997). Compared to the prior art, the present invention has the following advantages: The anti-CD47 monoclonal antibodies included herein can effectively block the binding of SIRPα to CD47 by specifically binding to CD47, thereby promoting the phagocytosis of tumor cells by macrophages. The affinity of the antibodies disclosed herein (such as 6F7H1L1) for CD47 on tumor cells is comparable to that of the control antibody Hu5F9-G4, but their affinity for normal human RBCs is lower than that of the control antibody Hu5F9-G4. Therefore, the antibodies disclosed herein have better potential for treating tumors by minimizing the impact on normal human red blood cells. [Brief explanation of the drawings]

[0092] [Figure 1] 1 shows the results of an assay for the binding activity of 6F7H1L1 (hG4) to human CD47IgVTEV-His. [Figure 2] 1 shows the results of an assay for the binding activity of 6F7H1L1(G1M) to human CD47IgVTEV-His, where 6F7H1L1(hG1DM) is 6F7H1L1(G1M). [Figure 3] 1 shows the results of an assay for the competitive binding activity of 6F7H1L1 (hG4) to human SIRPα ECD-hFc-biotin for human CD47IgVTEV-His. [Figure 4] 1 shows the results of an assay for the competitive binding activity of 6F7H1L1(G1M) to human SIRPα ECD-hFc-biotin in human CD47IgVTEV-His, where 6F7H1L1(hG1DM) is 6F7H1L1(G1M). [Figure 5] 1 shows the results of measuring the affinity constant of mouse antibody 6F7 for human CD47. [Figure 6] 1 shows the results of measuring the affinity constant of 6F7H1L1 (hG4) for human CD47. [Figure 7] 1 shows the results of measuring the affinity constant of Hu5F9-G4 for human CD47. [Figure 8]Binding curve of 6F7H1L1(G1M) to human RBCs (FACS). [Figure 9] 6 shows the binding activity of 6F7H1L1(G1M) to tumor cells Raji (FACS). [Figure 10] 1 shows an assay for the competitive binding activity of 6F7H1L1(G1M) to SIRP for LOVO (FACS). [Figure 11] 1 shows agglutination of human red blood cells by the 6F7H1L1(G1M) antibody. [Figure 12] Binding curve of 6F7H1L1 (hG4) to human RBCs (FACS). [Figure 13] 6 shows the binding activity of 6F7H1L1 (hG4) to tumor cells Raji (FACS). [Figure 14] 1 shows the competitive binding activity of 6F7H1L1 (hG4) to SIRP on tumor cells Raji (FACS). [Figure 15] The binding curve of 6F7H1L1 (hG4) to tumor cells LOVO is shown (FACS). [Figure 16] 1 shows an assay for competitive binding activity of 6F7H1L1 (hG4) to SIRP for LOVO (FACS). [Figure 17] 1 shows the agglutination of human red blood cells by anti-CD47 antibody. [Figure 18] 1 shows the therapeutic effect of 6F7H1L1(hG4) on subcutaneously implanted MDA-MB-231 tumors. [Figure 19] 1 shows the change in hemoglobin concentration after a single administration of 6F7H1L1(hG4) and Hu5F9-G4 to cynomolgus monkeys. [Figure 20] 1 shows the change in hematocrit after a single administration of 6F7H1L1(hG4) and Hu5F9-G4 to cynomolgus monkeys.

[0093] Notes on the Deposit of Biological Materials The hybridoma cell line LT012 was deposited with the China Center for Type Culture Collection (CCTCC) under CTCC number C2018135 on June 21, 2018. The deposit location was Wuhan University, Wuhan, China, postal code: 430072.

[0094] Detailed Description Hereinafter, the embodiments of the present invention will be described in detail with reference to examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. If the techniques or conditions are not specified, the examples were carried out according to the techniques or conditions described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., Third Edition, Science Press) or according to the product manuals. If the manufacturers of the reagents or equipment used are not specified, they are conventional products that are commercially available.

[0095] In the following examples of the present invention, BALB / C mice were purchased from Guangdong Medical Laboratory Animal Center.

[0096] The control antibody drug used was Hu5F9-G4 (synthesized by Zhongshan Akeso Biopharma Ltd., using the sequence of the CD47 antibody Hu5F9-G4 obtained from Forty Seven, Inc., i.e., SEQ ID NO: 37 of U.S. Patent No. 20150183874 as the heavy chain variable region, SEQ ID NO: 42 as the light chain variable region, and Ig γ-4 chain constant region (Genbank ID number P01861.1)).

[0097] Example 1: Preparation of anti-human CD47 antibody 6F7

[0098] 1. Preparation of Hybridoma Cell Line 6F7 The antigen used to generate the anti-CD47 antibody of the hybridoma cell line 6F7 was C47IgVTEV-His (a fusion protein containing the human CD47 mature peptide from positions 19 to 141 of GenBank ID number NP942088.1 and TEV (amino acid sequence: ENLYFQG, SEQ ID NO: 74)-His tag, synthesized by Zhongshan Akeso Biopharma Ltd.) and 3T3-CD47 cells (NIH / 3T3, manufacturer: ATCC, catalog number CRL-1658; the human CD47 mature peptide was transfected into NIH / 3T3-based cells to generate a stable 3T3-CD47 cell line). Spleen cells from immunized mice were fused with mouse myeloma cells to generate hybridoma cells. Using separately collected CD47IgVTEV-His and 3T3-CD47 cells as antigens, hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies capable of specifically binding to CD47. Hybridoma cells obtained by ELISA screening were screened by competitive ELISA to obtain hybridoma cell lines capable of secreting monoclonal antibodies capable of competing with CD47IgVTEV-His for binding to the receptor human SIRPα ECD-hFc-biotin ("SIRPα ECD" refers to the extracellular domain of SIRPα, which is positions 31-373 of the protein GenBank accession number NP_542970.1; hFc refers to the human IgGFc purification target, specifically the Igγ-1 chain C region, which is positions 114-330 of GenBank ID number P01857). These were then subjected to limiting dilution to obtain stable hybridoma cell lines. The hybridoma cell line was designated hybridoma cell line LT012, and the monoclonal antibody secreted by it was designated 6F7. The hybridoma cell line LT012 (CD47-6F7) was deposited with the China Center for Type Culture Collection (CCTCC) on June 21, 2018, under the CTCC number C2018135, and the deposit location was Wuhan University, Wuhan, China, postal code: 430072.

[0099] 2. Preparation of Anti-CD47 Antibody 6F7 The cell lines LT011, LT012, and LT015 prepared above were separately cultured in a chemically defined medium (CD medium; containing 1% streptomycin) in a 5% CO2, 37°C incubator. After 7 days, the supernatants were collected and purified by high-speed centrifugation and vacuum filtration through a microfiltration membrane, followed by passage through a HiTrap Protein A HP column to obtain antibody 6F7.

[0100] [Example 2] Sequence analysis of anti-CD47 antibody 6F7 mRNA was extracted from the cell line LT012 cultured in Example 1 according to the method described in the manual for the RNAprep pure Cell / Bacteria Kit (Tiangen, catalog number DP430). The cDNA was synthesized according to the manual of the Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR and amplified by PCR. The PCR-amplified product was subjected to direct TA cloning according to the manual for the pEASY-T1 Cloning Kit (Transgen CT101). The TA cloned product was directly sequenced, and the sequencing results are as follows: The nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1, which has a length of 351 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 2 having a length of 117 amino acids, and the sequences of the heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 5, 6 and 7, respectively. The nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 3, which has a length of 321 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 4 having a length of 107 amino acids, and the sequences of the light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 8, 9 and 10, respectively.

[0101] [Example 3] Design and preparation of the light and heavy chains of humanized anti-human CD47 antibodies 6F7H1L1 (hG4), 6F7H2L2 (hG4), and 6F7H3L3 (hG4)

[0102] 1. Design of the light and heavy chains of humanized anti-human CD47 antibodies 6F7H1L1 (hG4), 6F7H2L2 (hG4), and 6F7H3L3 (hG4) Based on the three-dimensional crystal structure of human CD47 protein (Hage T, Reinemer P, Sebald W., Crystals of a 1:1 Complex Between Human Interleukin-4 and the Extracellular Domain of Its Receptor Alpha Chain, Eur. J. Biochem., 1998; 258(2):831-6.) and the sequence of antibody 6F7 obtained in Example 2, the variable region sequences of antibodies 6F7H1L1, 6F7H2L2, and 6F7H3L3 were obtained by computer modeling and mutation design (antibody constant region sequences obtained from the NCBI database: the heavy chain constant region is the Ig γ-4 chain C region, accession number P01861.1; the light chain constant region is the Ig κ chain C region, accession number P01834). The designed variable region sequences are as follows: (1) Heavy and light chain variable region sequences of humanized monoclonal antibody 6F7H1L1 The nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 11, which has a length of 351 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 12 having a length of 117 amino acids, and the sequences of the heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 5, 6 and 7, respectively. The nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 13, which has a length of 321 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 14 having a length of 107 amino acids, and the sequences of the light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 8, 9 and 10, respectively. (2) Heavy and light chain variable region sequences of humanized monoclonal antibody 6F7H2L2 The nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 15, which has a length of 351 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 16 having a length of 117 amino acids, and the sequences of the heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 5, 6 and 7, respectively. The nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 17, which has a length of 321 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 18 having a length of 107 amino acids, and the sequences of the light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 8, 9 and 10, respectively. (3) Heavy and light chain variable region sequences of humanized monoclonal antibody 6F7H3L3 The nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 19, which has a length of 351 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 20 having a length of 117 amino acids, and the sequences of the heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 5, 6 and 7, respectively. The nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 21, which has a length of 321 bp. The encoded amino acid sequence is set forth in SEQ ID NO: 22 having a length of 107 amino acids, and the sequences of the light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 8, 9 and 10, respectively.

[0103] 2. Preparation of humanized antibodies 6F7H1L1(hG4), 6F7H2L2(hG4), and 6F7H3L3(hG4) The heavy chain constant region is the Ig gamma-4 chain C region, accession number P01861.1; the light chain constant region is the Ig kappa chain C region, accession number P01834. The heavy and light chain cDNAs of 6F7H1L1 (hG4), 6F7H2L2 (hG4), and 6F7H3L3 (hG4) were separately cloned into pUC57Simple (provided by Genscript) vectors to obtain pUC57Simple-6F7H1, pUC57Simple-6F7L1; pUC57Simple-6F7H2, pUC57Simple-6F7L2; and pUC57Simple-6F7H3, pUC57Simple-6F7L3. Referring to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the full-length heavy and light chain genes synthesized by digestion with EcoRI and HindIII were subcloned into the expression vector pcDNA3.1 by restricting digestion with EcoRI and HindIII to obtain expression plasmids pcDNA3.1-6F7H1, pcDNA3.1-6F7L1, pcDNA3.1-6F7H2, pcDNA3.1-6F7L2, pcDNA3.1-6F7H3, and pcDNA3.1-6F7L3, and the heavy and light chain genes of the recombinant expression plasmids were further sequenced. The designed gene combinations containing the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-6F7H1 / pcDNA3.1-6F7L1, pcDNA3.1-6F7H2 / pcDNA3.1-6F7L2, and pcDNA3.1-6F7H3 / pcDNA3.1-6F7L3) were then separately co-transfected into 293F cells, and the culture medium was collected and purified. After sequence confirmation, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. The culture medium was collected after 7 days and subjected to affinity purification on a Protein A column (MabSelectSURE (GE)) to obtain the humanized antibodies.

[0104] [Example 4] Design and preparation of the light and heavy chains of humanized anti-human CD47 antibodies 6F7H1L1(G1M), 6F7H2L2(G1M), and 6F7H3L3(G1M)

[0105] 1. Design of the light and heavy chains of humanized anti-human CD47 antibodies 6F7H1L1(G1), 6F7H2L2(G1), and 6F7H3L3(G1) Based on the three-dimensional crystal structure of human CD47 protein (Hage T, Reinemer P, Sebald W., Crystals of a 1:1 Complex Between Human Interleukin-4 and the Extracellular Domain of Its Receptor Alpha Chain, Eur. J. Biochem., 1998; 258(2):831-6.) and the sequence of antibody 6F7 obtained in Example 2, the variable region sequences of antibodies 6F7H1L1, 6F7H2L2, and 6F7H3L3 were obtained by computer modeling and mutation design (antibody constant region sequences obtained from the NCBI database: the heavy chain constant region is the Ig γ-1 chain C region, accession number P01857; the light chain constant region is the Ig κ chain C region, accession number P01834). To distinguish them from the humanized antibodies of Example 3, the above humanized antibodies were designated as 6F7H1L1(G1), 6F7H2L2(G1), and 6F7H3L3(G1). The variable region sequences of the humanized antibodies 6F7H1L1(G1), 6F7H2L2(G1), and 6F7H3L3(G1) designed in this example were identical to the variable region sequences of 6F7H1L1(hG4), 6F7H2L2(hG4), and 6F7H3L3(hG4) in Example 3.

[0106] 2. Preparation of humanized antibodies 6F7H1L1(G1), 6F7H2L2(G1), and 6F7H3L3(G1) All heavy chain constant regions were Igγ-1 chain C regions, accession number P01857, and all light chain constant regions were Igκ chain C regions, accession number P01834. The heavy and light chain cDNAs of 6F7H1L1, 6F7H2L2, and 6F7H3L3 were separately cloned into pUC57Simple (provided by Genscript) vectors to obtain pUC57Simple-6F7H1, pUC57Simple-6F7L1; pUC57Simple-6F7H2, pUC57Simple-6F7L2; and pUC57Simple-6F7H3, pUC57Simple-6F7L3, respectively. Referring to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the full-length heavy and light chain genes synthesized by digestion with EcoRI and HindIII were subcloned into the expression vector pcDNA3.1 by restricting digestion with EcoRI and HindIII to obtain expression plasmids pcDNA3.1-6F7H1, pcDNA3.1-6F7L1, pcDNA3.1-6F7H2, pcDNA3.1-6F7L2, pcDNA3.1-6F7H3, and pcDNA3.1-6F7L3, and the heavy and light chain genes of the recombinant expression plasmids were further sequenced. The designed gene combinations containing the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-6F7H1 / pcDNA3.1-6F7L1, pcDNA3.1-6F7H2 / pcDNA3.1-6F7L2, and pcDNA3.1-6F7H3 / pcDNA3.1-6F7L3) were then separately co-transfected into 293F cells, and the culture medium was collected and purified. After sequence confirmation, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. The culture medium was collected after 7 days and subjected to affinity purification on a Protein A column (MabSelectSURE (GE)) to obtain the humanized antibody.

[0107] 3. Design of non-variable region amino acid mutations based on humanized antibodies 6F7H1L1(G1), 6F7H2L2(G1), and 6F7H3L3(G1) Based on 6F7H1L1(G1), 6F7H2L2(G1), and 6F7H3L3(G1), new humanized antibodies were obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A) and a leucine-to-alanine point mutation at position 235 (L235A) in the hinge region of the heavy chain, and designated 6F7H1L1(G1M), 6F7H2L2(G1M), and 6F7H3L3(G1M), respectively.

[0108] [Example 5] Assay of antibody binding activity to antigen by ELISA

[0109] 1. Assay of the binding activity of antibody 6F7H1L1 (hG4) to the antigen human CD47IgVTEV-His by ELISA Experimental Procedure: Microplates were coated with 2 μg / mL human CD47IgV-TEV-His and incubated for 12 hours at 4°C. The antigen-coated microplates were washed three times with PBS and then blocked with 1% BSA in PBS for 2 hours. The microplates were washed three times with PBS. Serially diluted antibodies in PBST solution were added to the microplate wells, with the antibody dilution gradient detailed in Table 2. The microplates containing the test antibodies were incubated for 30 minutes at 37°C and then washed three times with PBST. A 1:5000 diluted secondary antibody working solution of HRP-labeled goat anti-human IgG (H+L) (purchased from Jackson ImmunoResearch Inc., catalog number 109-035-088) was added, and the microplates were incubated for 30 minutes at 37°C. The microplates were washed three times with PBST. TMB (Neogen, 308177) was added for 5 minutes in the dark to allow color development, and then stop solution was added to stop the color development. The microplate was then immediately placed in a microplate reader, and the OD value of each well in the microplate was read at 450 nm. Data were analyzed using SoftMaxPro 6.2.1. The results of detecting the binding of antibody 6F7H1L1 (hG4) to the antigen human CD47IgVTEV-His are shown in Figure 1. The OD values at all doses are shown in Table 1. The antibody binding EC50 was calculated by curve fitting using antibody concentration on the horizontal axis and absorbance value on the vertical axis, and the results are shown in Table 1 below. The results show that the binding EC50 of 6F7H1L1(hG4) to human CD47IgVTEV-His was 0.078 nM, which is comparable to that of Hu5F9-G4.

[0110] [Table 1]

[0111] 2. Assay of the binding activity of antibody 6F7H1L1(G1M) to the antigen human CD47IgVTEV-His by ELISA Experimental Procedure: Microplates were coated with 2 μg / mL human CD47IgV-TEV-His and incubated for 12 hours at 4°C. The antigen-coated microplates were washed three times with PBS and then blocked with 1% BSA in PBS for 2 hours. The microplates were washed three times with PBS. Serially diluted antibodies in PBST solution were added to the microplate wells, with the antibody dilution gradient detailed in Table 2. The microplates containing the test antibodies were incubated for 30 minutes at 37°C and then washed three times with PBST. A 1:5000 diluted secondary antibody working solution of HRP-labeled goat anti-human IgG (H+L) (purchased from Jackson ImmunoResearch Inc., catalog number 109-035-088) was added, and the microplates were incubated for 30 minutes at 37°C. The microplates were washed three times with PBST. TMB (Neogen, 308177) was added for 5 minutes in the dark to allow color development, followed by the addition of stop solution to stop the color development. The microplate was then immediately placed in a microplate reader, and the OD of each well in the microplate was read at 450 nm. The data were analyzed using SoftMaxPro 6.2.1. The results of detecting the binding of antibody 6F7H1L1(G1M) to the antigen human CD47IgVTEV-His are shown in Figure 2. The OD values at all doses are shown in Table 2. The antibody binding EC50 was calculated by curve fitting using the antibody concentration on the horizontal axis and the absorbance value on the vertical axis, and the results are shown in Table 2 below.

[0112] [Table 2] The results show that the binding EC50 of 6F7H1L1(G1M) to human CD47IgVTEV-His was 0.037 nM, which is slightly higher than that of Hu5F9-G4.

[0113] 3. Assay of competitive binding activity of antibody 6F7H1L1 (hG4) to human SIRPα ECD-hFc-biotin for human CD47IgVTEV-His by competitive ELISA Experimental Procedure: Microplates were coated with 2 μg / mL human CD47IgV-TEV-His at 50 μL per well and incubated at 4°C for 16 hours. The microplates were washed once, tapped dry, blocked with 1% BSA in PBS at 300 μL per well, incubated at 37°C for 2 hours, washed three times, and tapped dry. The antibody was diluted to an initial concentration of 3 μg / mL (final concentration: 1.5 μg / mL) and serially diluted 1:3 to give a total of seven concentrations, in addition to a blank control. Two replicate wells were set at the above concentrations in a final volume of 50 μL per well, and the plate was incubated for 10 minutes. 50 μL of 0.2 μg / mL (final concentration: 0.1 μg / mL) human SIRPα ECD-hFc-biotin (synthesized by Zhongshan Akeso Biopharma Ltd.) was added to the microplate at a volume of 50 μL per well and gently mixed with the antibody at a 1:1 volume ratio. The microplate was then incubated at 37°C for 30 minutes. The microplate was washed three times and gently tapped dry. 50 μL of SA-HRP (KPL, 14-30-00) working solution was added to each well, and the microplate was incubated at 37°C for 30 minutes. The microplate was washed four times and gently tapped dry. 50 μL of TMB color development solution was added to each well in the dark for 5 minutes at room temperature, and then 50 μL of stop solution was added to each well to stop the color development. The microplate was then immediately placed in a microplate reader, and the OD of each well in the microplate was read at 450 nm. Data were analyzed and processed with SoftMaxPro 6.2.1. The results are shown in Figure 3. The OD values at all doses are shown in Table 3. Quantitative analysis of the absorbance intensity of the bound antibody allowed curve simulation to provide the antibody binding efficiency EC50 (Table 3). The results show that 6F7H1L1(hG4) can effectively block the binding of the antigen human CD47IgVTEV-His to its receptor human SIRPαECD-hFc-biotin, and the blocking efficiency is dose-dependent. The blocking EC50 of 6F7H1L1(hG4) is 0.194 nM, the same as that of Hu5F9-G4.

[0114] [Table 3]

[0115] 4. Assay of competitive binding activity of antibody 6F7H1L1(G1M) to human SIRPα ECD-hFc-biotin in human CD47IgVTEV-His by competitive ELISA Experimental Procedure: Microplates were coated with 2 μg / mL human CD47IgV-TEV-His at 50 μL per well and incubated at 4°C for 16 hours. The microplates were washed once, tapped dry, and blocked with 1% BSA in PBS at 300 μL per well, incubated at 37°C for 2 hours, washed three times, and tapped dry. The antibody was diluted to an initial concentration of 3 μg / mL (final concentration: 0.5 μg / mL) and serially diluted 1:3 to give a total of seven concentrations, in addition to a blank control. Two replicate wells were set at the above concentrations in a final volume of 50 μL per well, and the plate was incubated for 10 minutes. 50 μL of 0.2 μg / mL (final concentration: 0.1 μg / mL) human SIRPα ECD-hFc-biotin was added to the microplate at a volume of 50 μL per well and gently mixed with the antibody at a 1:1 volume ratio. The microplate was then incubated at 37°C for 30 minutes. The microplate was washed three times and gently tapped dry. 50 μL of SA-HRP (KPL, 14-30-00) working solution was added to each well, and the microplate was incubated at 37°C for 30 minutes. The microplate was washed four times and gently tapped dry. 50 μL of TMB color development solution was added to each well in the dark for 5 minutes at room temperature, followed by the addition of 50 μL of stop solution to each well to stop the color development. The microplate was then immediately placed in a microplate reader, and the OD of each well in the microplate was read at 450 nm. Data were analyzed and processed using SoftMaxPro 6.2.1. The results are shown in Figure 4. The OD values at all doses are shown in Table 4. Quantitative analysis of the absorbance intensity of the bound antibody allowed curve simulation to provide the antibody binding efficiency EC50 (Table 4).

[0116] [Table 4] The results show that 6F7H1L1(G1M) can effectively block the binding of the antigen human CD47IgVTEV-His to its receptor human SIRPαECD-hFc-biotin, and the blocking efficiency is dose-dependent. The blocking EC50 of 6F7H1L1(G1M) is 0.274 nM, which is comparable to that of Hu5F9-G4.

[0117] [Example 6] Measurement of the affinity constant of mouse antibody 6F7 for human CD47 The kinetic parameters of binding of the murine antibody 6F7 to the antigen human CD47IgVTEV-His were measured using a Fortebio system (Forteio, model: QKe). An AR2G sensor (Forteio, catalog no. 18-5092) was activated with EDC / NHS, and antibodies were immobilized on the activated AR2G sensor by amine coupling. The sensor was equilibrated in PBST for 300 seconds. The antigen immobilized on the sensor was allowed to bind to the antibody at concentrations ranging from 3.125 to 100 nM (serial two-fold dilutions) for 420 seconds. The antigen and antibody were allowed to dissociate in PBST for 600 seconds. The affinity constants of mouse antibodies 6F7 and Hu5F9-G4 (as a control antibody) for human CD47IgVTEV-His were measured, and the results are shown in Table 5 and FIG.

[0118] [Table 5] The results, as shown in Table 5 and Figure 5, indicate that the affinity constants of murine antibodies 6F7 and Hu5F9-G4 for human CD47IgVTEV-His were equivalent, 6.52E-10 M and 6.38E-10 M, respectively, suggesting that the CDR region of 6F7 has a high CD47-binding ability comparable to that of the CDR region of Hu5F9-G4.

[0119] [Example 7] Measurement of the affinity constant of antibody 6F7H1L1 (hG4) for human CD47 The affinity constant of antibody 6F7H1L1 (hG4) for human CD47IgVTEV-His was measured using a Biacore system (Forteio, model: QKe) according to the manufacturer's instructions. The buffer was PBST. Human CD47 IgG-TEV-His was immobilized on the surface of a CM5 chip by amine coupling with an immobilization signal of 171.6 RU. The antibody was bound to human CD47 at concentrations ranging from 0.78 to 12.5 nM (two-fold dilutions) for 120 seconds at a flow rate of 30 μL / min. The antibody and human CD47 were allowed to dissociate for 300 seconds. The chip was regenerated with 3 M MgCl2 for 30 seconds at a flow rate of 30 μL / min. The data were analyzed using a 1:1 model fitting to obtain the affinity constant. Data were acquired using BiacoreControl 2.0 software and analyzed using BiacoreT200Evaluation 2.0 software. The affinity constants of 6F7H1L1 (hG4) and Hu5F9-G4 (as a control antibody) for human CD47IgVTEV-His were measured, and the results are shown in Table 6 and FIGS. As shown in the figure, the affinity constants of 6F7H1L1(hG4) and Hu5F9-G4 for human CD47IgVTEV-His were 1.52E-10 M and 4.42E-11 M, respectively, suggesting that 6F7H1L1(hG4) has a higher ability to bind to human CD47.

[0120] [Table 6]

[0121] [Example 8] Study on the cellular biological activity of 6F7H1L1(G1M) 1. Detection of 6F7H1L1(G1M) binding to normal human RBCs by FACS Normal human red blood cells were isolated in a biosafety cabinet: Blood buffer A and B were mixed well in a ratio of 1:9 to obtain blood buffer; 20 mL of fresh blood was mixed well with 60 mL of blood buffer; 15 mL of Ficoll Paque reagent was added to a 50 mL centrifuge tube, and then diluted fresh blood was slowly added to the surface of the reagent in a ratio of 3:4, i.e., 20 mL of diluted blood was added to each tube; Centrifugation was performed at 1550 rpm for 30 minutes; RBCs at the bottom of the centrifuge tube were slowly collected with a pipette, washed three times with PBS, and centrifuged; the cell pellet was collected in 500 μL of 1% The RBCs were resuspended in PBSA and counted; the RBC concentration was adjusted, and the cells were transferred to 1.5 mL centrifuge tubes at 300,000 cells per tube; centrifuged at 5600 rpm for 5 minutes, and the supernatant was discarded; 100 μL of antibody with the corresponding concentration (final concentration: 100, 10, 1, 0.1, 0.01, 0.001 nM) was added to each tube according to the experimental design to design a blank group (PBSA + cells) and an isotype control (hIgG) group, and then incubated on ice for 1 hour; 500 μL of 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded; 100 μL of FITC goat anti-human IgG (1:500) was added to each tube, and the resulting mixture was mixed well and incubated on ice in the dark for 30 minutes; 500 μL of 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded; 200 μL of 1% washing buffer was added to each tube, the cells were resuspended, and the fluorescent signal was detected in the FITC channel on the flow cytometer. The results were analyzed using Flowing software, curve fitting was performed separately using MFI and sample concentration on GraphPad prism 5, and EC50 was calculated. The binding results of 6F7H1L1(G1M) to CD47 on the cell membrane surface of normal human RBCs are shown in Figure 8. The results showed that both 6F7H1L1(G1M) and the commercial drug Hu5F9-G4, which targets the same target, could specifically bind to CD47 on the cell membrane surface of normal human RBCs, with corresponding binding EC50 values of 0.077 nM and 0.057 nM, respectively.

[0122] 2. Assay of the binding activity of 6F7H1L1(G1M) to Raji by FACS Log-phase Raji cells were harvested, centrifuged, and washed. The cell pellets were resuspended in 1% PBSA, counted, and viability was measured. Cells were transferred to 1.5 mL tubes at 3.0 x 105 cells / 500 μL / tube and centrifuged at 5600 rpm for 5 minutes. The supernatant was discarded. 100 μL of serially diluted corresponding antibodies was added to each tube according to the experimental design. A blank group (PBSA + cells) and an isotype control (hIgG) group were then designed, and the tubes were incubated on ice for 1 hour. 1% PBSA was then added, followed by centrifugation at 5600 rpm for 5 minutes. The supernatant was discarded. 100 μL of FITC goat anti-human IgG (1:500) was added to each tube, the mixture was mixed well, and the tubes were incubated on ice for 30 minutes in the dark. 500 μL of 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes. The supernatant was discarded. 1% PBSA was added to each tube to resuspend the cells, and the fluorescent signal was detected in the FITC channel on the flow cytometer. Curve fitting was performed using the MFI and sample concentrations separately to calculate the EC50. The binding activity of 6F7H1L1(G1M) to Raji is shown in Figure 9. As shown in the figure, the results indicate that both 6F7H1L1(G1M) and Hu5F9-G4 can specifically bind to CD47 on the cell membrane surface of Raji cells, with corresponding binding EC50 values of 0.013 nM and 0.012 nM, respectively.

[0123] 3. Assay of competitive binding bioactivity of 6F7H1L1(G1M) against SIRP against LOVO by FACS Log-phase LOVO cells (Chinese Academy of Sciences Cell Bank accession number bio-73085) were routinely harvested, centrifuged, and washed. The cell pellet was resuspended in 1% PBSA, counted, and viability was measured. The cell concentration was adjusted to the appropriate range using 1% PBSA, and the cells were grouped into 1.5 mL tubes at 500 μL per tube for a total of 300,000 cells. The cells were centrifuged at 5600 rpm for 5 minutes, and the supernatant was discarded. Serially diluted antibodies (final concentrations (descending): 300, 100, 10, 1, 0.3, 0.1, 0.01, 0.001, 0.0001 nM), a blank control (100 μL of 1% PBSA + cells), and an isotype control (human hIgG) were added and then incubated on ice for 30 minutes. 100 μL of SIRPα-mFc was added to each tube, and the mixture was mixed well for a final concentration of 20 nM and incubated on ice for 1 hour. 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 100 μL of FITC goat anti-mouse IgG (1:500 dilution) was added to each tube, followed by incubation on ice in the dark for 40 minutes. 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 200 μL of 1% PBSA was added to resuspend the cell pellet, and the suspension was transferred to a flow cytometry tube. Fluorescence signals were detected in the FITC channel on the flow cytometer. Curve fitting was performed separately using the MFI and sample concentrations to calculate the EC50. The competitive binding activity of 6F7H1L1(G1M) for SIRP in LOVO was assayed. The results are shown in Figure 10. As shown in the figure, both 6F7H1L1(G1M) and Hu5F9-G4 were able to compete with SIRP for binding to CD47 on the membrane surface of LOVO, thereby blocking SIRP binding to CD47, with corresponding competitive binding EC50 values of 0.16 nM and 0.24 nM, respectively.

[0124] 4. Effect of 6F7H1L1(G1M) on aggregation of normal human red blood cells Preparation of normal human red blood cells: Human blood PBMCs were isolated according to the Ficoll Paque Plus reagent manual, and the red blood cells that settled at the bottom were used in this experiment. Red blood cells were diluted to 1 × 10 in PBS. 7 The red blood cell suspension was obtained by diluting the red blood cells to a concentration of 1 / mL, which was then added to a round-bottom 96-well plate. The positive antibody with the corresponding concentration was added, 0.1 g / mL of dextran T500 was added as a control, and the corresponding hIgG or PBS was added as a negative control, and then the plate was incubated at 37°C for 4 hours. The red blood cell agglutination was examined and photographed. The effect of 6F7H1L1(G1M) on the aggregation of normal human red blood cells is shown in Figure 11. As shown in the figure, when the antibody concentration is lower than 20 μg / mL, 6F7H1L1(G1M) and the control antibody Hu5F9-G4 have no effect on red blood cell aggregation; however, when the antibody concentration is higher than 20 μg / mL, Hu5F9-G4 significantly promotes red blood cell aggregation, while 6F7H1L1(G1M) has no effect on red blood cell aggregation.

[0125] [Example 9] Study on the cellular biological activity of 6F7H1L1 (hG4) 1. Detection of 6F7H1L1 (hG4) binding to normal human RBCs by FACS Experimental Procedure: Blood buffer solution A (D-(+)-glucose: 1 g; CaCl2: 0.0056 g; MgCl2·6H2O: 0.1992 g; KCl: 0.4026 g; Tris: 17.5650 g; dissolved in 1 L of ultrapure water) and B (NaCl: 8.19 g, dissolved in 1 L of ultrapure water) were mixed in a 1:9 ratio to obtain blood buffer solution. Fresh blood was thoroughly mixed with the blood buffer solution (blood dilution ratio after concentration: 1:3). 15 mL of Ficoll Paqueplus reagent (GE, catalog no. 1440-02) was added to a 50 mL centrifuge tube, and diluted fresh blood was slowly added to the surface of the reagent in a volume ratio of 3:4 (i.e., 20 mL of diluted blood was added to each tube). After equilibration, the tubes were centrifuged at 1550 rpm for 30 minutes. PBMCs in the central buffy coat layer were collected with a pipette. Blood buffer was added at a volume ratio of cells to blood buffer of 1:4. The resulting mixture was mixed well and centrifuged at 950 rpm for 15 minutes, and the supernatant was discarded. 20 mL of blood buffer was added to resuspend the PBMCs. The resulting suspension was centrifuged and the supernatant was discarded. Centrifugation was followed by two washes. Cells were washed once with 10 mL of RPMI-1640 (without FBS). Centrifugation was followed by discarding the supernatant. Cells were resuspended in 5 mL of RPMI-1640 (with 10% FBS) and 3 × 10 per sample. 5Cells were counted. 500 μL of 1% PBSA was added to each tube and then centrifuged at 5600 rpm for 5 minutes. The supernatant was discarded. 100 μL of antibody with the corresponding concentration (final concentrations: 300, 100, 10, 1, 0.1, 0.01, 0.001 nM) was added to each tube to designate blank (PBSA + cells) and isotype controls, and then incubated on ice for 1 hour. 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 100 μL of FITC goat anti-human IgG (Jacson, catalog no. 109-095-098, 1:500 dilution) or FITC goat anti-mouse IgG (BD bioscience, catalog no. 555988) (1:500 dilution) was added, and the resulting mixture was mixed well and incubated on ice in the dark for 30 minutes. 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 200 μL of 1% wash buffer was added to each tube to resuspend the cells, and the fluorescent signal was detected in the FITC channel on the flow cytometer. Results were analyzed using Flowcytometer software, and curve fitting was performed separately using MFI and sample concentration on GraphPad Prism 5 to calculate the EC50. The binding results of 6F7H1L1(hG4) to CD47 on the cell membrane surface of normal human RBCs are shown in Figure 12 and Table 7. The results show that both 6F7H1L1(hG4) and the commercial drug Hu5F9-G4, which target the same target, can specifically bind to CD47 on the cell membrane surface of normal human RBCs, with binding EC50s of 0.60 nM and 0.06 nM, respectively, indicating that the affinity of Hu5F9-G4 for RBCs is 10-fold higher than that of 6F7H1L1(hG4).

[0126] [Table 7]

[0127] 2. Assay of the binding activity of 6F7H1L1 (hG4) to Raji by FACS The binding bioactivity of CD47 antibody to tumor cells Raji (Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences Cell Center, Catalog No. TCHU44) was assayed by flow cytometry. Raji cells were counted and viability was assessed at 3 x 10 per sample. 5 The assay was performed using 100 μL of cells. 500 μL of 1% PBSA was added to each tube, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. Serially diluted antibodies were added according to the experimental design, including a blank (PBSA + cells) group and an isotype control group (human IgG), and then incubated on ice for 1 hour. 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 100 μL of FITC goat anti-human IgG (1:500) or FITC goat anti-mouse IgG (1:500) was added, the resulting mixture was mixed well, and incubated on ice for 30 minutes in the dark. 500 μL of 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 200 μL of wash buffer was added to each tube to resuspend the cells, and the fluorescent signal was detected in the FITC channel on the flow cytometer. The results of the binding activity of 6F7H1L1(hG4) to Raji are shown in Figure 13 and Table 8. As shown in the figure and table, the results show that both 6F7H1L1(hG4) and Hu5F9-G4 can specifically bind to CD47 on the cell membrane surface of Raji cells, with corresponding binding EC50 values of 0.32 nM and 0.22 nM, respectively.

[0128] [Table 8]

[0129] 3. Assay of competitive binding activity of 6F7H1L1(hG4) to SIRP in Raji by FACS Log-phase Raji cells were routinely harvested, centrifuged, and washed. The cell pellets were resuspended in 1% PBSA, counted, and viability was measured. The cell concentration was adjusted to the appropriate range using 1% PBSA, and the cells were grouped into 1.5 mL tubes at 500 μL per tube for a total of 300,000 cells. The cells were centrifuged at 5600 rpm for 5 minutes, and the supernatant was discarded. Serially diluted antibodies (final concentrations (descending): 1, 0.3, 0.1, 0.01, 0.001, 0.0001 nM), as well as a blank control (100 μL of 1% PBSA + cells) and an isotype control (human hIgG), were added and then incubated on ice for 30 minutes. 100 μL of SIRPα-ECD-mFc (the sequence of the mFc is set forth in SEQ ID NO: 71) was added to each tube, and the mixture was mixed well to a final concentration of 20 nM and incubated on ice for 1 hour. 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 100 μL of FITC goat anti-mouse IgG (1:500 dilution) was added to each tube, followed by incubation on ice for 40 minutes in the dark. 500 μL of 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 200 μL of 1% PBSA was added to resuspend the cell pellet. Fluorescent signals were detected in the FITC channel on the flow cytometer. The results of competitive binding of 6F7H1L1(hG4) to SIRP in tumor cells Raji are shown in Figure 14 and Table 9. As shown in the figure and table, both 6F7H1L1(hG4) and Hu5F9-G4 can compete with SIRP for binding to CD47 on the membrane surface of Raji, thereby blocking the binding of SIRP to CD47, with corresponding competitive binding EC50 values of 0.017 nM and 0.014 nM, respectively.

[0130] [Table 9]

[0131] 4. Assay of binding activity of 6F7H1L1 (hG4) to LOVO by FACS Log-phase LOVO cells (Chinese Academy of Sciences Cell Bank accession number bio-73085) were harvested, centrifuged, and washed. The cell pellet was resuspended in 500 μL of 1% PBSA, counted, and viability was determined. Cells were cultured at 3.0 × 10 5 The cells were transferred to 1.5 mL tubes according to the cell / 500 μL / tube ratio, centrifuged at 5600 rpm for 5 minutes, and the supernatant was discarded. 100 μL of serially diluted corresponding antibody was added to each tube according to the experimental design, designating a blank group (PBSA + cells) and an isotype control group (human hIgG, whose heavy chain sequence is SEQ ID NO: 72 and whose light chain sequence is SEQ ID NO: 73), and then incubated on ice for 1 hour. Next, 500 μL of 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 100 μL of FITC goat anti-human IgG (1:500) was added to each tube, the mixture was mixed well, and incubated on ice for 30 minutes in the dark. 500 μL of 1% PBSA was added, followed by centrifugation at 5600 rpm for 5 minutes, and the supernatant was discarded. 200 μL of 1% PBSA was added to each tube to resuspend the cells, and the suspension was transferred to a flow cytometry tube and detected using a flow cytometer (DFACSCalibur). The results were analyzed using Flowing software, and curve fitting was performed separately using MFI and sample concentration on GraphPad Prism 5 to calculate the EC50. The binding results of 6F7H1L1(hG4) to LOVO are shown in Figure 15 and Table 10. As shown in the figure and table, both 6F7H1L1(hG4) and Hu5F9-G4 can specifically bind to CD47 on the cell membrane surface of LOVO cells, with binding EC50s of 0.02 nM and 0.06 nM, respectively. The binding activity of 6F7H1L1(hG4) was slightly higher than that of Hu5F9-G4.

[0132] [Table 10]

[0133] 5. Assay of competitive binding bioactivity of 6F7H1L1(hG4) against SIRP in LOVO by FACS The experimental procedure was the same as in Example 3, except that Raji cells were replaced with LOVO cells. The results of the assay for the competitive binding activity of 6F7H1L1(hG4) to SIRP on LOVO are shown in Figure 16 and Table 11. As shown in the figure and table, both 6F7H1L1(hG4) and Hu5F9-G4 can compete with SIRP for binding to CD47 on the membrane surface of LOVO, thereby blocking the binding of SIRP to human CD47, with competitive binding EC50s of 0.10 nM and 0.24 nM, respectively. The competitive binding activity of 6F7H1L1(hG4) was slightly higher than that of Hu5F9-G4.

[0134] [Table 11]

[0135] 6. Effect of anti-CD47 antibody on aggregation of normal human RBCs Preparation of normal human RBCs: Human blood PBMCs were isolated according to the manual of Ficoll Paque Plus reagent (GE, Cat. No. 17-1440-02), and the red blood cells that settled at the bottom were used in this experiment. Red blood cells were diluted to 1 × 10 in PBS. 7 The red blood cell suspension was diluted to a concentration of 1 / mL to obtain a red blood cell suspension, which was then added to a round-bottom 96-well plate. The corresponding concentration of positive antibody was added, 0.1 g / mL of Dextran T500 was added as a control, and the corresponding human IgG1 (Akeso Biopharma) or PBS was added as a negative control, and then the plate was incubated at 37°C for 4 hours. Red blood cell agglutination was examined and photographed. The effect of 6F7H1L1(hG4) on the aggregation of normal human red blood cells is shown in Figure 17. As shown in the figure, 6F7H1L1(hG4) did not induce red blood cell aggregation at any tested concentration, and the control antibody Hu5F9-G4 did not induce red blood cell aggregation at concentrations of 3.3 μg / mL or less. At concentrations of 10 μg / mL or more, a significant enhancement of red blood cell aggregation by Hu5F9-G4 could be observed.

[0136] [Example 10] Therapeutic effect of 6F7H1L1 (hG4) on subcutaneously transplanted MDA-MB-231 tumors The in vivo activity of 6F7H1L1(hG4) was investigated by measuring the volume of human breast cancer cell MDA-MB-231 tumors subcutaneously implanted in SCID / beige mice after administration of 6F7H1L1(hG4). Harvested MDA-MB-231 (ATCC, Cat. No. HTB-26) cells were implanted in SCID / beige mice at 5 × 10 in the right flank for a total of 40 mice. 6 Cells were implanted subcutaneously per mouse. Tumor volumes were approximately 100–120 mm. 3 At the time of tumor volume reaching 100 μg / day, the mice were equally divided into five groups of seven mice (model group, Hu5F9-G4 high dose group, Hu5F9-G4 low dose group, 6F7H1L1 (hG4) high dose group, and 6F7H1L1 (hG4) low dose group) according to the mean tumor volume, where the high dose group was treated with a dose of 0.2 mg / kg and the low dose group was treated with a dose of 0.02 mg / kg. The day of grouping is designated as D0, and administration was performed on D0, D3, D7, D10, D1, and D17. After grouping using a caliper, the size of the tumor was measured twice a week, and the tumor volume was calculated using the formula: TV = 0.5 × ab 2where a is the longest diameter of the tumor, b is the shortest diameter of the tumor, and TV is the tumor volume. TGI (%) (tumor growth inhibition rate) was calculated from the tumor volume by the formula: %TGI = (1-(Ti-T0) / (Ci-C0)) × 100%, where Ti and Ci are the average tumor volumes on day i of the treatment group and the model group, respectively, and T0 and C0 are the average tumor volumes on day 0 of the treatment group and the model group, respectively. The results were evaluated by one-way analysis of variance after intergroup comparison processed by GraphPad software. The results are shown in Figure 18. On day 24 after grouping, MDA-MB-231 tumor growth was effectively inhibited in both the high-dose control antibody Hu5F9-G4 group and the high-dose 6F7H1L1 (hG4) group (P<0.01), and the inhibition of MDA-MB-231 tumor growth by Hu5F9-G4 and 6F7H1L1 (hG4) showed a dose-dependent relationship. The TGI values (%) for the control antibody Hu5F9-G4 high-dose group, the 6F7H1L1 (hG4) high-dose group, and the 6F7H1L1 (hG4) low-dose group were 67%, 63%, and 25%, respectively. Compared with the control antibody group, the 6F7H1L1 (hG4) low-dose group showed significantly higher efficacy than the Hu5F9-G4 low-dose group, while the 6F7H1L1 (hG4) high-dose group and the control antibody high-dose group showed similar efficacy (P>0.05).

[0137] [Example 11] Effect of single administration of 6F7H1L1 (hG4) and Hu5F9-G4 on hemoglobin and hematocrit in cynomolgus monkeys Four cynomolgus monkeys were randomly assigned to two groups of two monkeys according to body weight and sex (half male and half female). 6F7H1L1(hG4) and Hu5F9-G4 were administered intravenously at a dose of 10 mg / kg. Hemoglobin and hematocrit were detected using a hematology analyzer. The results are shown in Figures 19 and 20 and Table 12. The results showed that after a single administration of H1L1(hG4) and Hu5F9-G4 at 10 mg / kg to cynomolgus monkeys, hemoglobin and hematocrit decreased to different degrees, reaching the lowest anemia point 2 to 7 days later, and the anemia level in the Hu5F9-G4 group was higher than that in the 6F7H1L1(hG4) group; the monkeys were able to spontaneously recover from anemia caused by both antibodies to baseline levels approximately 20 days after administration.

[0138] [Table 12] Although the embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Those skilled in the art can make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are included in the scope defined by the claims of this application.

[0139] Array List 6F7 heavy chain variable region: [ka] [ka] 6F7 light chain variable region: [ka] [ka] 6F7CDR HCDR1: GYTFTSYW (SEQ ID NO: 5) HCDR2: IDPSDSET (SEQ ID NO: 6) HCDR3: ARLYRWYFDV (SEQ ID NO: 7) LCDR1:EIVGTY (SEQ ID NO: 8) LCDR2:GAS (SEQ ID NO: 9) LCDR3: GQSYNFPYT (SEQ ID NO: 10) 6F7H1: [ka] [ka] 6F7L1: [ka] [ka] 6F7H2: [ka] [ka] 6F7L2: [ka] [ka] 6F7H3: [ka] [ka] 6F7L3: [ka] [ka] 6F7 heavy chain framework region FR-H1: QVQLQQPGAELVRPGASVKLSCKAS (SEQ ID NO: 23) FR-H2:MNWVKQRPGQGLEWIGM (SEQ ID NO: 24) FR-H3:HNNQMFKDKATLTVDKSSNTAYMHLSSLTSEDSAVYHC (SEQ ID NO: 25) FR-H4: WGAGTTVTVSS (SEQ ID NO: 26) 6F7 light chain framework region FR-L1: NIVMTQSPKSMSMSLGERVTLSCKAS (SEQ ID NO: 27) FR-L2: VSWFQQKPHQSPKLLIY (SEQ ID NO: 28) FR-L3: NRYTGVPDRFTGSGSATDFTLTISNVQAEDLADYHC (SEQ ID NO: 29) FR-L4: FGGGTKLEIK (SEQ ID NO: 30) 6F7H1 Framework Area FR-H1: QVQLVQSGAEVVKPGASVKLSCKAS (SEQ ID NO: 31) FR-H2:MNWVRQRPGQGLEWIGM (SEQ ID NO: 32) FR-H3: HNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYC (SEQ ID NO: 33) FR-H4: WGAGTTVTVSS (SEQ ID NO: 34) 6F7L1 framework area FR-L1: NIVMTQSPATMSMSPGERVTLSCRAS (SEQ ID NO: 35) FR-L2: VSWFQQKPGQAPRLLIY (SEQ ID NO: 36) FR-L3: NRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHC (SEQ ID NO: 37) FR-L4: FGGGTKLEIK (SEQ ID NO: 38) 6F7H2 Framework Area FR-H1: QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 39) FR-H2:MNWVRQRPGQGLEWIGI (SEQ ID NO: 40) FR-H3: SNAQKFQGRVTLTVDKSTSTAYMHLSSLRSEDTAVYYC (SEQ ID NO: 41) FR-H4: WGAGTTVTVSS (SEQ ID NO: 42) 6F7L2 Framework Area FR-L1: NIVMTQSPATLSLSPGERVTLSCRAS (SEQ ID NO: 43) FR-L2: VSWFQQKPGQAPRLLIY (SEQ ID NO: 44) FR-L3: NRATGIPARFSGSGSGTDFTLTISSLQPEDLADYYC (SEQ ID NO: 45) FR-L4: FGGGTKLEIK (SEQ ID NO: 46) 6F7H3 Framework Area FR-H1: QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 47) FR-H2:MNWVRQAPGQGLEWIGI (SEQ ID NO: 48) FR-H3: SYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 49) FR-H4: WGAGTTVTVSS (SEQ ID NO: 50) 6F7L3 Framework Area FR-L1: NIVMTQSPATLSLSPGERVTLSCRAS (SEQ ID NO: 51) FR-L2: LSWYQQKPGQAPRLLIY (SEQ ID NO: 52) FR-L3: TRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYC (SEQ ID NO: 53) FR-L4: FGGGTKLEIK (SEQ ID NO: 54) IgG1M heavy chain constant region [ka] Heavy chain constant region Igγ-4 chain C region [ka] Light chain constant region Igκ chain C region [ka] Heavy chain constant region Igγ-1 chain C region [ka] Amino acid sequence of the 6F7H1L1(G1M) heavy chain [ka] Amino acid sequence of the 6F7H1L1(G1M) light chain [ka] Amino acid sequence of the 6F7H2L2(G1M) heavy chain [ka] Amino acid sequence of the 6F7H2L2(G1M) light chain [ka] Amino acid sequence of the 6F7H3L3(G1M) heavy chain [ka] Amino acid sequence of the 6F7H3L3(G1M) light chain [ka] Amino acid sequence of 6F7H1L1 (hG4) heavy chain [ka] 6F7H1L1(hG4) light chain amino acid sequence [ka] Amino acid sequence of the 6F7H2L2 (hG4) heavy chain [ka] 6F7H2L2(hG4) light chain amino acid sequence [ka] 6F7H3L3(hG4) heavy chain amino acid sequence [ka] Amino acid sequence of the 6F7H3L3 (hG4) light chain [ka] mFc tag sequence: (SEQ ID NO: 71) [ka] hIgG heavy chain sequence (SEQ ID NO: 72) [ka] hIgG light chain sequence (SEQ ID NO: 73) [ka] The amino acid sequence of TEV is ENLYFQG (SEQ ID NO: 74).

Claims

1. An antibody that specifically binds to CD47 or an antigen-binding fragment thereof, The aforementioned antibody is as follows: HCDR1 containing or consisting of the sequence described in Sequence ID No. 5; HCDR2 comprising or consisting of the sequence described in Sequence ID No. 6; HCDR3 containing or consisting of the sequence described in Sequence ID No. 7; LCDR1 containing or consisting of the sequence described in Sequence ID No. 8; LCDR2 containing or consisting of the sequence described in Sequence ID No. 9; and LCDR3 containing or consisting of the sequence described in Sequence ID No. 10 It includes, and The aforementioned antibody is as follows: (1) A sequence having at least 90% sequence identity with the heavy chain variable region described in Sequence ID No. 2 or Sequence ID No. 2, and a sequence having at least 90% sequence identity with the light chain variable region described in Sequence ID No. 4 or Sequence ID No. 4; (2) A heavy chain variable region described in SEQ ID NO: 16 or a sequence having at least 90% sequence identity with SEQ ID NO: 16, and a light chain variable region described in SEQ ID NO: 18 or a sequence having at least 90% sequence identity with SEQ ID NO: 18; or (3) A sequence having at least 90% sequence identity with the heavy chain variable region described in Sequence ID No. 20 or Sequence ID No. 20, and a sequence having at least 90% sequence identity with the light chain variable region described in Sequence ID No. 22 or Sequence ID No.

22. An antibody or its antigen-binding fragment, including an antibody.

2. The antibody or antigen-binding fragment thereof according to Claim 1, The aforementioned antibody is as follows: (1) FR in the heavy chain variable region, comprising FR-H1, FR-H2, FR-H3 and FR-H4, where, FR-H1 contains or consists of the amino acid sequence described in SEQ ID NO: 23; FR-H2 contains or consists of the amino acid sequence described in SEQ ID NO: 24; FR-H3 contains or consists of the amino acid sequence described in Sequence ID No. 25; and FR-H4 contains or consists of the amino acid sequence described in SEQ ID NO:

26. FR in the heavy chain variable region, and FR in the light chain variable region, comprising FR-L1, FR-L2, FR-L3 and FR-L4, where, FR-L1 contains or consists of the amino acid sequence described in SEQ ID NO: 27; FR-L2 contains or consists of the amino acid sequence described in Sequence ID No. 28; FR-L3 contains or consists of the amino acid sequence described in Sequence ID No. 29; and FR-L4 contains or consists of the amino acid sequence described in SEQ ID NO:

30. FR in the light chain variable region; (2) FR in the heavy chain variable region, comprising FR-H1, FR-H2, FR-H3 and FR-H4, where, FR-H1 contains or consists of the amino acid sequence described in Sequence ID No. 39; FR-H2 contains or consists of the amino acid sequence described in Sequence ID No. 40; FR-H3 contains or consists of the amino acid sequence described in SEQ ID NO: 41; and FR-H4 contains or consists of the amino acid sequence described in SEQ ID NO:

42. FR in the heavy chain variable region, and FR in the light chain variable region, comprising FR-L1, FR-L2, FR-L3 and FR-L4, where, FR-L1 contains or consists of the amino acid sequence described in SEQ ID NO: 43; FR-L2 contains or consists of the amino acid sequence described in SEQ ID NO: 44; FR-L3 contains or consists of the amino acid sequence described in Sequence ID No. 45; and FR-L4 contains or consists of the amino acid sequence described in SEQ ID NO:

46. FR in the light chain variable region; and (3) FR in the heavy chain variable region, comprising FR-H1, FR-H2, FR-H3 and FR-H4, where, FR-H1 contains or consists of the amino acid sequence described in Sequence ID No. 47; FR-H2 contains or consists of the amino acid sequence described in Sequence ID No. 48; FR-H3 contains or consists of the amino acid sequence described in Sequence ID No. 49; and FR-H4 contains or consists of the amino acid sequence described in Sequence ID No.

50. FR in the heavy chain variable region, and FR in the light chain variable region, comprising FR-L1, FR-L2, FR-L3 and FR-L4, where, FR-L1 contains or consists of the amino acid sequence described in Sequence ID No. 51; FR-L2 contains or consists of the amino acid sequence described in Sequence ID No. 52; FR-L3 contains or consists of the amino acid sequence described in Sequence ID No. 53; and FR-L4 contains or consists of the amino acid sequence described in Sequence ID No.

54. FR in the light chain variable region An antibody or its antigen-binding fragment, further comprising a combination of FR in the heavy chain variable region and FR in the light chain variable region, selected from the group consisting of the above.

3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, The aforementioned antibody is as follows: (1) A heavy chain variable region containing or consisting of the amino acid sequence described in SEQ ID NO: 2, and Light chain variable regions containing or consisting of the amino acid sequence described in Sequence ID No. 4; (2) A heavy chain variable region comprising or consisting of the amino acid sequence described in SEQ ID NO: 16, A light chain variable region containing or consisting of the amino acid sequence described in SEQ ID NO: 18; or (3) Heavy chain variable regions comprising or consisting of the amino acid sequence described in SEQ ID NO: 20, Light chain variable region containing or consisting of the amino acid sequence described in SEQ ID NO: 22 An antibody or its antigen-binding fragment, including an antibody.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, and the constant region is derived from a species other than mouse.

5. An antibody or antigen-binding fragment thereof according to Claim 4, wherein the constant region is derived from a human antibody.

6. An antibody or antigen-binding fragment thereof according to Claim 4, wherein the constant region is derived from human IgG or IgM.

7. An antibody or antigen-binding fragment thereof according to Claim 4, wherein the constant region is derived from IgG1.

8. An antibody or antigen-binding fragment thereof according to Claim 4, wherein the heavy chain constant region is the Igγ-1 chain C region, accession number P01857 (SEQ ID NO: 58) or the Igγ-4 chain C region, accession number P01861.1 (SEQ ID NO: 56); and the light chain constant region is the Igκ chain C region, accession number P01834 (SEQ ID NO: 57).

9. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody further comprises an amino acid mutation introduced at positions 234 and / or 235 according to the EU numbering system.

10. An antibody or antigen-binding fragment antibody according to any one of claims 1 to 9, wherein the antibody contains mutations L234A and / or L235A according to the EU numbering system.

11. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, The aforementioned antibody is as follows: (1) The heavy chain described in Sequence ID No. 61 and the light chain described in Sequence ID No. 62; (2) The heavy chain described in Sequence ID No. 63 and the light chain described in Sequence ID No. 64; (3) The heavy chain described in Sequence ID No. 67 and the light chain described in Sequence ID No. 68; and (4) Heavy chain described in Sequence ID No. 69 and light chain described in Sequence ID No. 70 An antibody or its antigen-binding fragment comprising a combination of a heavy chain and a light chain selected from the group consisting of the following.

12. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, Fab / c, a single-chain antibody, and a bivalent antibody.

13. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antigen-binding fragment is scFv.

14. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody.

15. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody is a bispecific antibody.

16. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody binds to the human CD47 protein with a KD of less than 10⁻⁵ M.

17. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody binds to the human CD47 protein at an EC50 of less than 100 nM.

18. (1) A polypeptide comprising the sequence described in Sequence ID No. 2 and the sequence described in Sequence ID No. 4; (2) A polypeptide comprising the sequence described in SEQ ID NO: 16 and the sequence described in SEQ ID NO: 18; or (3) polypeptides containing the sequence described in Sequence ID No. 20 and the sequence described in Sequence ID No. 22 An isolated polynucleotide that codes for [something].

19. An isolated polynucleotide according to claim 18, The aforementioned polynucleotide molecule (1) The nucleotide sequence described in Sequence ID No. 1, and the nucleotide sequence described in Sequence ID No. 3; (2) The nucleotide sequence described in Sequence ID No. 15 and the nucleotide sequence described in Sequence ID No. 17; or (3) The nucleotide sequence described in Sequence ID No. 19 and the nucleotide sequence described in Sequence ID No. 21 Isolated polynucleotides containing or consisting of the following.

20. A vector comprising the polynucleotide molecule according to claim 18 or 19.

21. A host cell comprising the polynucleotide molecule according to claim 18 or 19, or the vector according to claim 20.

22. A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17, comprising culturing a host cell according to claim 21 under appropriate conditions, and isolating the antibody or an antigen-binding fragment thereof from the cell culture.

23. An antibody conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17 and a conjugate portion bound thereto, wherein the conjugate portion is a purified tag, a cytotoxic drug, or a detectable label.

24. An antibody conjugate according to claim 23, wherein the conjugate portion is a His tag, a radioisotope, a luminescent substance, a coloring substance, an enzyme, or polyethylene glycol.

25. A multispecific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, and an antibody or antigen-binding fragment against another antigen and / or another antigen epitope.

26. A fusion protein comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17.

27. ​​A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or an antibody conjugate according to claim 23 or 24, a multispecific antibody according to claim 25, or a fusion protein according to claim 26.

28. The kit according to claim 27, further comprising a second antibody for specifically identifying the antibody or its antigen-binding fragment.

29. The kit according to claim 28, wherein the second antibody further comprises a detectable label.

30. The kit according to claim 29, wherein the detectable label is a radioisotope, a luminescent substance, a coloring substance, an enzyme, or polyethylene glycol.

31. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, an antibody conjugate according to claim 23 or 24, a multispecific antibody according to claim 25, or a fusion protein according to claim 26 in detecting the presence or level of human CD47 in a sample, or in preparing a kit for detecting the presence or level of human CD47 in a sample.

32. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, an antibody conjugate according to claim 23 or 24, a multispecific antibody according to claim 25, or a fusion protein according to claim 26.

33. A pharmaceutical composition according to claim 32, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

34. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, an antibody conjugate according to claim 23 or 24, a multispecific antibody according to claim 25, or a fusion protein according to claim 26, for treating a tumor.

35. The antibody or antigen-binding fragment thereof according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the tumor is a tumor expressing CD47.

36. The antibody or antigen-binding fragment thereof according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the tumor is cancer.

37. The antibody or antigen-binding fragment thereof according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the tumor is a hematological malignancy or a solid tumor.

38. The antibody or antigen-binding fragment thereof according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the tumor is lymphoma, colon cancer, or breast cancer.

39. The antibody or antigen-binding fragment thereof according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the tumor is a non-Hodgkin lymphoma.

40. The antibody or antigen-binding fragment thereof according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the tumor is a B-cell lymphoma.

41. The antibody or antigen-binding fragment according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the antibody or antigen-binding fragment thereof is in a form suitable for injection.

42. The antibody or antigen-binding fragment according to claim 34, the antibody conjugate according to claim 34, the multispecific antibody according to claim 34, or the fusion protein according to claim 34, wherein the antibody or antigen-binding fragment thereof is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intrafocal injection.

43. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, an antibody conjugate according to claim 23 or 24, a multispecific antibody according to claim 25, or a fusion protein according to claim 26, the following: Blocking the binding of CD47 to human SIRPα, Blocking the activity of human CD47 or downregulating the level of human CD47, and Blocking the cellular response mediated by the binding of human SIRPα to CD47. An antibody or its antigen-binding fragment, antibody conjugate, multispecific antibody, or fusion protein for any one of the following uses.

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