CN117886936A

CN117886936A - CD47 antibody and medical application thereof

Info

Publication number: CN117886936A
Application number: CN202211250976.5A
Authority: CN
Inventors: 李冠英; 廖诗骅; 李花利; 汪敬佩; 王旭鹏; 李健友
Original assignee: Shanghai Hailu Biological Technology Co ltd; Yangtze River Pharmaceutical Group Co Ltd
Current assignee: Shanghai Hailu Biological Technology Co ltd; Yangtze River Pharmaceutical Group Co Ltd
Priority date: 2022-10-13
Filing date: 2022-10-13
Publication date: 2024-04-16

Abstract

The application provides a CD47 antibody and medical application thereof. The antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; wherein, HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO. 39, SEQ ID NO. 40 and SEQ ID NO. 41 respectively, or have the amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47 respectively, as defined by the Kabat numbering system; LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown as SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44, respectively, or have the amino acid sequences shown as SEQ ID NO. 48, SEQ ID NO. 49 and SEQ ID NO. 50, respectively; at least one of SEQ ID NOS.39-50 may be replaced with a variant having 1, 2 or 3 amino acid differences therefrom. The CD47 antibody provided by the application has high binding affinity with the extracellular region of CD47, good specificity and strong stability; and has the activity of blocking the binding of CD47 to signal regulator protein alpha (SIRP alpha) on immune cells.

Description

CD47 antibody and medical application thereof

Technical Field

The application relates to the technical field of biological medicine, in particular to a CD47 antibody and medical application thereof.

Background

CD47 is a transmembrane protein, a cell surface glycoprotein molecule belonging to the immunoglobulin superfamily that binds to a variety of proteins, including integrin, thrombospondin-1, and signal-regulating protein alpha (sirpa). CD47 is an important tumor antigen, and CD47 is overexpressed in different types of tumors, including myeloma, leiomyosarcoma, acute lymphoblastic leukemia, non-hodgkin lymphoma, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, etc., and is associated with the occurrence and development of various cancers, and high expression levels of CD47 are associated with therapeutic response and prognosis of cancer exacerbation. CD47 signals "eat me" by binding to the N-terminus of signal regulatory protein α (sirpa) on immune cells, inhibiting phagocytosis by macrophages, thereby protecting healthy cells from damage by the immune system. In contrast, when the surface expression of CD47 is reduced, the CD 47-sirpa signaling pathway is diminished and macrophages can migrate to these cells and swallow them. CD47 on normal erythrocytes binds to sirpa on the surface of macrophages, producing an inhibitory signal that prevents phagocytosis, but when erythrocytes age, the expression level of CD47 decreases, and aging erythrocytes deficient in CD47 are considered as foreign and rapidly cleared by macrophages in the spleen. CD47 and its ligands not only regulate immune responses, but also mediate various pathophysiological processes such as neutrophil chemotaxis and nervous system development, and play a regulatory role in immune tolerance and T cell activation. There is currently less development of CD47 antibodies, and there is a need in the art for CD47 antibodies with good stability and high binding affinity, good specificity.

Disclosure of Invention

The purpose of the application is to provide an antibody or an antigen binding fragment thereof capable of specifically binding with CD47, which has high binding affinity, good specificity and strong stability.

In a first aspect the present application provides an antibody or antigen binding fragment thereof capable of specifically binding to CD47 comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; wherein:

(1) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 39, SEQ ID NO. 40 and SEQ ID NO. 41, or respectively have amino acid sequences shown as SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44, or respectively have amino acid sequences shown as SEQ ID NO. 48, SEQ ID NO. 49 and SEQ ID NO. 50; the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 39-50 are defined according to the Kabat numbering system; or (b)

(2) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53, or respectively have amino acid sequences shown as SEQ ID NO. 57, SEQ ID NO. 58 and SEQ ID NO. 59; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 54, SEQ ID NO. 55 and SEQ ID NO. 56, or respectively have amino acid sequences shown as SEQ ID NO. 60, SEQ ID NO. 61 and SEQ ID NO. 62; the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 51-62 are defined according to the IMGT numbering system; or (b)

(3) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 63, SEQ ID NO. 64 and SEQ ID NO. 65, or respectively have amino acid sequences shown as SEQ ID NO. 69, SEQ ID NO. 70 and SEQ ID NO. 71; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 66, SEQ ID NO. 67 and SEQ ID NO. 68, or respectively have amino acid sequences shown as SEQ ID NO. 72, SEQ ID NO. 73 and SEQ ID NO. 74; the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 63-74 are defined according to the CGC numbering system; or (b)

(4) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 75, SEQ ID NO. 76 and SEQ ID NO. 77 or respectively have amino acid sequences shown as SEQ ID NO. 81, SEQ ID NO. 82 and SEQ ID NO. 83; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 78, SEQ ID NO. 79 and SEQ ID NO. 80, or respectively have amino acid sequences shown as SEQ ID NO. 84, SEQ ID NO. 85 and SEQ ID NO. 86; the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 75-86 are defined according to the chothia numbering system; or (b)

(5) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 87, SEQ ID NO. 88 and SEQ ID NO. 89, or respectively have amino acid sequences shown as SEQ ID NO. 93, SEQ ID NO. 94 and SEQ ID NO. 95; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 90, SEQ ID NO. 91 and SEQ ID NO. 92, or respectively have amino acid sequences shown as SEQ ID NO. 96, SEQ ID NO. 97 and SEQ ID NO. 98; the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 87-98 are defined according to the North numbering system; or (b)

(6) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 99, SEQ ID NO. 100 and SEQ ID NO. 101, or respectively have amino acid sequences shown as SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 102, SEQ ID NO. 103 and SEQ ID NO. 104, or respectively have amino acid sequences shown as SEQ ID NO. 108, SEQ ID NO. 109 and SEQ ID NO. 110; the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO 99-110 are defined according to the AbM numbering system;

at least one of the SEQ ID NOS.39-110 may be replaced with a variant having 1, 2 or 3 amino acid differences thereto.

In some embodiments of the present application, the antibody or antigen binding fragment thereof capable of specifically binding to CD 47:

(1) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 39, SEQ ID NO. 40 and SEQ ID NO. 41, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44; or (b)

(2) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 48, SEQ ID NO. 49 and SEQ ID NO. 50; or (b)

(3) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 54, SEQ ID NO. 55 and SEQ ID NO. 56; or (b)

(4) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 57, SEQ ID NO. 58 and SEQ ID NO. 59; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 60, SEQ ID NO. 61 and SEQ ID NO. 62; or (b)

(5) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 63, SEQ ID NO. 64 and SEQ ID NO. 65, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 66, SEQ ID NO. 67 and SEQ ID NO. 68; or (b)

(6) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 69, SEQ ID NO. 70 and SEQ ID NO. 71; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 72, SEQ ID NO. 73 and SEQ ID NO. 74; or (b)

(7) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 75, SEQ ID NO. 76 and SEQ ID NO. 77, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 78, SEQ ID NO. 79 and SEQ ID NO. 80; or (b)

(8) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 81, SEQ ID NO. 82 and SEQ ID NO. 83; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 84, SEQ ID NO. 85 and SEQ ID NO. 86; or (b)

(9) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 87, SEQ ID NO. 88 and SEQ ID NO. 89, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 90, SEQ ID NO. 91 and SEQ ID NO. 92; or (b)

(10) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 93, SEQ ID NO. 94 and SEQ ID NO. 95; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 96, SEQ ID NO. 97 and SEQ ID NO. 98; or (b)

(11) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 99, SEQ ID NO. 100 and SEQ ID NO. 101, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 102, SEQ ID NO. 103 and SEQ ID NO. 104; or (b)

(12) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107; the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 108, SEQ ID NO. 109 and SEQ ID NO. 110;

At least one of the SEQ ID NOS.39-110 may be mutated to a variant having 1, 2 or 3 amino acid differences therefrom.

In some embodiments of the present application, the antibody or antigen binding fragment thereof capable of specifically binding to CD 47: and SEQ ID NO: 42. SEQ ID NO: 66. SEQ ID NO: 78. SEQ ID NO:90 or SEQ ID NO:102, the 5 th amino acid of LCDR1 is mutated to either one of Q, A, G and D; or with SEQ ID NO:54, the amino acid at position 2 of LCDR1 is mutated to either one of Q, A, G and D;

and/or

And SEQ ID NO: 44. SEQ ID NO: 56. SEQ ID NO: 68. SEQ ID NO: 80. SEQ ID NO:92 or SEQ ID NO:104, the amino acid at position 5 of LCDR3 is mutated to Q.

In some embodiments of the present application, an antibody or antigen binding fragment thereof capable of specifically binding to CD47 comprises a mutation as set forth in any one of the following:

(1) And SEQ ID NO: 42. SEQ ID NO: 66. SEQ ID NO: 78. SEQ ID NO:90 or SEQ ID NO:102, the 5 th amino acid of LCDR1 is mutated to Q; or (b)

(2) And SEQ ID NO: 42. SEQ ID NO: 66. SEQ ID NO: 78. SEQ ID NO:90 or SEQ ID NO:102, the 5 th amino acid of LCDR1 is mutated to a; or (b)

(3) And SEQ ID NO:42, SEQ ID NO: 66. SEQ ID NO: 78. SEQ ID NO:90 or SEQ ID NO:102, wherein the 5 th amino acid of LCDR1 is mutated to G; or (b)

(4) And SEQ ID NO: 42. SEQ ID NO: 66. SEQ ID NO: 78. SEQ ID NO:90 or SEQ ID NO:102, the 5 th amino acid of LCDR1 is mutated to D; or (b)

(5) And SEQ ID NO:42, and the 5 th amino acid of LCDR1 is mutated to G and is identical to SEQ ID NO:44, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(6) And SEQ ID NO:66, the amino acid at position 5 of LCDR1 is mutated to G and is identical to SEQ ID NO:68, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(7) And SEQ ID NO:78, and the amino acid at position 5 of LCDR1 is mutated to G and is identical to SEQ ID NO:80, wherein the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(8) And SEQ ID NO:90, and the 5 th amino acid of LCDR1 is mutated to G and is identical to SEQ ID NO:92, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(9) And SEQ ID NO:102, and the 5 th amino acid of LCDR1 is mutated to G and is identical to SEQ ID NO:104, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(10) And SEQ ID NO:42, and the 5 th amino acid of LCDR1 is mutated to a and is identical to SEQ ID NO:44, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(11) And SEQ ID NO:66, the amino acid at position 5 of LCDR1 is mutated to a and is identical to SEQ ID NO:68, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(12) And SEQ ID NO:78, and the amino acid at position 5 of LCDR1 is mutated to a and is identical to SEQ ID NO:80, wherein the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(13) And SEQ ID NO:90, and the amino acid at position 5 of LCDR1 is mutated to a and is identical to SEQ ID NO:92, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(14) And SEQ ID NO:102, and the amino acid at position 5 of LCDR1 is mutated to a and is identical to SEQ ID NO:104, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(15) And SEQ ID NO:54, the amino acid at position 2 of LCDR1 is mutated to Q; or (b)

(16) And SEQ ID NO:54, the amino acid at position 2 of LCDR1 is mutated to a; or (b)

(17) And SEQ ID NO:54, the amino acid at position 2 of LCDR1 is mutated to G; or (b)

(18) And SEQ ID NO:54, the amino acid at position 2 of LCDR1 is mutated to D; or (b)

(19) And SEQ ID NO:54, and the amino acid at position 2 of LCDR1 is mutated to G and is identical to SEQ ID NO:56, the 5 th amino acid of LCDR3 is mutated to Q; or (b)

(20) And the sequence of SEQ ID NO:54, and the amino acid at position 2 of LCDR1 is mutated to a and is identical to SEQ ID NO:56, the 5 th amino acid of LCDR3 is mutated to Q.

In some embodiments of the present application, an antibody or antigen binding fragment thereof capable of specifically binding to CD47 comprises: a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 8, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 5 or SEQ ID NO. 7; or a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18; or a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 or SEQ ID NO. 34.

In some embodiments of the present application, an antibody or antigen binding fragment thereof capable of specifically binding to CD47 comprises: a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 6, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 5; or a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 8, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 7; or a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18; or a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 22, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 or SEQ ID NO. 34; or a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 21, and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 34.

In some embodiments of the present application, an antibody or antigen-binding fragment thereof capable of specifically binding to CD47 further comprises a heavy chain constant region and a light chain constant region; the sequence of the heavy chain constant region comprises any one constant region sequence selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, or a variant thereof; the sequence of the light chain constant region comprises any one constant region sequence selected from a lambda light chain or a kappa light chain, or a variant thereof.

In some embodiments of the present application, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO. 35, SEQ ID NO. 36 or SEQ ID NO. 38 in an antibody or antigen binding fragment thereof capable of specifically binding to CD 47; and the light chain constant region has an amino acid sequence as shown in SEQ ID NO. 37.

In some embodiments of the present application, the antibody comprises at least one of a monoclonal antibody and a multispecific antibody, and the antigen-binding fragment comprises at least one of a Fab, fab ', F (ab') 2, fv, VHH, single domain antibody, scFv, and sdAb.

In a second aspect the present application provides a nucleic acid comprising at least one of a nucleotide sequence encoding an antibody or antigen binding fragment thereof as described in the first aspect of the present application or a complement thereof.

In a third aspect the present application provides an expression vector comprising a nucleic acid as described in the second aspect of the present application.

In a fourth aspect the present application provides a host cell comprising a nucleic acid as described in the second aspect of the present application, or comprising an expression vector as described in the third aspect of the present application.

In a fifth aspect the present application provides a method of preparing an antibody or antigen binding fragment thereof as described in the first aspect of the present application comprising: culturing the host cell of the fourth aspect of the present application under suitable culture conditions; and recovering the expressed antibody or antigen binding fragment thereof from the culture medium or from the cultured host cells.

A sixth aspect of the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the present application or a nucleic acid according to the second aspect of the present application or an expression vector according to the third aspect of the present application or a host cell according to the fourth aspect, and at least one of a pharmaceutically acceptable excipient, diluent or carrier.

A seventh aspect of the present application provides the use of an antibody or antigen binding fragment thereof according to the first aspect of the present application or a nucleic acid according to the second aspect of the present application or an expression vector according to the third aspect of the present application or a host cell according to the fourth aspect of the present application or a pharmaceutical composition according to the sixth aspect of the present application in the manufacture of a medicament for the treatment or prophylaxis of a CD47 mediated disease.

In some embodiments of the present application, the CD 47-mediated disease is selected from at least one of non-hodgkin's lymphoma (NHL), acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), multiple Myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, smooth muscle tumor, leiomyosarcoma, glioma, glioblastoma, and the like.

In an eighth aspect the present application provides a kit comprising an antibody or antigen binding fragment thereof according to the first aspect of the present application or a nucleic acid according to the second aspect of the present application or an expression vector according to the third aspect of the present application or a host cell according to the fourth aspect of the present application or a pharmaceutical composition according to the sixth aspect of the present application.

The beneficial effects of the application are that:

the CD47 antibody or the antigen binding fragment thereof provided by the application can be specifically combined with the extracellular region of CD47, and has high binding affinity, good specificity and strong stability; and has the activity of blocking the binding of CD47 to signal regulator protein alpha (SIRP alpha) on immune cells.

Of course, not all of the above-described advantages need be achieved simultaneously in practicing any one of the products or methods of the present application.

Drawings

For a clearer description of the technical solutions of the present application or of the prior art, reference will be made below to the accompanying drawings used in the embodiments or in the description of the prior art, which are, obviously, only some embodiments of the present application, from which it is possible for a person skilled in the art to obtain other embodiments.

FIG. 1 shows the effect of CD47 antibodies on hemagglutination in example 5;

FIG. 2a shows the binding of the CD47 antibody to CD47+ Raji cells in example 6;

FIG. 2b shows the binding of the CD47 antibody to CD47+ Raji cells in example 6;

FIG. 3 shows that the CD47 antibody of example 7 blocks SIPRa binding to CD47+ Raji cells;

FIG. 4a shows the binding of CD47 antibodies to human erythrocytes in example 8;

FIG. 4b shows the binding of CD47 antibodies to human erythrocytes in example 8;

FIG. 5 shows the competitive binding of the CD47 antibody of example 9 to free CD47 and human erythrocytes;

FIG. 6 shows that the CD47 antibody of example 10 promotes phagocytosis of tumor cells by human macrophages;

FIG. 7 shows the effect of CD47 antibodies in Raji xenograft models in example 11;

FIG. 8 shows the trend of body weight change of animals after administration in example 11;

figure 9 shows the tumor growth trend after administration in example 11.

Detailed Description

The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. Based on the embodiments herein, a person of ordinary skill in the art would be able to obtain all other embodiments based on the disclosure herein, which are within the scope of the disclosure herein.

In the present application, the term "antibody" refers to a protein that binds to a specific antigen, which broadly refers to all proteins and protein fragments, particularly full length antibodies or antibody functional fragments, that comprise complementarity determining regions (CDR regions). "full length antibodies" include polyclonal antibodies as well as monoclonal antibodies, and the term "antibody functional fragment" refers to a substance comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. In some embodiments, the antibody functional fragment has the effect of specifically recognizing and binding CD 47. In some embodiments, the antibody functional fragment is a fragment that has the function of blocking the binding of CD47 to signal regulatory protein alpha (sirpa).

In some embodiments, the antibody functional fragment may block or reduce the activity of CD 47. In some embodiments, such fragments will comprise a single heavy chain and a single light chain, or portions thereof. The fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

Where a term is used and/or accepted in the art, the definition of a term used in this application is intended to include all meanings unless explicitly stated to the contrary. For example, the term "complementarity determining region" ("CDR") is used to describe a discontinuous antigen binding site present in both the variable regions of a heavy chain and a light chain polypeptide. For the precise amino acid sequence boundaries of the CDRs of an antibody, they can be defined according to well-known methods, e.g., chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al, nature 342:877-883, 1989; al-Lazikani et al, journal of Molecular Biology,273:927-948, 1997); or Kabat based on antibody sequence variability (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health, 1987), abM (University of Bath), contact (University College London), and IMGT (the international ImMunoGeneTics database,1999 Nucleic Acids Research,27,209-212); or a North CDR definition based on neighbor-propagation clusters (affinity propagation clustering) that utilize a large number of crystal structures; the entire contents of which are incorporated by reference into the present application.

There are three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) in this application. Herein, the terms "CDR" and "CDRs" are used to refer to regions comprising one or more or even all of the amino acid residues that play a major role in the binding affinity of an antibody to an antigen or epitope that it recognizes. In another embodiment, CDR regions or CDRs refer to highly variable regions of heavy and light chains of CGC-defined immunoglobulins.

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 ^-6 M, e.g. less than about 10 ^-7 M、10 ^- ⁸ M、10 ^-9 M or 10 ^-10 Affinity of M or less (K _D ) And (5) combining.

Variants of antibodies are also within the scope of the present application, e.g., variants having 1, 2 or 3 amino acid differences from the respective CDRs; or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% identical to the amino acid or nucleotide sequence of each CDR or Framework Region (FR), or variable region (heavy chain variable region VL and/or light chain variable region VH), or the full length of an antibody described herein. In some cases, the variant of the antibody comprises at least the 6 CDRs described above; in some cases, variants of an antibody include at least one heavy chain and one light chain, while in other cases, variant forms contain two identical light chains and two identical heavy chains (or sub-portions thereof). In some cases, the variant of an antibody is obtained by conservative modifications or conservative substitutions or substitutions in the antibody sequences provided herein. "conservative modifications" or "conservative substitutions or substitutions" refer to amino acids in other amino acid substituted proteins that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. Those skilled in The art are aware that in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) Molecular Biology of The Gene, the Benjamin/Cummings pub.co., page 224, (4 th edition)), which is incorporated herein by reference in its entirety. In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. In some cases, the variant retains the ability to block CD47 from binding to signal regulatory protein α (sirpa). One of ordinary skill in the art will be able to determine suitable variants of antigen binding molecules as set forth herein using well known techniques. In certain embodiments, one of skill in the art can identify suitable regions of a molecule that can be altered by targeting regions that are believed to be unimportant to activity without disrupting activity. For nucleotide and amino acid sequences, the term "identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

The antibodies described herein may be mouse-derived antibodies, human-mouse chimeric antibodies, or humanized antibodies.

The term "mouse-derived antibody" is herein a monoclonal antibody against human CD47 prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with CD47 antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In some embodiments of the present application, the murine CD47 antibody or antigen binding fragment thereof may further comprise a light chain constant region of murine kappa chain, lambda chain or variants thereof, or further comprise a heavy chain constant region of murine IgG1, igG2, igG3 or variants thereof.

The term "chimeric antibody" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce the immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In some embodiments of the present application, the antibody light chain of the CD47 chimeric antibody further comprises a light chain constant region of a human kappa chain, lambda chain, or variant thereof. The antibody heavy chain of the CD47 chimeric antibody further comprises a heavy chain constant region of human IgG1, igG2, igG3, igG4, or variants thereof.

The term "humanized antibody (humanized antibody)", in the present invention, refers to a CDR-grafted antibody (CDR-grafted antibody), i.e., an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. To avoid a decrease in immunogenicity while causing a decrease in functional activity, such as binding to CD47, the human antibody variable region framework sequences may be subjected to minimal back or back mutations to maintain activity. Humanized antibodies of the present application also include humanized antibodies that are further affinity matured for CDRs by phage display.

and/or with SEQ ID NO: 44. SEQ ID NO: 56. SEQ ID NO: 68. SEQ ID NO: 80. SEQ ID NO:92 or SEQ ID NO:104, the amino acid at position 5 of LCDR3 is mutated to Q.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

In some embodiments of the present application, the antibody or antigen binding fragment thereof comprises any one of the following:

(3) And SEQ ID NO: 42. SEQ ID NO: 66. SEQ ID NO: 78. SEQ ID NO:90 or SEQ ID NO:102, the 5 th amino acid of LCDR1 is mutated to G; or (b)

(1) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 8, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 5 or SEQ ID NO. 7; or (b)

(2) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18; or (b)

(3) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 or SEQ ID NO. 34.

(1) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 6, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 5; or (b)

(2) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 8, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 7; or (b)

(3) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18; or (b)

(4) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 22, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 or SEQ ID NO. 34; or (b)

(5) A heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 21, and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 34.

Those skilled in the art can assemble the light and heavy chain variable regions of an antibody by the various CDRs provided herein with framework region 1 (FR 1), framework region 2 (FR 2), framework region 3 (FR 3) and framework region 4 (FR 4) in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In some embodiments of the present application, the sequence of the heavy chain constant region comprises any one constant region sequence selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, or a variant thereof; the sequence of the light chain constant region comprises any one constant region sequence selected from a lambda light chain or a kappa light chain, or a variant thereof. In some embodiments, the species from which the constant region is derived is selected from the group consisting of cattle, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, cocks, or humans.

In some embodiments of the present application, the heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO. 35, SEQ ID NO. 36 or SEQ ID NO. 38; and the light chain constant region has an amino acid sequence as shown in SEQ ID NO. 37.

In some embodiments of the present application, the antibody comprises at least one of a monoclonal antibody and a multispecific antibody, and the antigen-binding fragment comprises at least one of a Fab, fab ', F (ab') 2, fv, VHH, single domain antibody, scFv, and sdAb. The antibodies also include fusion proteins in which the antibody fragment is linked to another functionalized fragment, or ADC drugs in which the antibody is conjugated to a small molecule toxin.

The term "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region VH) and an antibody light chain variable domain (or region VL) connected by a linker. Such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers may for example consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in this application are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Rovers et al (2001), cancer Immunol.

The term "fusion protein" refers to a molecule of linked antibody fragment and another functionalized fragment, which may be the recombinant expression product of two genes obtained by DNA recombination technology or by hydrogen bonding, or a protein obtained by mediating fusion of two cytoplasmic membranes.

The term "ADC" refers to an antibody coupled drug, which is formed by coupling a monoclonal antibody and a small molecular drug, specifically recognizes an antigen on the surface of a tumor cell through the targeting function of the monoclonal antibody, and then utilizes the endocytosis of the in vivo cell to enable a chemical drug to enter the tumor cell so as to achieve the aim of killing the tumor cell in vivo, and has the characteristics of high specificity, good safety and the like.

The nucleic acid is typically RNA or DNA, and the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are preferably used when they are incorporated into vectors. Furthermore, since antibodies are membrane proteins, nucleic acids typically carry signal peptide sequences.

The term "expression vector" refers to a nucleic acid vector into which a polynucleotide can be inserted and which allows the expression of the protein encoded by the inserted polynucleotide. The expression vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Expression vectors are well known to those of skill in the art and include, but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, and the like. In some embodiments, expression vectors described herein comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

In some embodiments, the expression vector further comprises a reporter gene, which may be selected from the group consisting of metabolic markers, catalytic reporter genes, antibiotic markers, antibiotic resistance genes, herbicide resistance genes, auxotrophic reporter genes, compound detoxification enzyme genes, and carbohydrate metabolic enzyme selection marker genes, as known to those skilled in the art; in some preferred embodiments, for ease of observation and detection, the expression product of the reporter gene is a substance that emits light or produces a color change by catalyzing the substrate reaction itself, or emits light or produces a color change by catalyzing the substrate reaction, or produces an emitted light or produces a color change upon irradiation with excitation light. Such materials include more typically fluorescent proteins, luciferases and LacZ. Both fluorescent proteins and luciferases belong to the class of photoproteins, and the fluorescent expression can be detected with a camera or similar device. Fluorescent proteins function by absorbing light of one color (excitation) and then emitting light of a different color (emission) at a lower energy. In contrast, luciferases (and other bioluminescent enzymes) emit light by catalyzing a chemical reaction of a substrate (i.e., luciferin). Unlike the two labels described above, lacZ does not emit light. The product β -galactosidase of the LacZ gene catalyzes the conversion of X-gal to an opaque blue compound resembling indigo. Further, the fluorescent protein may be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein. The green fluorescent protein can adopt common GFP, and also can adopt modified GFP genes, such as enhanced GFP gene EGFP and the like; the blue fluorescent protein may be selected from EBFP, azuritc, tagBFP and the like; the yellow fluorescent protein may be selected from EYFP, ypct, phiYFP and the like; the orange fluorescent protein may be selected from mKO, mOrange, mBanana and the like; the red fluorescent protein may be selected from TagRFP, mRuby, mCherry, mKate and the like.

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include their progeny. Thus, "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It will also be appreciated that due to deliberate or unintentional mutation, all offspring may not be exactly identical in terms of DNA content, including mutant offspring that have the same function or biological activity as screened in the original transformed cells.

Host cells or cell lines suitable for expressing the antigen binding proteins of the present application include: mammalian cells such as NS0, sp2/0, CHO, COS, HEK, fibroblasts, myeloma cells, and the like. Human cells can be used, thus allowing the molecule to be modified with a human glycosylation pattern; alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product generation and purification, are known in the art. Bacterial cells may be used as host cells suitable for expressing the recombinant Fab or other embodiments of the present application. However, since proteins expressed in bacterial cells tend to be in an unfolded or incorrectly folded form or in a non-glycosylated form, any recombinant Fab produced in bacterial cells must be screened to preserve antigen binding capacity. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell will be the desired host, or in alternative embodiments, the molecule may be expressed in a bacterial host and then refolded. For example, various E.coli strains for expression are well known host cells in the biotechnology field. Various strains of Bacillus subtilis, streptomyces, other Bacillus, and the like may also be used in the method. Yeast cell strains known to those skilled in the art, as well as insect cells, such as Drosophila and lepidopteran insects and viral expression systems, can also be used as host cells.

The present application may be directed to the use of vectors as described above for insertion into a host cell, or direct transfer of nucleic acid into a host without the attachment of the vector (e.g., liposome-mediated transfection techniques).

The general methods for constructing the vector, the transfection methods required for the production of the cells of the present application, and the culture methods necessary for the production of the antibodies of the present application from the cells may all be conventional techniques. Likewise, once the antibodies of the present application are produced, they can be purified from the cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Such techniques are well known in the art. Another method of expressing antibodies may utilize expression in animals, particularly transgenic animals or nude mice. This involves an expression system that utilizes animal casein promoters, which when transgenically incorporated into a mammal, allow a female to produce the desired recombinant protein in its milk. The antibody-secreting culture may be purified using conventional techniques. For example, purification with an A or G Sepharose FF column containing adjusted buffers to remove non-specifically bound components; eluting the combined antibody by a pH gradient method, detecting antibody fragments by SDS-PAGE, and collecting; the antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is immediately frozen, e.g., lyophilized at-70 ℃.

A sixth aspect of the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the present application or a nucleic acid according to the second aspect of the present application or an expression vector according to the third aspect of the present application or a host cell according to the fourth aspect of the present application, and at least one of a pharmaceutically acceptable excipient, diluent or carrier.

The term "pharmaceutically acceptable excipient, diluent or carrier" refers to an excipient, diluent or carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, and is well known in the art, including but not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, and the like. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

The ninth aspect of the present application also relates to an antibody or antigen binding fragment thereof according to the first aspect of the present application or a nucleic acid according to the second aspect of the present application or an expression vector according to the third aspect of the present application or a host cell according to the fourth aspect of the present application or a pharmaceutical composition according to the sixth aspect of the present application or a kit as provided in the eighth aspect of the present application for use as a medicament or for use in therapy.

Embodiments of the present application will be described in detail below with reference to examples. The experimental method without specific conditions being noted in the examples of the present application is generally carried out according to conventional conditions, such as an antibody technical laboratory manual, a molecular cloning manual, etc. of cold spring harbor; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1: preparation of antibodies to CD47 antigen

1. Protein design and expression

The ECD (Extra-cellular domain) portion (Gln 19-Pro 139) of human CD47 sequence (NCBI: NP-942088) was cloned into pHL vector (Shanghai sea Biotechnology Co., ltd.) as a base, and the recombinant DNA was transiently expressed in 293 cells or transiently expressed and purified in CHO-S cells to obtain proteins for immunization and detection.

hCD47-ECD: the extracellular portion of human CD47 is the following sequence:

QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSP(SEQ ID NO:1)。

hCD47-ECD-his: the human CD47 extracellular segment with his tag is used for immunizing mice and screening, and the sequence is as follows:

MEFGLSWLFLVAILKGVQCQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPHHHHHH(SEQ ID NO:2)；

wherein, the cross line is signal peptide, and the italic is his-tag label.

hCD47-ECD-hFc: fusion proteins of Human CD47 extracellular region and Human IgG1 Fc were used for detection or immunization and the sequences were as follows:

Wherein, the cross line is signal peptide, the italic is hFC.

hCD47-ECD-mFc: fusion proteins of Human CD47 extracellular region and mouse IgG1 Fc were used for detection or immunization, and the sequences were as follows:

wherein the cross line is a signal peptide, and the italic is mFC.

2. Purification of CD 47-associated recombinant proteins, hybridoma antibodies, and purification of recombinant antibodies

(1) Purification of His tagged proteins

The cell expression liquid was centrifuged at high speed, and the supernatant was collected and the pellet was discarded. The HisTrap FF pre-packed column was equilibrated with PBS (phosphate buffer) for 5-10 column volumes. Cell expression supernatants were loaded at a rate of 2 mL/min. The pre-packed column was washed with PBS until mAu reading reached baseline, then the target protein was eluted with 20mM, 50mM, 250mM imidazole buffer (pH 7.4) and collected, and finally the target protein solution eluted with 250mM imidazole buffer was transferred to a concentration tube, centrifuged, and the target protein was replaced in PBS for storage for subsequent experiments.

(2) Purification of hybridomas, recombinant antibodies and Fc fusion proteins

Centrifuging the cell expression supernatant sample at a high speed to remove impurities, and purifying the hybridoma expression supernatant, the recombinant antibody and the Fc fusion protein by using a ProteinA column; the column was washed with PBS to A ₂₈₀ The reading drops to baseline. The target protein was eluted with 100mM acetate buffer (pH 3.0) and neutralized with 1M Tris-HCl (pH 8.0). According to the situation, the eluted sample is further purified by gel chromatography Superdex200 (GE) balanced by PBS after being properly concentrated, and the sample is collected and split-packed for standby.

Example 2: preparation of anti-human CD47 hybridoma monoclonal antibody

1. Immunization

Anti-human CD47 monoclonal antibodies were generated by immunizing mice. The mice used in this example were Balb/C mice, females, 6 weeks old (Beijing Vitolihua laboratory animal technologies Co., ltd., animal production license number: SCXK 2012-0001); feeding environment: SPF stage. Raising the purchased laboratory environment of the mice for 1 week, regulating the light/dark period of 12/12 hours, and controlling the temperature to be 20-25 ℃; humidity is 40-60%. The acclimatized mice were immunized as follows, with the immunizing antigen being the human CD47 extracellular segment tagged with either hFc or mFc or his.

Conventional immunization was performed with CFA (sigma & F5506-10 mL) and IFA (sigma & F5881-10 mL). The mixing volume ratio of the antigen for the first immunization and the adjuvant CFA (sigma & F5506-10 mL) is 1:1, the mixing volume ratio of the antigen for the booster immunization and the adjuvant IFA (sigma & F5881-10 mL) is 1:1, and the dosage is 50 mug/time (first immunization) and 25 mug/time (booster immunization). The antigen is inoculated after emulsification, and the time is respectively 0 day, 14 day and 28 day, namely 50 mug/dose of emulsified antigen is injected Intraperitoneally (IP) on day 0; day 14 Intraperitoneal (IP) injection of 25 μg/g post-emulsified antigen; day 28 Intraperitoneal (IP) injection of 25 μg/g post-emulsification antigen. Blood was taken on days 21 and 35, respectively, and antibody titers in mouse serum were determined by ELISA (enzyme-linked immunosorbent assay). After three immunizations, mice with high and plateau titers in serum were selected for spleen cell fusion. The immunization was boosted 3 days before spleen cell fusion, which was an antigen solution prepared by Intraperitoneal (IP) injection of 50. Mu.g/saline.

2. Spleen cell fusion

Spleen lymphocytes and myeloma Sp2/0 cells were fused with optimized PEG (polyethylene glycol) mediated fusion procedureAnd (5) fusing to obtain the hybridoma cells. Fused hybridoma cells were obtained in a form of (0.5-1). Times.10 ⁶ Density per mL was resuspended in complete medium (DMEM medium containing 20v/v% FBS (fetal bovine serum), 1 XHAT (hypoxanthine, aminopterin and thymidine) medium, 1 XOPI), 100. Mu.L/well in 96-well plates, 37 ℃, 5% CO ₂ After 3-4 days of incubation (volume fraction, remainder air), HAT complete medium 100. Mu.L/well was supplemented and incubation was continued for 3-4 days until needle-tip-like clones were formed. The supernatant was removed and 200. Mu.L/well of HT complete medium (RPMI-1640 medium containing 20v/v% FBS, 1 XHT and 1 XOPI), 37℃and 5% CO was added ₂ ELISA assays were performed after 3 days of incubation.

3. Hybridoma cell screening

According to the growth density of the hybridoma cells, detecting the supernatant of the hybridoma culture by a combined ELISA method, and carrying out a cell blocking experiment and a red blood cell agglutination experiment on the supernatant of positive hole cells detected by the combined ELISA method. The cells of the hole which are positive in combination and blocking are timely amplified, frozen and preserved, and subcloned for two to three times until single cell clone is obtained.

Each batch of subcloned cells was tested by CD47 binding ELISA and cell blocking assay. Hybridoma clones are obtained through the experimental screening, antibodies are further prepared through a serum-free cell culture method, and the antibodies are purified according to a purification example and are used for subsequent experiments.

Binding of CD47 antibodies to antigen CD 47: plates were coated with 0.15. Mu.g/well of CD47-ECD-his protein overnight at 4℃and then gradient diluted CD47 antibody was added and reacted at room temperature for 1 hour; BSA (bovine serum albumin) or milk was blocked, and after washing, goat anti-mouse IgG-Fc horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, horseradish peroxidase chromogenic solution was added, and the absorbance was read at 405 nm. Data processing and mapping analysis are carried out by using software SotfMax Pro v5.4, and a CD47 binding curve and EC of the antibody are obtained through four-parameter fitting ₅₀ (half maximum effect concentration) value.

4. Hybridoma positive clone sequence determination

Cloning sequences from positive hybridomas, comprising: collecting hybridoma cells in logarithmic growth phase, lysing the cell fluid with Trizol (Invitrogen, cat No. 15596-018), and extracting RNA, followed by PrimeScript ^TM Reverse Transcriptase the kit was subjected to reverse transcription (Takara, cat No. 2680A). PCR amplification and sequencing are carried out on cDNA obtained by reverse transcription by adopting a mouse Ig-Primer Set (Novagen, TB326Rev. B0503), and the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the obtained hybridoma clones 397 and 427 are shown as SEQ ID NO. 5-8, and the corresponding DNA sequences are shown as SEQ ID NO. 9-12.

397VL amino acid sequence: DVQITQSPSYLAASPGETITINCRTSRNISKFLAWYQEKPGKTYKLLIYSGSTLQSGIPSRFSGSGFSTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELK (SEQ ID NO: 5).

397VH amino acid sequence: QVQLQQPGAELVRPGSSVKLSCKASGYTFTNYWMHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSNTAYMQLSSLTSEDSAVYFCAKTRLGPFDYWGQGTTLTVSS (SEQ ID NO: 6).

427VL amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK (SEQ ID NO: 7).

427VH amino acid sequence: QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVNQRPGRGLEWIGRIDPNTGGTEYNEKFKNRATLTVDKPSSTAHMQLSSLTSEDSAVYYCAKGGISGLGYWGQGTTLTVSS (SEQ ID NO: 8).

397VL DNA sequence: gatgtccagataacccagtctccatcttatcttgctgcatctcctggagaaaccattactattaattgcaggacaagtaggaacattagcaaatttttagcctggtatcaagagaaacctgggaaaacttataagcttcttatctattctggatccactttgcaatctggaattccatcaaggttcagtggcagtggatttagtacagatttcactctcaccatcagtagcctggagcctgaagattttgcaatgtattactgtcaacagcataatgaatacccgctcacgttcggtgctgggaccaaactggagctgaaa (SEQ ID NO: 9).

397VH DNA sequence: caggtccaactgcagcagcctggggctgagctggtgaggcctgggtcttcagtgaagctgtcctgcaaggcttctggctacaccttcaccaactactggatgcattgggtgaaacagaggcctatacaaggccttgaatggattggtaatattgacccttctgatagtgaaactcactacaatcaaaagttcaaggacaaggccacattgactgtagacaaatcctccaacacagcctacatgcagctcagcagcctgacatctgaggactctgcggtctatttctgtgcaaaaaccagactgggaccgtttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 10).

427VL DNA sequence: gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagaacattgttcatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgctttcaaggttcacatgttcctcggacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID NO: 11).

427VH DNA sequence: caggtccaactgcagcagcctggggctgagcttgtgaagcctggggcttcagtgaagctgtcctgcaaggcttctggctacaccttcaccaactactggatgcactgggtgaaccagaggcctggacgaggccttgagtggattggaaggattgatcctaatactggtggtactgagtacaatgagaagttcaagaacagggccacactgactgttgacaaaccctccagcacagcccacatgcagctcagcagcctgacatctgaggactctgcggtctattattgtgcaaaggggggcatctcaggccttggctactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 12).

397. 427, the CDR regions (complementarity determining regions) of the heavy chain variable region, the light chain variable region are according to different coding rules, the sequences are shown in table 1, the rules in table 1 apply to the CDRs of all antibodies, variants of the present application.

TABLE 1 CDR region sequences for each heavy and light chain

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Example 3: preparation of CD47 humanized antibody

The antibody is selected from the group consisting of human heavy chain IgG 4/light chain kappa constant regions in combination with variable regions, and S228P mutations in the Fc region are used to increase the stability of the IgG4 antibody, and other mutations may be used to increase its performance.

In the application, the constant region of the heavy chain IgG 1/light chain kappa is also selected to be combined with each variable region, and L234A, L235A, G237A and N297A are mutated in the Fc segment to reduce the effect function of Fc, and meanwhile, in order to prevent heterogeneity caused by loss of lysine at the tail of the heavy chain, the lysine at the tail is removed during construction; other mutations may also be selected to increase their performance.

Heavy chain constant region (where mutation sites are underlined):

(1) human IgG4 (S228P), abbreviation: igG4, sequence as follows:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:35)。

(2) human IgG1 (L234A, L235A, G237A, N297A), abbreviation: igG1-MT, sequence as follows:

(3) Wild type, abbreviated as: igG1-WT, sequence as follows:

light chain constant region:

the sequence of human Kappa is as follows:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:37)。

1. molecular cloning of recombinant antibodies

And sequencing positive antibody molecules obtained by hybridoma screening to obtain a variable region coding gene sequence. Designing head and tail primers by using sequences obtained by sequencing, constructing VH/VK gene fragments of each antibody by using a sequencing gene as a template through PCR, and carrying out homologous recombination with an expression vector pHL (with a signal peptide and hIgG4/hkappa constant region gene (CH 1-FC/CL) fragments) to construct a recombinant antibody full-length expression plasmid VH-CH1-FC-pHL/VL-CL-pHL.

2. Expression and purification of recombinant and humanized antibodies

Plasmids containing the antibody light and heavy chains were expressed separately and then at 1:2, after 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified by Protein A column. The column was washed with PBS to A ₂₈₀ The reading drops to baseline. Eluting target protein with acidic eluent with pH of 3.0, neutralizing with 1M Tris-HCl (pH 8.0), and substituting eluted sample into PBS; further purification was performed by gel chromatography Superdex200 (GE) if necessary.

Example 4: humanization of anti-human CD47 murine hybridoma monoclonal antibody 397

The humanization method is generally performed by using a frame grafting method and a protein surface amino acid humanization method, and comprises the following steps: by comparing the germline gene database of the heavy and light chain variable region of the IMGT human antibody with MOE software, the germline gene of the heavy and light chain variable region with high homology with a female parent is respectively selected as a template, and the CDR of the murine antibody is respectively transplanted into a corresponding humanized template and subjected to corresponding back mutation to form variable region sequences with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Wherein the amino acid residues are identified and annotated using the CGC numbering system.

1. Hybridoma clone 397 humanized design and optimization

Humanized light chain templates of murine antibody 397 are IGKV1-9 x 01, IGKV1-39 x 01 and IGKJ2 x 01, and humanized heavy chain templates are IGHV1-46 x 01 and IGHJ4 x 01. The amino acid sequence of the humanized variable region sequence is shown in SEQ ID NOS 13-22, and is specifically as follows.

H397VL_1(1-9)：DIQLTQSPSFLSASVGDRVTITCRTSRNISKFLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKLEIK(SEQ ID NO:13)；

H397VL_2(1-9)：DVQLTQSPSFLSASVGDRVTITCRTSRNISKFLAWYQQKPGKAYKLLIYSGSTLQSGIPSRFSGSGFGTEFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKLEIK(SEQ ID NO:14)；

H397VL_3(1-9)：DVQITQSPSFLSASVGDRITINCRTSRNISKFLAWYQQKPGKAYKLLIYSGSTLQSGIPSRFSGSGFSTEFTLTISSLQPEDFATYYCQQHNEYPLTFGAGTKLEIK(SEQ ID NO:15)；

H397VL_1(1-39)：DIQMTQSPSSLSASVGDRVTITCRTSRNISKFLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKLEIK(SEQ ID NO:16)；

H397VL_2(1-39)：DVQMTQSPSSLSASVGDRVTITCRTSRNISKFLAWYQQKPGKAYKLLIYSGSTLQSGIPSRFSGSGFGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKLEIK(SEQ ID NO:17)；

H397VL_3(1-39)：DVQITQSPSSLSASVGDRITINCRTSRNISKFLAWYQQKPGKAYKLLIYSGSTLQSGIPSRFSGSGFSTDFTLTISSLQPEDFAMYYCQQHNEYPLTFGQGTKLEIK(SEQ ID NO:18)；

H397VH_1：QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGNIDPSDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARTRLGPFDYWGQGTLVTVSS(SEQ ID NO:19)；

H397VH_2：QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWIGNIDPSDSETHYNQKFKDRVTMTVDTSTSTVYMELSSLRSEDTAVYFCAKTRLGPFDYWGQGTLVTVSS(SEQ ID NO:20)；

H397VH_3：QVQLVQSGAEVVKPGASVKVSCKASGYTFTNYWMHWVKQRPGQGLEWIGNIDPSDSETHYNQKFKDRVTLTVDTSTSTVYMELSSLRSEDTAVYFCAKTRLGPFDYWGQGTLVTVSS(SEQ ID NO:21)；

H397VH_4：QVQLVQSGAEVVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGNIDPSDSETHYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYFCAKTRLGPFDYWGQGTLVTVSS(SEQ ID NO:22)。

In the study of murine antibodies, the inventors found that the motif (NIS) of one glycosylation site is present in the light chain CDR1 of the 397 antibody, and that mutational engineering thereof can affect the binding of the antibody to human erythrocyte surface CD 47. The inventors have made a series of mutations and combinations of mutations for the 397 humanized light chain H397VL_3 (1-39), H397VL_2 (1-39), H397VL_3 (1-9) and H397VL_2 (1-9), as shown in Table 2.

TABLE 2 mutation of humanized antibody light chain

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The specific amino acid sequence of the mutated variable region is shown below, wherein the underlined nucleotides are the mutated nucleotides, which are bolded.

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The mutated light chain was combined with the humanized heavy chains H397VH_3 and H397VH_4, respectively, as shown in Table 3, and a series of mutated antibodies were prepared by the method described in example 3, and the results after purification are shown in Table 4.

TABLE 3 mutant antibody names and sequences

TABLE 4 antibody purification results

Example 5: effect of CD47 antibodies on hemagglutination

Washing human red blood cells (Shanghai sea goose medical science Co., ltd.) with PBS for 3 times, centrifuging at 3000rpm for 5min, sucking the precipitate, diluting with PBS 200 times, and adding 100 μl per well to 96-well U-shaped plate; centrifuging at 3000rpm (2000 g) for 5min, and discarding the supernatant; diluting 20. Mu.g/mL of antibody by 3 times to 8 gradients (dilution method: starting at 20. Mu.g/mL, diluting 3 times to 8 gradients, i.e., 1 st concentration is 20. Mu.g/mL, 2 nd concentration is 20/3. Mu.g/mL, 3 rd concentration is 20/3/3. Mu.g/mL, and so on), adding diluted antibodies to the above U-shaped plates, respectively, and resuspending red blood cells; incubation at 37 ℃ for 2 hours; photographing and recording a result.

Experiments were performed with 1F8 (from the astronomical biotechnology (Shanghai) limited), 5F9 (from the university of Tenfu, small Li Lansi, the institute of custody), ALX148 (from the ALX tumor Biotechnology company) and 397 chimeric antibody (CH 397, variable region murine sequence, constant region human sequence) as controls. The experimental group settings are shown in Table 5, wherein, taking the first group as an example, the first half of the antibody name "H397H4L3 (1-39 QIS)", the sequence of which is the same as that of the mutant antibody variable region in Table 4, the second half of the antibody name "hIgG1 (MT)" is the sequence selection of the constant region, and the Fc in groups 1-13 is hIgG1 (MT), which means that the constant region is the sequence of the mutant Fc segment with the nucleotide sequence shown in SEQ ID NO: 35; the hIgG1 (WT) of group 14 shows that the constant region is the wild-type Fc segment sequence shown in SEQ ID NO. 38.

TABLE 5 hemagglutination assay group settings

Group setting	Antibody name
		1	H397H4L3(1-39QIS)-hIgG1(MT)
2	H397H4L3(1-39AIS)-hIgG1(MT)
		3	H397H4L3(1-9GIS-Q)-hIgG1(MT)
4	H397H4L3(1-9QIS)-hIgG1(MT)
		5	H397H4L3(1-9AIS-Q)-hIgG1(MT)
6	H397H4L2(1-9DIS)-hIgG1(MT)
		7	H397H4L2(1-9QIS)-hIgG1(MT)
8	H397H4L2(1-9GIS)-hIgG1(MT)
		9	H397H4L2(1-9AIS)-hIgG1(MT)
10	H397H4L2(1-39QIS)-hIgG1(MT)
		11	H397H4L3(1-39GIS)-hIgG1(MT)
12	H397H3L2(1-9AIS)-hIgG1(MT)
		13	H397H4L2(1-39AIS)-hIgG1(MT)
14	H397H4L3(1-9QIS)-hIgG1(WT)
		15	CH397
16	5F9
		17	1F8
18	IgG4(Isotype)

The non-aggregated red blood cells precipitate to form clear red spots, and the aggregated red blood cells form larger plaques. The experimental results are shown in fig. 1, and it can be seen from fig. 1 that the 16 th group (control molecule 5F 9) shows serious coagulation reaction, i.e. does not meet the safety requirement; the experimental groups except the groups 2, 6 and 13 have slight coagulation reaction, and the rest groups have no coagulation phenomenon, so that the overall safety requirements are met.

Example 6: dose-response of binding of CD47 antibodies to cd47+ Raji cells

Raji cells expressing human CD47 endogenously on the surface were allowed to stand for binding for 60 min at 4 ℃ with different concentrations of antibodies and control antibodies 1F8, 5F9 and ALX148 (control antibody sources are the same as in example 5); cells were washed 3 times with 1w/v% BSA (PBS formulation); then mixed with Phycoerythrin (PE) -labeled anti-human Fc-specific antibody (manufacturer Jackson immune, cat No. 109-115-098) and incubated at 4 ℃ for 30 min; the antibody binding was detected using a flow cytometer.

In this example, the group numbering convention is the same as that of example 5, and the constant regions are all hIgG1 (MT), which means that the constant regions are mutant Fc segment sequences having the nucleotide sequences shown in SEQ ID NO. 35.

The test results are shown in fig. 2a and 2b, and the test conditions of fig. 2a and 2b are the same; in fig. 2a and 2b, the abscissa indicates the antibody concentration and the ordinate indicates the MFI value of antibody binding. As can be seen from fig. 2a and 2b, the CD47 antibody binds to the tumor cells cd47+ Raji to some extent and has a drug concentration dose effect.

Example 7: dose-response of CD47 antibodies blocking binding of cd47+ Raji cells to sipra

CD47 signals "eat me" by binding to the N-terminus of signal regulatory protein α (sirpa) on immune cells, inhibiting phagocytosis by macrophages, thereby protecting healthy cells from damage by the immune system. After the antibody binds to CD47, the interaction of CD 47-SIRPalpha is blocked, so that the signal of 'do not eat me' is relieved, and the macrophage can recover the anti-tumor function.

Raji cells (source same as example 6) expressing human CD47 endogenously on the surface were used, incubated with different concentrations of antibodies and biotin-labeled sipra-Fc protein, and allowed to stand at 4 ℃ for 60 min for binding; cells were washed 3 times with 1w/v% BSA (PBS formulation); then mixed with PE-labeled streptavidin (manufacturer Jackson immune., cat. No. 016-030-084) and incubated at 4℃for 30 minutes; and detecting by using a flow cytometry analyzer.

The test results are shown in fig. 3, wherein the abscissa indicates the antibody concentration and the ordinate indicates the MFI value of antibody binding. As can be seen from fig. 3, the CD47 antibody can effectively block binding of cd47+ Raji cells to sipra.

Example 8: dose effect of binding of CD47 antibodies to human erythrocytes

Since erythrocytes also express CD47, if CD47 antibodies bind to erythrocytes, it is possible to destroy erythrocytes by causing them to agglutinate or by attack by macrophages, thereby causing toxic side effects. However, not all antibodies cause erythrocyte agglutination, and by sequencing and screening, antibodies that do not cause erythrocyte agglutination and do not bind or weakly bind to erythrocytes can be selected. The more strongly the antibody binds to erythrocytes, the greater the toxicity.

Human erythrocytes (from the same source as in example 5) were washed 3 times with PBS, centrifuged at 3000rpm for 5min, the pellet was aspirated, diluted 1000-2000 fold with PBS and added to a 96-well U-plate at 100. Mu.L per well; centrifuging at 3000rpm for 5min, and discarding supernatant; adding antibodies with different concentrations and a control antibody, and standing at 4 ℃ for binding for 30 minutes; cells were washed 3 times with 1w/v% BSA (PBS formulation); then mixed with PE-labelled anti-human Fc specific antibody (source same as example 6), incubated for 30 min at 4 ℃; the antibody binding was detected using a flow cytometer.

In this example, the group numbering scheme is the same as that of example 5, and the first half of the antibody name, "H397H4L3 (1-39 QIS)", which has the same sequence as that of the mutant antibody variable region shown in Table 4, and the second half of the antibody name, "hIgG1 (MT)", which shows the constant region as a mutant Fc segment sequence having the nucleotide sequence shown in SEQ ID NO. 35, and "hIgG1 (WT)", which shows the constant region as a wild-type Fc segment sequence having the nucleotide sequence shown in SEQ ID NO. 38.

The test results are shown in fig. 4a and 4b, wherein the abscissa indicates the antibody concentration and the ordinate indicates the MFI value of antibody binding. As can be seen from fig. 4a and 4b, the CD47 antibody has only weak binding to human erythrocytes, and it can be seen that the toxic and side effects are quite weak, and has obvious advantages compared with the control antibody 5F 9.

Example 9: competitive binding of CD47 antibodies to free CD47 and human erythrocytes

Human CD47-his protein, 0.1. Mu.g/well coated overnight at 4 ℃; PBST (Tween-20-containing phosphate buffer) was washed 3 times; sealing 5w/v% milk, and standing at 37 ℃ for 1.5h; PBST is washed 3 times for standby. Human erythrocytes 1E 8/well were plated, and gradient diluted antibody was added and incubated for 30min at 37 ℃. Red blood cells and antibodies were transferred together to a blocked Elisa plate and incubated at 37 ℃ for 60min; PBST wash 5 times; adding 100 mu L of erythrocyte lysate, and incubating for 20min at 37 ℃; PBST washing; incubation with HRP-labeled goat anti-human secondary antibody (manufacturer Jackson immune, cat No. 109-035-003) at 37deg.C for 30min; TMB (3, 3', 5' -tetramethylbenzidine) developed after PBST wash and read with an ELISA reader.

The test results are shown in FIG. 5, in which the abscissa represents the antibody concentration and the ordinate represents the developed OD ₄₅₀ And reading the value. As can be seen from FIG. 5, the antibodies of the present application retain high binding capacity (EC) for CD47-his binding after incubation with human erythrocytes ₅₀ 0.072 μg/mL and 0.076 μg/mL, respectively) relative to control molecules 5F9 and ALX148 (EC ₅₀ 1.157 μg/mL and 3.155 μg/mL, respectively) with 2 orders of magnitude lifting. The results show that the antibodies provided by the application can achieve better curative effect in vivo with lower dosage.

Example 10: CD47 antibodies promote phagocytosis of tumor cells by human macrophages

PBMCs were isolated from human blood and monocytes differentiated into macrophages over 6 days. The monocyte-derived macrophages were scraped off and resuspended in a 24-well plate and attached for 24 hours. The human tumor cell line Raji expressing CD47 endogenously was selected as target cells and labeled with 1. Mu.M CFSE (carboxyfluorescein diacetate succinimidyl ester) for 10min, followed by the addition of macrophages at a ratio of 5:1, 5 tumor cells per macrophage, and different doses of CD47 antibody. Incubation for 3h, washing off target cells which are not phagocytized with PBS, scraping off the remaining phagocytes, staining with macrophage marker CD14 antibody, and analyzing with flow cytometry. Cd14+ cells were measured and the percentage of cfse+ cells in the cd14+ cells was analyzed for characterization of phagocytosis.

In this example, the group numbering convention is the same as that of example 5, and the constant regions are all hIgG1 (MT), which means that the constant regions are the mutant Fc segment sequences having the nucleotide sequences shown in SEQ ID NO. 36.

The results of the test are shown in FIG. 6, in which the abscissa indicates the concentration of antibodies and the ordinate indicates the proportion of phagocytic macrophages occurring. As can be seen from fig. 6, the antibodies of the present application can effectively promote phagocytosis of tumor cells by macrophages, and the activation effect is concentration-dependent.

Example 11: detection of in vivo efficacy of CD47 antibodies using fluorescein-labeled Raji xenograft model (CDX)

Raji-Luc cells resuspended in PBS were cultured at 5X 10 ⁵ A concentration of 0.2mL was inoculated into the tail vein of the B-NDG mice at an inoculum size of 0.2 mL/min. Tumor imaging signal values were measured using a small animal imager on day 0, 3 post-inoculation, when the average imaging signal intensity reached 3.26E+06p/sec/cm ² At/sr, the appropriate animals were selected into groups according to tumor imaging signal values and animal weights, and were equally distributed into 10 experimental groups of 6 animals each.

The dosing was started on the day of the group, 1mpk dose, i.p. (intraperitoneal injection) route, 2 times per week (BIW) for 3 weeks; the specific dosing regimen is shown in table 6. The growth of tumors in mice was measured by in vivo imaging system analysis of fluorescence in mice. The animals were stopped when their body weight was reduced to less than 85% of their body weight at the beginning of the drug treatment, and continued administration was resumed until their body weight was 90% of their body weight at the beginning of the drug treatment.

During the course of the experiment, animals were euthanized if any one or more of the following occurred: 1) The animal has abnormal movement or paralysis; 2) The animals lost more than 20% of their body weight at the beginning of the drug treatment and lasted for more than 72 hours.

Table 6 specific dosing regimen

Group of

Test article

Animal number (only)

Dosage (mg/kg)

Route of administration

Frequency of administration

Number of administrations

G1

IgG4(isotype)

6

1

i.p.

BIW

6

G2

5F9

6

1

i.p.

BIW

6

G3

CH397

6

1

i.p.

BIW

6

G4

H397H4L2(1-39AIS)

6

1

i.p.

BIW

6

G5

H397H4L3(1-39GIS)

6

1

i.p.

BIW

6

G6

H397H4L2(1-39QIS)

6

1

i.p.

BIW

6

G7

H397H3L2(1-9AIS)

6

1

i.p.

BIW

6

G8

H397H4L2(1-9GIS)

6

1

i.p.

BIW

6

G9

H397H4L2(1-9QIS)

6

1

i.p.

BIW

6

G10

H397H4L3(1-9AIS-Q)

6

1

i.p.

BIW

6

Note that: the constant regions of the antibodies are IgG4 (S228P), and the nucleotide sequence is shown as SEQ ID NO. 35.

On day 17 after grouping, mice in the isotype control (isotype) group developed severe weight loss and paralysis, and the experiment was terminated.

The body weight changes of the mice are shown in Table 7 and FIG. 8, and the tumor growth conditions are shown in Table 8, FIG. 7 and FIG. 9.

TABLE 7 influence of CD47 antibodies on Raji-Luc cell transplantation B-NDG mice body weight

Note that: a: mean ± standard error;

b: statistical comparison of dosing group body weight with hIgG4 control group body weight at day 17 of group dosing, T-test analysis, <0.05, <0.01, < P; "-" means absent;

the constant regions of the antibodies are IgG4 (S228P), and the nucleotide sequence is shown as SEQ ID NO. 35.

TABLE 8 Effect of test substances on tumor volume of Raji-Luc cell transplantation B-NDG mice

Note that: a: mean ± standard error;

b: statistically comparing tumor imaging signal intensities of the dosing group with the solvent control group at day 17 of group dosing, t-test, P <0.01, P <0.0001;

"-" means absent;

From the above results, a large number of tumor signals were observed in mice of the isotype control group, whereas no tumor signals were observed in the G2, G3, G8 groups, and only individual mice in the G4, G5, G6, G7, G9, and G10 groups were detected. The results show that the antibodies provided by the application have positive anti-tumor effects in vivo.

In order to verify the stability and efficacy of the antibody, the present application set up the groups of examples 5-11, and conducted two sets of experiments on the constant region of the antibody, igG4 (S228P) with the nucleotide sequence of the constant region of the antibody shown as SEQ ID NO:35, and antibody constant region hIgG1 (MT) with the nucleotide sequence of the constant region of the antibody shown as SEQ ID NO:36 or antibody constant region hIgG1 (WT) with the nucleotide sequence of SEQ ID NO:38, respectively, and as a result, it was found that both in vivo and in vitro medicinal effects could be achieved, and all experimental results were not shown in the present application. The inventors believe that the test results shown in examples 5-11 are sufficient to demonstrate that a CD47 antibody comprising the CDRs or variable regions described herein is capable of achieving the technical effects described herein.

In this specification, each embodiment is described in a related manner, and identical and similar parts of each embodiment are all referred to each other, and each embodiment mainly describes differences from other embodiments.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the present application. Any modifications, equivalent substitutions, improvements, etc. that are within the spirit and principles of the present application are intended to be included within the scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CD47 comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; wherein:

(1) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 39, SEQ ID NO. 40 and SEQ ID NO. 41, or respectively have amino acid sequences shown as SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47;

the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44, or respectively have amino acid sequences shown as SEQ ID NO. 48, SEQ ID NO. 49 and SEQ ID NO. 50;

the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 39-50 are defined according to the Kabat numbering system; or (b)

(2) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53, or respectively have amino acid sequences shown as SEQ ID NO. 57, SEQ ID NO. 58 and SEQ ID NO. 59;

the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 54, SEQ ID NO. 55 and SEQ ID NO. 56, or respectively have amino acid sequences shown as SEQ ID NO. 60, SEQ ID NO. 61 and SEQ ID NO. 62;

the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 51-62 are defined according to the IMGT numbering system; or (b)

(3) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 63, SEQ ID NO. 64 and SEQ ID NO. 65, or respectively have amino acid sequences shown as SEQ ID NO. 69, SEQ ID NO. 70 and SEQ ID NO. 71;

the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 66, SEQ ID NO. 67 and SEQ ID NO. 68, or respectively have amino acid sequences shown as SEQ ID NO. 72, SEQ ID NO. 73 and SEQ ID NO. 74;

the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 63-74 are defined according to the CGC numbering system; or (b)

(4) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 75, SEQ ID NO. 76 and SEQ ID NO. 77 or respectively have amino acid sequences shown as SEQ ID NO. 81, SEQ ID NO. 82 and SEQ ID NO. 83;

The LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 78, SEQ ID NO. 79 and SEQ ID NO. 80, or respectively have amino acid sequences shown as SEQ ID NO. 84, SEQ ID NO. 85 and SEQ ID NO. 86;

the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 75-86 are defined according to the chothia numbering system; or (b)

(5) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 87, SEQ ID NO. 88 and SEQ ID NO. 89, or respectively have amino acid sequences shown as SEQ ID NO. 93, SEQ ID NO. 94 and SEQ ID NO. 95;

the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 90, SEQ ID NO. 91 and SEQ ID NO. 92, or respectively have amino acid sequences shown as SEQ ID NO. 96, SEQ ID NO. 97 and SEQ ID NO. 98;

the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO. 87-98 are defined according to the North numbering system; or (b)

(6) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 99, SEQ ID NO. 100 and SEQ ID NO. 101, or respectively have amino acid sequences shown as SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107;

The LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 102, SEQ ID NO. 103 and SEQ ID NO. 104, or respectively have amino acid sequences shown as SEQ ID NO. 108, SEQ ID NO. 109 and SEQ ID NO. 110;

the HCDR1-3 and LCDR1-3 corresponding to the amino acid sequences shown in SEQ ID NO 99-110 are defined according to the AbM numbering system;

2. The antibody or antigen-binding fragment thereof of claim 1, wherein:

(12) The HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107; the LCDR1, LCDR2 and LCDR3 have amino acid sequences shown as SEQ ID NO. 108, SEQ ID NO. 109 and SEQ ID NO. 110, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein:

and SEQ ID NO: 42. SEQ ID NO: 66. SEQ ID NO: 78. SEQ ID NO:90 or SEQ ID NO:102, the 5 th amino acid of LCDR1 is mutated to either one of Q, A, G and D; or with SEQ ID NO:54, the amino acid at position 2 of LCDR1 is mutated to either one of Q, A, G and D;

And/or

4. The antibody or antigen-binding fragment thereof of claim 3, wherein:

5. The antibody or antigen-binding fragment thereof of claim 1, comprising:

a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 8, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 5 or SEQ ID NO. 7; or (b)

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18; or (b)

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 21 or SEQ ID NO. 22, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 or SEQ ID NO. 34.

6. The antibody or antigen-binding fragment thereof of claim 5, comprising:

a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 6, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 5; or (b)

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 8, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 7; or (b)

A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 22, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 or SEQ ID NO. 34; or (b)

A heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 21, and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 34.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, further comprising a heavy chain constant region and a light chain constant region;

the sequence of the heavy chain constant region comprises any one constant region sequence selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, or a variant thereof;

the sequence of the light chain constant region comprises any one constant region sequence selected from a lambda light chain or a kappa light chain, or a variant thereof.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein,

the heavy chain constant region has an amino acid sequence shown as SEQ ID NO. 35, SEQ ID NO. 36 or SEQ ID NO. 38; and

the light chain constant region has an amino acid sequence as shown in SEQ ID NO. 37.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the antibody comprises at least one of a monoclonal antibody and a multispecific antibody, and the antigen-binding fragment comprises at least one of a Fab, fab ', F (ab') 2, fv, VHH, single domain antibody, scFv, and sdAb.

10. A nucleic acid comprising at least one of a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9 or a complement thereof.

11. An expression vector comprising the nucleic acid of claim 10.

12. A host cell comprising the nucleic acid of claim 10, or comprising the expression vector of claim 11.

13. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-9, comprising:

culturing the host cell of claim 12 under suitable culture conditions; and

recovering the expressed antibody or antigen binding fragment thereof from the culture medium or from the cultured host cells.

14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 or the nucleic acid of claim 10 or the expression vector of claim 11 or the host cell of claim 12, and at least one of a pharmaceutically acceptable excipient, diluent or carrier.

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-9 or the nucleic acid of claim 10 or the expression vector of claim 11 or the host cell of claim 12 or the pharmaceutical composition of claim 14 in the manufacture of a medicament for the treatment or prevention of a CD 47-mediated disease.

16. The use of claim 15, wherein the CD 47-mediated disease is selected from at least one of non-hodgkin's lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphoblastic leukemia, chronic myelogenous leukemia, multiple myeloma, breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, smooth muscle tumor, leiomyosarcoma, glioma, and glioblastoma.

17. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 or the nucleic acid of claim 10 or the expression vector of claim 11 or the host cell of claim 12 or the pharmaceutical composition of claim 14.