CN118459587A

CN118459587A - Anti-CD 47 antibodies and uses thereof

Info

Publication number: CN118459587A
Application number: CN202410486983.8A
Authority: CN
Inventors: 王岩; 郑倩倩; 高新
Original assignee: Qichen Baitai Beijing Pharmaceutical Technology Co ltd
Current assignee: Qichen Baitai Beijing Pharmaceutical Technology Co ltd
Priority date: 2019-02-01
Filing date: 2019-02-01
Publication date: 2024-08-09
Also published as: CN111518208A; CN111518208B

Abstract

The present application provides recombinant anti-CD 47 antibodies and uses thereof. The anti-CD 47 antibody can be specifically combined with CD47, has the effects of blocking the combination of CD47 and SIRP alpha, promoting the phagocytosis of tumor cells by macrophages, inhibiting the growth of tumors and the like, and can be used for treating CD47 related diseases, such as CD47 related tumors.

Description

Anti-CD 47 antibodies and uses thereof

The application relates to a division application of 201910104772.2 Chinese patent application, and the application date of 201910104772.2 Chinese patent application is 2019, 2 and 1.

Technical Field

The present application relates to the field of antibodies, more specifically, the application relates to antibodies against CD47 and uses thereof.

Background

CD47 is a transmembrane glycoprotein that is widely expressed on the cell surface (particularly highly expressed on the surface of tumor cells). CD47 is a self-recognizing protein that activates signaling pathways by binding to ligand signaling regulatory protein alpha (sirpa) expressed on macrophages and dendritic cells, thereby preventing phagocytosis by macrophages, allowing tumor cells to evade immune monitoring. The use of an anti-CD 47 antibody can prevent the binding of CD47 to sirpa, thereby restoring phagocytic function of macrophages to tumor cells.

Therefore, the development of an anti-CD 47 antibody capable of specifically blocking the binding of CD47 to sirpa is expected to be useful for the treatment of CD 47-related diseases including tumors.

Summary of The Invention

In a first aspect, the application provides an antibody or antigen binding portion thereof that specifically binds CD47, comprising HCDR1, HCDR2 and/or HCDR3 of the heavy chain variable region.

In some embodiments, the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-8. In some embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 9-17. In some embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18-25. In alternative embodiments, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment, an Fv fragment, an scFv fragment, or an Fd fragment.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds CD47 further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and/or LCDR3.

In some embodiments, the LCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26-33. In some embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 34-38. In some embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 39-43.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding portion thereof that specifically binds CD47 is selected from the amino acid sequences set forth in any one of SEQ ID NOs 44-53, or an amino acid sequence having at least 80% homology thereto.

In some specific embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding portion thereof that specifically binds CD47 is selected from the amino acid sequences set forth in any one of SEQ ID NOs 44-49.

In some embodiments, the amino acid sequence of the light chain variable region of the antibody or antigen binding portion thereof that specifically binds CD47 is selected from the amino acid sequences set forth in any one of SEQ ID NOs 54-62, or an amino acid sequence having at least 80% homology thereto.

In some specific embodiments, the amino acid sequence of the light chain variable region of the antibody or antigen binding portion thereof that specifically binds CD47 is selected from the amino acid sequences set forth in any one of SEQ ID NOs 54-59.

In some embodiments, the antibody that specifically binds CD47 comprises a heavy chain variable region selected from the amino acid sequences set forth in any one of SEQ ID NOs 44-53 and a light chain variable region selected from the amino acid sequences set forth in any one of SEQ ID NOs 54-62.

In some embodiments, the antibody or antigen binding portion thereof that specifically binds CD47 is capable of specifically binding to human CD 47.

In some embodiments, the antibody that specifically binds CD47 is a full length antibody, a single chain antibody, a single domain antibody, or a bispecific antibody.

In some embodiments, the antibody or antigen binding portion thereof that specifically binds CD47 is capable of blocking binding of CD47 to sirpa.

In some embodiments, the antibody or antigen binding portion thereof that specifically binds CD47 is capable of promoting phagocytosis of tumor cells by macrophages.

In some embodiments, the antibody that specifically binds CD47 is a monoclonal antibody.

In some embodiments, the antibody that specifically binds CD47 is murine or humanized.

In some embodiments, the antibody or antigen binding portion thereof that specifically binds CD47 further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, an IgG3 subtype, or an IgG4 subtype. In some preferred embodiments, the heavy chain constant region is of the IgG1 subtype or the IgG2a subtype.

In some embodiments, the antibody or antigen binding portion thereof that specifically binds CD47 further comprises a light chain constant region selected from the group consisting of kappa type or lambda type. In some preferred embodiments, the light chain constant region is kappa-type.

In a second aspect, the application provides a nucleic acid molecule encoding an antibody or antigen-binding portion thereof according to the first aspect that specifically binds CD 47.

In a third aspect, the application provides an expression vector comprising a nucleic acid molecule according to the second aspect.

In a fourth aspect, the application provides a host cell comprising a nucleic acid molecule according to the second aspect or an expression vector according to the third aspect.

In a fifth aspect, the application provides a pharmaceutical composition comprising an antibody or antigen-binding portion thereof that specifically binds CD47 according to the first aspect and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients. In some embodiments, the active ingredient is an anti-tumor drug.

In some embodiments, the pharmaceutical composition is for treating a CD 47-associated disease.

In a sixth aspect, the present application provides the use of an antibody or antigen-binding portion thereof that specifically binds CD47 as described in the first aspect, or a pharmaceutical composition as described in the fifth aspect, for the manufacture of a medicament for the prevention and/or treatment of a CD 47-associated disease, such as a tumor.

In some embodiments, the drug is an antibody-conjugated drug.

In some embodiments, the tumor is selected from one or more of the following: leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.

In other aspects, the application provides a detection reagent or kit comprising an antibody or antigen-binding portion thereof that specifically binds CD47 according to the first aspect.

In other aspects, the application also provides a method of preventing and/or treating a CD 47-associated disease comprising administering to a subject in need thereof an antibody or antigen-binding portion thereof that specifically binds CD47 according to the first aspect, or a pharmaceutical composition according to the fifth aspect.

The antibodies or antigen binding portions thereof of the application that specifically bind CD47 are capable of binding to CD47 with one or more of the following effects: can be combined with human CD47 expressed by membrane cells in natural conformation to promote phagocytosis of CD47 expression cells by macrophages; the binding between CD47 and SIRP alpha can be effectively blocked, and phagocytosis of tumor cells by macrophages can be promoted; blocking and inhibiting the binding of CD47 and SIPR alpha without causing severe erythrocyte aggregation, and promoting phagocytosis of macrophages; and/or can inhibit tumor growth, etc.

Drawings

FIG. 1 shows the ability of each anti-human CD47 monoclonal antibody to bind to CD47 on the cell surface using FACS analysis.

FIG. 2 shows FACS analysis of whether each anti-human CD47 monoclonal antibody has a blocking effect on SIRPalpha binding to CD47 on the cell surface.

FIG. 3 shows the ability of each anti-human CD47 monoclonal antibody to promote phagocytosis of macrophages by ATP assay.

FIG. 4 shows the ability of each anti-human CD47 monoclonal antibody to inhibit tumor growth.

Detailed Description

The present application provides novel anti-CD 47 antibodies or antigen-binding portions thereof that specifically bind CD 47. In a preferred embodiment, the antibodies of the application, or antigen binding portions thereof, bind to CD47 expressed on the surface of a cell membrane, blocking binding of CD47 to sipra, promoting phagocytosis of CD47 expressing cells by macrophages. The application also provides nucleic acids encoding the antibodies or antigen binding portions thereof, expression vectors comprising the nucleic acids, host cells comprising the nucleic acids or expression vectors, methods of making and purifying the antibodies, and medical and biological uses of the antibodies or antigen binding portions thereof, e.g., in the prevention and/or treatment of CD 47-associated diseases or disorders. The application also encompasses detection reagents or kits comprising the antibodies or antigen binding portions thereof.

For ease of understanding the present application, certain terms used herein are first defined.

As used herein, the term "antibody" refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., igM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises a domain (CL 1). VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are conserved, termed Framework Regions (FR).

As used herein, the term "antigen binding portion" of an antibody refers to a portion or segment of an intact antibody molecule responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. The antigen binding portion of an antibody may be prepared from the intact antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques and the like. Non-limiting examples of antigen binding moieties include: fab fragments; f (ab') 2 fragments; fd fragment; fv fragments; single chain Fv (scFv) molecules; a single domain antibody; dAb fragments and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues that mimic the hypervariable regions of the antibody. The term "antigen binding portion" also includes other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and the like.

It is well known to those skilled in the art that complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) are regions of the variable region that have the greatest influence on the affinity and specificity of an antibody. There are two common definitions of CDR sequences for VH or VL, namely the kabat definition and the Chothia definition, see for example Kabat et al.,"Sequences of Proteins of Immunological Interest",National Institutes of Health,Bethesda,Md.(1991);A1-Lazikani et al.,J.Mol.Biol.273:927-948(1997); and Martin et al, proc.Natl. Acad.Sci.USA86:9268-9272 (1989). For a given antibody variable region sequence, CDR sequences in VH and VL sequences may be determined according to the Kabat definition or Chothia definition. In an embodiment of the application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2, and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2, and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

For a given antibody variable region sequence, the CDR sequences in the variable region sequence can be analyzed in a number of ways, for example, as determined using on-line software Abysis (http:// www.abysis.org /).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as binding of an antibody to an epitope of an antigen, e.g., the ability of an antibody to bind to a specific antigen with an affinity that is at least twice greater than its affinity for a non-specific antigen. However, it will be appreciated that antibodies can specifically bind to two or more sequence-related antigens. For example, the antibodies of the invention can specifically bind to CD47 of humans and non-humans (e.g., non-human primates).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for the naturally occurring mutations that may be present in a small number of individuals. The monoclonal antibodies described herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. No. 4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

As used herein, the term "homology" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and introduction of gaps, if desired, to achieve a maximum percentage of homology. Methods and computer programs for alignment are well known in the art. As used herein, "at least 80% homology" refers to any number of homology of 80% to 100%, such as 85%, 90%, 95% or 99%, etc.

As used herein, the term "CD 47-associated disease" includes diseases and/or symptoms associated with the CD47 signaling pathway. Exemplary CD 47-associated diseases or disorders include tumors, such as leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, melanoma, and the like.

As used herein, the term "EC50" refers to the half maximal effect concentration (concentration for 50%of maximal effect,EC50), which refers to the concentration that causes 50% of the maximal effect.

As used herein, the term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The use of degenerate bases (in addition to A, T, C, G conventional bases) in the nucleic acid sequences given herein is referred to in the sense as commonly understood by those skilled in the art. For example, R represents A or G; y represents C or T; m represents A or C; k represents G or T; s represents C or G; w represents A or T; h represents A or C or T; b represents C or G or T; v represents A or C or G; d represents A or G or T; n represents A or C or G or T.

In a first aspect, the application provides an antibody or antigen binding portion thereof that specifically binds CD47, comprising HCDR1, HCDR2 and/or HCDR3 of the heavy chain variable region. The CDRs, heavy chain variable region amino acid sequences and light chain variable region amino acid sequences suitable for use in the antibodies of the present disclosure are illustratively set forth in tables 1-4 below. In certain embodiments, the anti-CD 47 antibody, or antigen-binding portion thereof, comprises HCDR1, HCDR2, and/or HCDR3, independently selected from any one of the HCDR1, HCDR2, or HCDR3 sequences shown in table 1. In certain embodiments, an anti-CD 47 antibody of the application may further comprise LCDR1, LCDR2, and/or LCDR3, independently selected from any of the LCDR1, LCDR2, or LCDR3 sequences shown in table 2. For example, an anti-CD 47 antibody of the application can comprise any of the heavy chain variable regions shown in table 3, optionally paired with any of the light chain variable regions shown in table 4.

Table 1: sequence numbers of heavy chain CDR amino acid sequences of exemplary anti-CD 47 antibodies

Antibody numbering	Sequence number corresponding to HCDR1	Sequence number corresponding to HCDR2	Sequence number corresponding to HCDR3
				A1	SEQ ID NO:1	SEQ ID NO:9	SEQ ID NO:18
A2	SEQ ID NO:2	SEQ ID NO:10	SEQ ID NO:19
				A3	SEQ ID NO:3	SEQ ID NO:11	SEQ ID NO:20
A4	SEQ ID NO:4	SEQ ID NO:12	SEQ ID NO:21
				A5	SEQ ID NO:5	SEQ ID NO:13	SEQ ID NO:22
A6	SEQ ID NO:6	SEQ ID NO:14	SEQ ID NO:23
				A7	SEQ ID NO:5	SEQ ID NO:15	SEQ ID NO:22
A8	SEQ ID NO:7	SEQ ID NO:16	SEQ ID NO:24
				A9	SEQ ID NO:8	SEQ ID NO:17	SEQ ID NO:25
A10	SEQ ID NO:5	SEQ ID NO:13	SEQ ID NO:22

Table 2: sequence numbers of light chain CDR amino acid sequences of exemplary anti-CD 47 antibodies

Antibody numbering	Sequence number corresponding to LCDR1	Sequence number corresponding to LCDR2	Sequence number corresponding to LCDR3
				A1	SEQ ID NO:26	SEQ ID NO:34	SEQ ID NO:39
A2	SEQ ID NO:27	SEQ ID NO:35	SEQ ID NO:40
				A3	SEQ ID NO:28	SEQ ID NO:36	SEQ ID NO:41
A4	SEQ ID NO:29	SEQ ID NO:37	SEQ ID NO:42
				A5	SEQ ID NO:30	SEQ ID NO:35	SEQ ID NO:39
A6	SEQ ID NO:31	SEQ ID NO:38	SEQ ID NO:43
				A7	SEQ ID NO:32	SEQ ID NO:35	SEQ ID NO:39
A8	SEQ ID NO:28	SEQ ID NO:36	SEQ ID NO:41
				A9	SEQ ID NO:33	SEQ ID NO:35	SEQ ID NO:42
A10	SEQ ID NO:30	SEQ ID NO:35	SEQ ID NO:39

Table 3: heavy chain variable region amino acid sequences of exemplary anti-CD 47 antibodies and sequence numbers of coding sequences thereof

Table 4: exemplary anti-CD 47 antibody light chain variable region amino acid sequence and sequence number of its coding sequence

In some embodiments, an antibody disclosed herein, or antigen binding portion thereof, HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 1-8. In some embodiments, an antibody disclosed herein, or an antigen binding portion thereof, HCDR2 comprises an amino acid sequence of any one of SEQ ID NOs 9-17. In some embodiments, the HCDR3 sequences of antibodies or antigen-binding portions thereof disclosed herein comprise the amino acid sequences set forth in any one of SEQ ID NOs: 18-25.

In alternative embodiments, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment, an Fv fragment, an scFv fragment, or an Fd fragment.

The antibodies or antigen binding portions thereof disclosed herein may further comprise a light chain variable region in addition to the heavy chain variable region.

In some embodiments, an antibody or antigen binding portion thereof disclosed herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26-33. In some embodiments, an antibody or antigen binding portion thereof disclosed herein comprises an amino acid sequence set forth in any one of SEQ ID NOs 34-38. In some embodiments, the LCDR3 sequences of antibodies or antigen-binding portions thereof disclosed herein comprise the amino acid sequences set forth in any one of SEQ ID NOs: 39-43.

In particular embodiments, the amino acid sequence of the heavy chain variable region of an antibody or antigen binding portion thereof disclosed herein has at least 80% homology, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology, to an amino acid sequence selected from any of SEQ ID NOs 44-53. In a specific embodiment, the amino acid sequence of the heavy chain variable region of an antibody or antigen binding portion thereof disclosed herein is selected from the amino acid sequences set forth in any one of SEQ ID NOs 44-53. In a more specific embodiment, the amino acid sequence of the heavy chain variable region of an antibody or antigen binding portion thereof disclosed herein is selected from the amino acid sequences set forth in any one of SEQ ID NOs 44-49.

In particular embodiments, the amino acid sequence of the light chain variable region of an antibody or antigen binding portion thereof disclosed herein has at least 80% homology, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology, to an amino acid sequence selected from any of SEQ ID NOs 54-62. In a specific embodiment, the amino acid sequence of the light chain variable region of an antibody or antigen binding portion thereof disclosed herein is selected from the amino acid sequences set forth in any one of SEQ ID NOs 54-62. In a more specific embodiment, the amino acid sequence of the light chain variable region of an antibody or antigen binding portion thereof disclosed herein is selected from the amino acid sequences set forth in any one of SEQ ID NOs 54-59.

In some embodiments, the heavy chain variable region or the light chain variable region of an antibody disclosed herein can be substituted, deleted, or added with at least one amino acid based on the respective specific amino acid sequences recited above, and the resulting variant still retains CD47 binding activity.

In certain embodiments, the number of amino acid substitutions, deletions or additions described above is any number between 1 and 30 or between 1 and 30, preferably 1 to 20, more preferably 1 to 10. In preferred embodiments, the sequence variants differ from the original amino acid sequence by about 1,2,3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions, and/or additions. In more preferred embodiments, the sequence variant differs from the original amino acid sequence by about 1,2,3, 4, or 5 amino acid substitutions, deletions, or additions. In specific embodiments, the amino acid substitution is a conservative substitution.

In some embodiments, the antibodies disclosed herein are full length antibodies, single chain antibodies, single domain antibodies, or bispecific antibodies.

In some embodiments, the antibodies disclosed herein are monoclonal antibodies.

In some embodiments, the antibodies disclosed herein are murine or humanized.

In some embodiments, an antibody or antigen binding portion thereof disclosed herein is capable of specifically binding to CD47. In specific embodiments, the antibodies, or antigen binding portions thereof, disclosed herein specifically bind primate CD47, or CD47 of any species having a high homology to primate CD47. In a preferred embodiment, the antibodies, or antigen binding portions thereof, disclosed herein specifically bind human CD47. In some embodiments, an antibody or antigen binding portion thereof disclosed herein specifically binds monkey CD47.

In some embodiments, an antibody or antigen binding portion thereof disclosed herein is capable of blocking binding of CD47 to sirpa. In some embodiments, the antibody or antigen binding portion thereof that specifically binds CD47 is capable of promoting phagocytosis of tumor cells by macrophages.

For example, the inventors have performed in vitro and in vivo biological experiments on the anti-CD 47 antibodies disclosed herein, and the results indicate that the antibodies can well bind to CD47 molecules, block the binding of CD47 to sirpa, promote phagocytosis of tumor cells by macrophages, and/or inhibit tumor growth.

The application also provides nucleic acid molecules encoding the antibodies or antigen binding portions thereof disclosed herein, expression vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or expression vectors, and methods of making and purifying the antibodies.

In some embodiments, the nucleic acid encoding the antibody or antigen binding portion thereof is operably linked to regulatory sequences that can be recognized by a host cell transformed with the expression vector.

In some embodiments, any suitable expression vector may be used in the present application. For example, the expression vector may be any one of pcDNA3.3-TOPO, pTT5, pUC57, pDR1, pcDNA3.1 (+), pDHFF and pCHO 1.0. Fusion DNA sequences linked to appropriate transcriptional and translational regulatory sequences may be included in the expression vector.

In some embodiments, the host cells that can be used are cells containing the expression vectors described above, and can be eukaryotic cells, such as yeast, insect cells, or mammalian cell culture systems, all of which can be used for expression of the antibodies or antigen-binding portions thereof of the application. For example, HEK293E cells, CHO cells, and the like are suitable for use in the present application. The host cell may be a prokaryotic cell containing the above expression vector, and may be, for example, E.coli. In some specific embodiments, the mammalian cell is preferably a HEK293E cell or CHO cell.

In some embodiments, the methods of making an anti-CD 47 monoclonal antibody disclosed herein comprise: culturing the host cell under expression conditions to express the anti-CD 47 monoclonal antibody; isolation and purification of the expressed anti-CD 47 monoclonal antibody. Using the above method, the recombinant protein can be purified to a substantially homogeneous material, e.g., as a single band on SDS-PAGE electrophoresis.

In some embodiments, the anti-CD 47 antibodies disclosed herein may be isolated and purified using affinity chromatography methods, and depending on the characteristics of the affinity column used, the anti-CD 47 antibodies bound to the affinity column may be eluted using conventional methods such as high salt buffers, pH change, and the like.

In specific embodiments, the murine anti-CD 47 monoclonal antibodies disclosed herein are obtained by the following method:

The method comprises the steps of immunizing mice and hybridomas by using human, mouse and monkey CD47 extracellular domain fusion proteins to obtain a hybridoma cell line capable of expressing anti-human CD47 antibodies, screening candidate hybridoma cell lines by using in-vitro ELISA and FACS methods, carrying out binding, blocking and cross reaction experiment verification on the antibodies expressed by the candidate hybridoma cell lines with human CD47 proteins, carrying out affinity and in-vitro function experiment verification, and carrying out subsequent in-vivo anti-mouse Raji lymphoma growth experiments. Based on the experimental results, a brand new murine anti-human CD47 antibody is finally obtained.

In an exemplary embodiment, a method of obtaining an anti-CD 47 monoclonal antibody of the invention comprises the steps of:

(1) Expression A human CD47 extracellular domain fusion protein (hCD 47-ECD-6 His) and a positive control antibody protein were prepared.

For example, the extracellular region amino acid sequence of the human CD47 protein is fused with the amino acid sequence of the connecting peptide-6 His. And (3) carrying out codon artificial optimization on an amino acid sequence corresponding to the human CD47 extracellular region fusion protein (hCD 47-ECD-6 His), artificially synthesizing a DNA sequence, inserting the DNA sequence into an expression vector through an enzyme cutting site, and finally obtaining an expression plasmid pcDNA3.3-hCD47-ECD-6His. Transient expression was performed in FreeStyle medium using FreeStyle ^TM F cells, followed by purification of the fusion protein using a nickel column (Ni), resulting in a purified human hCD47-ECD-6His fusion protein.

(2) Anti-human CD47 monoclonal antibodies were obtained by mouse immunization and hybridoma technology.

The purified antigen and Freund's complete adjuvant are mixed by dosage vortex, and after complete emulsification, balB/C female mice of 6 weeks old are immunized for the first time. One week later, a second immunization was performed and the antigen was emulsified with Freund's incomplete adjuvant, with 10 μg of antigen per mouse injected, with a weekly frequency of immunization. After 3 times of immunization, the mice are subjected to orbital blood sampling, a small amount of blood samples are taken for serum titer detection, and after the serum titer detection by an indirect ELISA method reaches 1:200000 or more, the mice are subjected to booster immunization. Three days after boost, mice were sacrificed, lymph node B cells were isolated and counted, uniformly mixed in a ratio of 1:2 with myeloma cells (p3x63ag8.653) prepared in advance, treated with pronase, subjected to high voltage electric fusion, and the fused cells were cultured in 1/2HA medium containing 20% FBS for 7 days and 10 days, respectively, and after the liquid exchange, subjected to ELISA screening of hybridoma lines, and the obtained positive lines were subcloned in hybridoma medium containing 20% FBS using a limiting dilution method. Monoclonal cell well supernatants were subjected to ELISA and FACS experiments to detect binding of monoclonal antibodies to human, murine, monkey CD47, respectively. Hybridoma cell lines which are detected as double positive by ELISA and FACS are transferred into 24 holes from 96 holes for culture, and transferred into a 25cm ² culture flask for expansion culture after the hybridoma cell lines grow fully.

(3) Anti-human CD47 monoclonal antibodies were prepared and purified.

Resuscitates the monoclonal hybridoma cell strain, expands the strain to about 200ml of cell culture supernatant, centrifugally collects the supernatant, filters the supernatant, purifies the supernatant by using a Protein A affinity chromatography method and carries out purity identification by SDS-PAGE electrophoresis.

The present application provides pharmaceutical compositions comprising an antibody or antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier. The anti-CD 47 antibodies (e.g., anti-human CD47 monoclonal antibodies) disclosed herein can be formulated into pharmaceutical formulations with pharmaceutically acceptable carriers to more stably exert therapeutic effects. In some embodiments, these formulations can ensure the conformational integrity of the amino acid core sequence of the anti-CD 47 antibodies disclosed herein (e.g., anti-human CD47 monoclonal antibodies), while also protecting the multifunctional groups of the protein from degradation (including, but not limited to, aggregation, deamidation, or oxidation). In some embodiments, for liquid formulations, stability may generally be maintained at 2 ℃ to 8 ℃ for at least one year. In some embodiments, for lyophilized formulations, stability is maintained at 30 ℃ for at least six months. In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients. In some embodiments, the active ingredient is an anti-tumor drug.

The application also provides methods of preventing and/or treating CD 47-associated diseases comprising administering to an individual an anti-CD 47 antibody, or a pharmaceutical composition comprising an anti-CD 47 antibody (e.g., an anti-human CD47 monoclonal antibody). In some embodiments, the anti-tumor effect is pronounced upon administration to animals, including humans. In particular, the anti-CD 47 antibodies disclosed herein are effective in preventing and/or treating tumors and can be used as anti-tumor agents.

The application also provides the use of an anti-CD 47 antibody, or a pharmaceutical composition comprising an anti-CD 47 antibody, in the manufacture of a medicament for the prevention and/or treatment of a CD 47-associated disease or condition. In some embodiments, the drug is an antibody-conjugated drug. In some embodiments, the CD 47-associated disease or condition is a tumor.

In some embodiments, the tumor is leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, melanoma, or the like.

When the anti-human CD47 antibody and the pharmaceutical composition thereof disclosed herein are administered to animals including humans, the administration amount varies depending on the age and weight of the individual, the disease characteristics and severity, and the administration route, and reference may be made to the results and comprehensive conditions of animal experiments, and the total administration amount cannot exceed a certain range.

The dosage and frequency of administration of the antibody or composition thereof may vary depending on the prevention and/or treatment of the disease. In prophylactic applications, a composition comprising an antibody or antigen-binding portion thereof of the application is administered to a patient not already in a disease state to enhance patient resistance, this amount being defined as a "prophylactically effective dose". In this application, the particular dosage will depend on the patient's health and systemic immunity. Relatively low doses are typically administered at relatively infrequent intervals for a longer period of time. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until the disease progression is slowed or terminated, and preferably until the patient shows a partial or complete improvement in the symptoms of the disease. Thereafter, a prophylactic regimen can be administered to the patient. One of ordinary skill in the art can readily grasp specific dosages and frequencies as desired.

The application also provides detection reagents or kits comprising the antibodies or antigen binding portions thereof disclosed herein.

As used herein, the term "individual" refers to mammals, including but not limited to primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or human. Particularly preferred mammals are humans.

In this specification and claims, the words "comprise", "comprising" and "includes" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the application may be applied to any other aspect, embodiment or example described herein unless contradicted by context.

The foregoing disclosure generally describes the present application and the following examples are further illustrative of the present application and are not to be construed as limiting the application. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, see for example Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning：A Laboratory Manual,2nd edition,Cold spring Harbor Laboratory Press.

Examples

Example 1: preparation of recombinant proteins

The extracellular region amino acid sequence of human, murine, and monkey CD47 protein was fused to the linker peptide-6 His amino acid sequence. The amino acid sequence of human, mouse and monkey CD47 protein extracellular region fusion protein (hCD 47-ECD-6 His) is subjected to codon artificial optimization, and a DNA sequence of the amino acid sequence of human, mouse and monkey CD47 protein extracellular region fusion protein (hCD 47-ECD-6 His) is synthesized. The DNA sequence of the amino acid sequence of the extracellular fusion protein (hCD 47-ECD-6 His) of the human, mouse and monkey CD47 protein is inserted into the expression vector pcDNA3.3-TOPO through the enzyme cutting site, and finally the recombinant expression vector pcDNA3-hCD 47-ECD-6His is obtained. The expression of FreeStyle ^TM 293F cells was transiently transfected with the recombinant expression vector pcDNA3.3-hCD47-ECD-6His in FreeStyle medium. Subsequently, the fusion protein hCD47-ECD-6His is purified by using a nickel column (Ni), and finally the purified human, mouse and monkey hCD47-ECD-6His fusion protein is obtained.

The amino acid sequence of the positive control antibody P02 is obtained by referring to the disclosure of U.S. patent application No. 2013/0224188, gene synthesis is carried out according to the amino acid sequence of the positive control antibody, and the gene is cloned to an expression vector pcDNA3.3-TOPO, and finally the recombinant expression vector pcDNA3.3-P02 is obtained. The expression of FreeStyle ^TM 293F cells was transiently transfected with the recombinant expression vector pcDNA3.3-P02 in FreeStyle medium. P02 was then purified using a Protein a column (Protein purification liquid chromatography system/AKTA Purifier10, GE) according to the protocol provided by the manufacturer to obtain a purified positive control antibody P02.

Example 2: preparation of monoclonal hybridomas

1. BalB/C mice were immunized:

The purified hCD47-ECD-6His fusion protein antigen and Freund's complete adjuvant are mixed uniformly by dosage vortex, and after complete emulsification, the female mice of 6 weeks old BalB/C are immunized for the first time. Each mouse was injected with 10 μg antigen at one plantar spot of hind limb, 2 groups of mice were immunized with 5 animals each. The mice were subjected to a second plantar immunization one week after the first immunization, with Freund's incomplete adjuvant, at the same dose as the first immunization. Mice were then plantarimmunized once a week, with the same adjuvant and antigen doses as the second immunization.

The mice were subjected to orbital small amounts of blood sampling every three weeks after the first immunization and serum titers were detected, and after the serum titers were detected to be 1:200000 or more by an indirect ELISA method, the mice used for fusion were subjected to booster immunization.

2. Preparation of cells for fusion (myeloma cells)

Myeloma cells P3X63Ag8.653 for fusion were cultured for two weeks with DMEM medium containing 1X 8-azaguanine and 10% fetal bovine serum, and then cultured for one week with DMEM medium containing 10% fetal bovine serum, maintaining the density of P3X63Ag8.653 at 70% -80% to the day of fusion.

3. Cell fusion and HAT screening:

Acquisition and preparation of B lymphocytes: 2 mice after the immunization in the step 1 are taken, and the mice are killed after immune serum is collected and soaked in 75% alcohol for 2-3 minutes. The skin on the ventral side and the inner side of hind limb of the immunized mouse was cut off, and the lymph nodes were exposed. Lymph nodes were picked with pointed forceps, ground with a grinding rod, filtered through a cell screen, and prepared into single cell suspension for viable cell counting.

Cell fusion pretreatment: myeloma cells P3X63Ag8.653 in the flask were collected, centrifuged at 1000rpm/5min, and the supernatant was discarded, and the viable myeloma cells were counted after resuspension.

Cell fusion: per B lymphocyte: P3X63Ag8.653=1:2, centrifuging the supernatant at 2000rpm/5min, shaking off the cell pellet, adding 1mg/ml Pronase (Pronase) to 400 μl/1×10 ⁸ B lymphocytes, incubating for 15 seconds, adding 10ml fetal bovine serum to terminate the reaction, supplementing the electrotransfer solution (ECF), centrifuging the supernatant, resuspension with ECF and performing viable cell count, and adjusting the B lymphocyte density to 2×10 ⁶ cells/ml. And adding the cell suspension with the regulated density into an electrofusion tank, and operating an electrotransport instrument to perform cell fusion to obtain the mouse hybridoma. The murine hybridoma cell suspension was transferred from the fusion tank to 1/2HAT medium, allowed to stand for 3 hours, and then cell plated.

HAT medium selection: HAT screening medium containing 1/2HAT, 1 Xpenicillin-streptomycin, 20% fetal bovine serum and 80% DMEM medium was prepared. The mouse hybridoma cells were resuspended using the HAT selection medium described above and thoroughly mixed. The cell suspension was added to a 96-well cell culture plate at a density of 1X 10 ⁶ B cells/plate, 200. Mu.l/well, and incubated in a 37℃cell incubator. After 1 week of culture, the first liquid exchange was performed with HAT screening medium, and after 3 days of culture, the second liquid exchange was performed with HAT screening medium after the continuous culture in a 37 ℃ cell incubator.

4. Screening of positive hybridoma cell strains

After 2 weeks of fusion, cell supernatants were taken for ELISA and FACS experiments, the binding of the cell supernatants to human CD47 protein was detected, hybridoma cells which had positive ELISA and FACS results and blocked SIRPalpha binding to CD47 were subcloned and expanded.

5. Expansion culture

Hybridoma cell lines positive in ELISA and FACS detection were transferred from a 96-well plate to a 24-well plate for culture, and transferred to a 25cm ² flask for culture after full growth.

6. Limiting dilution subcloning

After sucking a small number of positive hybridoma cells for viable cell count, about 200 positive hybridoma cell lines were added to 80ml of complete medium, mixed well and inoculated in 4 96-well plates at a density of 0.5 cells/well. In addition, about 400 positive hybridoma cells were aspirated, added to 80ml of complete medium, mixed well and seeded in 4 96-well plates at a density of 1 cell/well. In addition, about 1000 positive hybridoma cells were aspirated, added to 20mL of complete medium, mixed well and seeded in 1 96-well plate at a density of 10 cells/well. 96-well plates were incubated at 37℃in a 5% CO ₂ incubator.

7. Clone detection and expansion culture

Monoclonal cell well supernatants were taken for ELISA and FACS detection, and binding of the cell monoclonal antibodies to human CD47 was detected separately. Hybridoma cells positive in ELISA and FACS are transferred from a 96-well plate to a 24-well plate for culture, and transferred to a culture flask of 25cm ² for culture after being full of the hybridoma cells.

8. Identification of cell subclasses

96-Well plates were coated with goat anti-mouse IgG 1-type antibody, goat anti-mouse IgG2 a-type antibody, goat anti-mouse IgG2 b-type antibody, goat anti-mouse IgG2 c-type antibody, goat anti-mouse IgG 3-type antibody, goat anti-mouse IgM-type antibody and goat anti-mouse IgGA-type antibody, respectively, 50 ng/100. Mu.l/well, and blocked with BSA at room temperature after overnight at 4 ℃. Then adding the hybridoma cell supernatant to be detected, incubating for 2 hours at room temperature, then adding an enzyme-labeled secondary anti-goat anti-mouse IgG type antibody, a goat anti-mouse kappa type antibody and a goat anti-mouse lambda type antibody for color development, and after stopping the reaction, reading at 450nm to judge whether the subclass of the hybridoma cell strain to be detected is the IgG1 subtype, the IgG2a subtype or the kappa type.

9. Cell cryopreservation

The frozen stock solution contained 90% fetal bovine serum and 10% DMSO.

After cell counting, the cells in the flask were resuspended, the supernatant was discarded after centrifugation at 1000rpm for 5min, blown down with 10% DMSO in fetal bovine serum, frozen in a cryopreservation box at a density of 5X10 ⁶ cells/tube, overnight at-80℃and transferred into liquid nitrogen the next day.

10. Monoclonal hybridoma gene preservation

Positive monoclonal hybridoma cell strains are collected, added with Trizol lysate for cell lysis, RNA is extracted and reversely transcribed into cDNA, and the cDNA is preserved at-80 ℃.

Example 3: murine monoclonal antibody gene sequencing

After immunization, fusion and monoclonalization, 10 monoclonal antibody hybridoma cells numbered CD47-1, CD47-2, CD47-3, CD47-4, CD47-5, CD47-6, CD47-7, CD47-8, CD47-9 and CD47-10 were selected for total RNA extraction based on ELISA, FACS and blocking experimental results. cDNA synthesis was performed using TAKARA PRIMESCRIPTII 1st Srand cDNA synthesis kit, followed by G treatment of the 5 'end of the synthesized cDNA template, and PCR was performed through 5' end primers and heavy and light chain constant region primers (HC-R and LC-R) to amplify the heavy and light chain variable regions of the antibody. The sequences of HC-R and LC-R are as follows:

5' end primer: CCCCCCCCCCCCCCCCCC (SEQ ID NO: 82)

HC-R：CTCAGGGAARTARCCYTTGAC(SEQ ID NO:83)

LC-R：TCACTGCCATCAATCTTCCAC(SEQ ID NO:84)

And (3) detecting PCR products of the amplified heavy chain variable region and the amplified light chain variable region through agarose gel electrophoresis, and then carrying out sample feeding and sequencing to identify the primers and the amplification primers. Finally, the amino acid sequence of the heavy chain variable region is determined as SEQ ID NOs: 44-53; the amino acid sequence of the light chain variable region is shown as SEQ ID NOs: 54-62; encoding the amino acid sequence shown in SEQ ID NOs:44-53, and a heavy chain variable region amino acid sequence as set forth in SEQ ID no the nucleotide sequences of the heavy chain variable regions are shown in SEQ ID NOs respectively: 63-72; encoding the amino acid sequence shown in SEQ ID NOs:54-62, and a light chain variable region amino acid sequence as set forth in SEQ ID no the nucleotide sequences of the light chain variable regions are shown in SEQ ID NOs respectively: 73-81.

Example 4: ELISA binding assay of anti-human CD47 monoclonal antibody and human CD47 protein

Monoclonal antibody hybridoma cells numbered CD47-1 to CD47-10 were cultured in an expanded manner and expressed, each of which gave 1L of expression supernatant, and the anti-human CD47 monoclonal antibodies obtained by purification with ProteinA were numbered A1-A10, respectively.

HCD47-ECD-6His fusion protein was diluted with PBS buffer, pH7.4,0.01M, and 96-well plates were coated at 50ng/50 μl/well overnight at 4 ℃. Plates were washed 3 times with 200. Mu.l/well PBST (1% Tween 20), and blocked at 37℃for 2 hours with 250. Mu.l/well of 3% PBS-BSA. After washing the plate three times, the best 6 anti-human CD47 monoclonal antibodies A1-A6 (50. Mu.l/well) were added in gradient dilutions, the highest concentration of monoclonal antibodies was 10. Mu.g/ml, 2-fold gradient dilutions, 12 concentration gradients per monoclonal antibody were combined, and incubated for 1 hour at room temperature. The plate was then washed 5 times, and 3% BSA was added at 5000:1, and incubating the goat anti-mouse IgG-HRP secondary antibody diluted in proportion for 1h at room temperature. After washing the plate 4 times, the plate was developed with TMB development kit (50. Mu.l/well), developed for 2 minutes at room temperature in the dark, and the development was stopped with 2MH ₂SO₄ (50. Mu.l/well). The samples were read at 450nm and 630nm using a microplate reader, respectively. As shown in Table 5, the EC50 values of the binding curves of A1-A6 were 0.016, 0.007, 0.010, 0.076, 0.016 and 0.010. Mu.g/ml, respectively, and all the monoclonal antibodies tested bound well to human CD47 with little difference in affinity between the monoclonal antibodies.

TABLE 5 EC50 values for ELISA binding of anti-human CD47 monoclonal antibodies

Anti-human CD47 monoclonal antibody numbering	EC50(μg/ml)
		A1	0.016
A2	0.007
		A3	0.010
A4	0.076
		A5	0.016
A6	0.010

Example 5 binding experiments of anti-human CD47 monoclonal antibodies to cell membrane-expressed human CD47 protein

Jurkat cells expressing human CD47 protein on the cell membrane surface were cultured to the total amount of cells required for the experiment, diluted to 1X10 ⁶ cells/ml with 1% BSA/PBS solution, plated to 96-well U-shaped bottom plate at a density of 1X10 ⁵ cells/100. Mu.l/well, centrifuged at 1500rpm for 4 minutes, and the supernatant was discarded. The anti-human CD47 monoclonal antibodies A1-A6 to be tested and the positive control antibody P02 were each diluted in a 3-fold gradient at an initial concentration of 10. Mu.g/ml for 12 concentration spots, each of which was resuspended in Jurkat cells at 100. Mu.l per well, and incubated at 4℃for 1 hour. The cells were subsequently washed 2 times with 180. Mu.l 1% BSA/PBS by centrifugation at 1500rpm for 4 minutes, the supernatant being discarded. After the final wash the supernatant was discarded, 100. Mu.l of 100-fold diluted goat anti-mouse IgG Fc conjugated FITC-conjugated secondary antibody was added to each well and incubated at 4℃for 0.5 h. After the secondary antibody incubation was completed, the supernatant was discarded after centrifugation at 1500rpm for 4 minutes. Cells were washed 2 times with 180 μl 1% BSA/1xPBS per well, and the supernatant was discarded after centrifugation at 1500rpm for 4 minutes each time. After the last washing, cells were resuspended in 100. Mu.l/well of 1% BSA/PBS solution and the binding between the anti-human CD47 monoclonal antibody and the cell membrane surface-expressed human CD47 protein was determined on a BD flow cytometer. As a result, as shown in FIG. 1, the binding curves of A1-A6 have EC50 values of 0.016, 0.017, 0.018, 0.017, 0.013 and 0.015. Mu.g/ml, respectively, and the binding curve of P02 has EC50 values of 0.018. Mu.g/ml, so that the anti-human CD47 monoclonal antibody can bind to human CD47 in a natural conformation expressed by the cells.

Example 6 anti-human CD47 monoclonal antibody blocking human SIRPalpha binding experiments with cell membrane expressed human CD47 protein

Jurkat cells expressing human CD47 protein on the cell membrane surface were cultured to the total amount of cells required for the experiment, diluted to 1X 10 ⁶ cells/ml with 1% BSA/PBS solution, plated on 96-well U-shaped bottom plates at a density of 1X 10 ⁵ cells/100. Mu.l/well, centrifuged at 1500rpm for 4 minutes, and the supernatant was discarded. The anti-human CD47 monoclonal antibodies A1-A6 to be tested and the positive control antibody P02 were diluted in 3-fold gradients at 12 concentration spots with an initial concentration of 10. Mu.g/ml, respectively, and the anti-human CD47 monoclonal antibodies of each concentration gradient were premixed with SIRPalpha at a final concentration of 0.1. Mu.g/ml, while only 0.1. Mu.g/ml SIRPalpha was added to the parallel 12 wells, and JURKAT cells were resuspended at 100. Mu.l per well and incubated for 1 hour at 4 ℃. The cells were subsequently washed 2 times with 180. Mu.l 1% BSA/PBS by centrifugation at 1500rpm for 4 minutes, the supernatant being discarded. After the final wash and removal of the supernatant, 100. Mu.l of a 100-fold dilution of anti-His antibody conjugated PE fluorescent secondary antibody was added to each well and incubated at 4℃for 0.5 h. After the secondary antibody incubation was completed, the supernatant was discarded after centrifugation at 1500rpm for 4 minutes. Cells were washed 2 times with 180 μl 1% BSA/1xPBS per well, and the supernatant was discarded after centrifugation at 1500rpm for 4 minutes each time. After the last wash, cells were resuspended in 100 μl/well of 1% BSA/PBS solution and the anti-human CD47 monoclonal antibody was assayed on a BD flow cytometer to block binding of SIRPalpha to human CD47 protein expressed on the cell membrane surface. As a result, as shown in FIG. 2, the inhibition curves of A1-A6 have IC50 values of 0.08, 0.15, 0.25, 0.17, 0.07 and 0.18. Mu.g/ml, respectively, and the inhibition curve of P02 has IC50 values of 0.13. Mu.g/ml, and the anti-human CD47 monoclonal antibodies can effectively block the binding of SIRPalpha to CD47 on the cell surface.

Example 7 Cross-species experiments of anti-human CD47 monoclonal antibodies with human, murine, monkey CD47 protein

CT26-hCD47 cells, CT26-mCD47 cells and CT26-cCD47 cells expressing human, mouse and monkey CD47 proteins on the cell membrane surface were cultured respectively until the total amount of the cells reached the experimental requirements, the cells were diluted to 1X 10 ⁶ cells/ml with 1% BSA/PBS solution, plated at a density of 100. Mu.l (1X 10 ⁵ cells)/well, centrifuged at 1500rpm for 4 minutes, and the supernatant was discarded. The primary antibodies of the anti-human CD47 monoclonal antibodies (A1-A6, positive control antibody P02) to be tested were diluted to two concentration points of 5. Mu.g/ml and 1. Mu.g/ml, respectively, and the resuspended cells were added at a volume of 100. Mu.l per well and incubated for 1 hour at 4 ℃. Cells were then washed 2 times with 180 μl1% BSA/PBS solution, the supernatant was discarded after the last wash, 100 μl/well of 100-fold diluted goat anti-mouse IgG Fc conjugated FITC fluorescent secondary antibody was added and incubated for 0.5 hours at 4deg.C. After incubation, cells were washed 2 times, resuspended in 100 μl1% BSA/PBS solution per well, and the binding of anti-human CD47 monoclonal antibodies to human, murine, and monkey CD47 proteins expressed on the cell membrane surface was measured on a BD flow cytometer. Wherein NC is to replace primary antibody by PBS and add secondary antibody in parallel; the blank is the primary antibody, neither of which is added, and only the background fluorescence value of the cells is detected. As a result, as shown in Table 6, the anti-human CD47 antibody could bind to the human CD47 protein and monkey CD47 protein expressed on the cell membrane surface, but did not recognize the murine CD47 protein expressed on the cell membrane surface.

TABLE 6 Cross-species experiments of anti-human CD47 monoclonal antibodies with human, murine, monkey CD47

Example 8 determination of affinity of anti-human CD47 monoclonal antibody by FACS

CT26-hCD47 cells expressing human CD47 protein on the cell membrane surface were cultured until the total amount of the cells reached the experimental requirement, the cells were diluted to 1X10 ⁶ cells/ml with 1% BSA/PBS solution, plated at a density of 100. Mu.l (1X 10 ⁵ cells)/well, centrifuged at 1500rpm for 4 minutes, and the supernatant was discarded. The initial concentration of anti-human CD47 monoclonal antibody (A1-A6, positive control antibody P02) was 10. Mu.g/ml, 2-fold gradient dilution was 11 spots total, 100. Mu.l/well of monoclonal antibody was added to resuspend cells, two duplicate wells were set for each concentration of antibody, and incubation was performed for 1 hour at 4 ℃. The cells were then centrifuged at 1500rpm for 4 minutes, the supernatant was discarded, and after washing 2 times with 1% BSA/PBS solution, 100-fold dilutions of goat anti-mouse IgG Fc-conjugated FITC-fluorescent secondary antibodies were added at 100. Mu.l/well and incubated for 0.5 hours at 4 ℃. After incubation, centrifugation was carried out at 1500rpm for 4 minutes, and the supernatant was discarded. Cells were washed 1 time with 1% BSA/1xPBS solution, centrifuged to discard the supernatant, and the cells were resuspended in 100. Mu.l of 1% BSA/1xPBS solution per well and detected on a BD flow cytometer. The results are shown in Table 7, and the affinity of the detected anti-human CD47 monoclonal antibody is better than that of the positive control antibody P02.

TABLE 7 affinity between anti-human CD47 monoclonal antibodies and human CD47 proteins expressed on the surface of cell membranes

Example 9 anti-human CD47 monoclonal antibodies promote macrophage phagocytosis assay

Preparation of C57BL murine peritoneal primary macrophages

The C57BL mice are killed by neck-guiding, the whole mice are immersed in 75% alcohol for 5 seconds in a biosafety cabinet and placed on an dissecting table, the limbs are fixed by a needle, the hands are used for holding a surgical scissors and forceps to tear the skin open to two sides, the peritoneum is exposed, after the peritoneum wall is scrubbed by 75% alcohol, 8-10ml of precooled DPBS is injected into the peritoneum by a syringe, and meanwhile, the peritoneum wall is rubbed with an finger to enable the liquid to flow fully in the peritoneum. The abdominal wall was gently lifted with the needle while the C57BL mice were gently tilted, and the fluid in the abdominal cavity was collected under the needle and sucked into the needle tube. The needle was carefully withdrawn, the liquid was poured into a centrifuge tube, centrifuged at 1000rpm at 4℃for 3 minutes, the supernatant was removed, 2ml of DMEM medium (DMEM+10% FBS+1% PS) was added to count the cells, then centrifuged at 1000rpm for 3 minutes, the supernatant was removed, 2ml of DMEM medium was added, and the mixture was placed in a 5% CO ₂ incubator at 37℃for overnight, the next day of pipetting, digested with pancreatin after two days, and after cell counting, 96-well plates were inoculated, 4X 10 ⁴ cells/well (100. Mu.l) were incubated in a 5% CO ₂ incubator for 18 hours.

2. Macrophage phagocytosis assay

The anti-human CD47 monoclonal antibody is examined by an ATP method to promote phagocytosis of Raji cells by C57BL mice peritoneal primary macrophages. The supernatant was removed from the incubated macrophages, 100. Mu.l/well of 1640 serum-free medium was added and starved for 2 hours at 37 ℃. The anti-human CD47 monoclonal antibodies to be tested (A1-A6, positive control antibody P02) were added to the prepared starved macrophages (50. Mu.l/well) at a starting concentration of 10. Mu.g/ml at 10 concentration spots in total diluted with serum-free 1640 medium at a 3-fold concentration gradient. Raji cells were prepared in advance, mixed into macrophages and anti-human CD47 monoclonal antibodies at 2×10 ⁴ cells/well, and incubated in a 5% CO ₂ incubator at 37 ℃ for 24 hours or more. 100. Mu.l of ATP solution/well was added, mixed well in the dark for 2 minutes, and left to stand at room temperature for 8 minutes for detection. As shown in FIG. 3, the EC50 values of the phagocytosis curves of A1-A6 are 0.004394, 0.004622, 0.003910, 0.007386, 0.006648 and 0.005675 micrograms/ml, the EC50 value of the phagocytosis curve of P02 is 0.009951 micrograms/ml, and the detected anti-human CD47 monoclonal antibodies can promote phagocytosis of Raji cells by macrophages, and the effects of the antibodies are superior to those of positive control antibodies.

Example 10 animal tumor inhibition experiments with anti-human CD47 monoclonal antibody

32 Six week old immunodeficiency (NPG) mice of comparable body weight were randomly divided into four groups of eight positive control antibody group (P02), A1 antibody-administered group (A1), A5 antibody-administered group (A5) and blank control group (PBS), respectively. All immunodeficient mice were inoculated subcutaneously with 100 μl of Raji cells at a density of 1×10 ⁶ cells/ml, the mice were observed for tumor formation, and dosing was started on day 6 after tumor loading (tumor volume about 50-150mm ³). The administration concentrations of the positive control antibody and the anti-human CD47 monoclonal antibody to be detected are 20mg/kg, and the administration is carried out intraperitoneally once every 3 days. The body weight of each group of mice individuals and the volume of tumor in the body were measured every two days simultaneously. After the end of the administration, tumors in mice were removed, weighed and the tumor inhibition rate of the antibody administration group was calculated, and the results are shown in table 8 and fig. 4, and the tumor inhibition rate of the anti-human CD47 monoclonal antibody to be tested administered at the same dose was equivalent to that of the positive control antibody.

TABLE 8 tumor inhibition of anti-human CD47 monoclonal antibodies

It should be understood that while the application has been described in connection with the above specific forms, it is not intended to be limited to the specific form set forth herein. It will be obvious to those skilled in the art that various equivalent changes can be made to the technical features contained in the application as described without departing from the spirit of the application, and these changes shall fall within the scope of the application.

Claims

1. An antibody or antigen-binding portion thereof that specifically binds CD47 comprising HCDR1, HCDR2 and/or HCDR3 of a heavy chain variable region, wherein the HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 1 to 8; the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 9-17; and/or said HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 18-25; preferably, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment, an Fv fragment, an scFv fragment or an Fd fragment.

2. The antibody or antigen-binding portion thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is selected from the amino acid sequences set forth in any one of SEQ ID NOs 44-53, or an amino acid sequence having at least 80% homology thereto.

3. The antibody or antigen-binding portion thereof of claim 1 or 2, further comprising a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and/or LCDR3, wherein the LCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 26-33; the LCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs 34-38; and/or said LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 39-43.

4. An antibody or antigen-binding portion thereof according to claim 3, wherein the amino acid sequence of the light chain variable region is selected from the amino acid sequences set forth in any one of SEQ ID NOs 54-62, or an amino acid sequence having at least 80% homology thereto.

5. The antibody or antigen-binding portion thereof of any one of claims 1-4, wherein the antibody comprises a heavy chain variable region selected from the amino acid sequences set forth in any one of SEQ ID NOs 44-53 and a light chain variable region selected from the amino acid sequences set forth in any one of SEQ ID NOs 54-62.

6. The antibody or antigen-binding portion thereof of any one of claims 1-5, wherein the antibody or antigen-binding portion thereof is capable of specifically binding to human CD 47;

Optionally, the antibody is a full length antibody, a single chain antibody, a single domain antibody, or a bispecific antibody;

Preferably, the antibody or antigen binding portion thereof is capable of blocking binding of CD47 to sirpa and/or promoting phagocytosis of tumor cells by macrophages.

7. The antibody or antigen-binding portion thereof of any one of claims 1-6, wherein the antibody is a monoclonal antibody;

Optionally, the antibody is murine or humanized.

8. The antibody or antigen-binding portion thereof of any one of claims 1-7, further comprising a heavy chain constant region selected from an IgG1 subtype, an IgG2 subtype, an IgG3 subtype, or an IgG4 subtype, and/or further comprising a light chain constant region selected from a kappa type or a lambda type;

preferably, the heavy chain constant region is of the IgG1 subtype or the IgG2a subtype and the light chain constant region is of the kappa type.

9. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-8.

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

11. A host cell comprising the nucleic acid molecule of claim 9 or the expression vector of claim 10;

preferably, the host cell is a prokaryotic cell, yeast, insect cell, or mammalian cell;

more preferably, the prokaryotic cell is E.coli; and/or the mammalian cell is a HEK293E cell or CHO cell.

12. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-8, and a pharmaceutically acceptable carrier;

Optionally, the pharmaceutical composition further comprises one or more additional active ingredients.

13. Use of the antibody or antigen-binding portion thereof of any one of claims 1-8, or the pharmaceutical composition of claim 12, in the manufacture of a medicament for the prevention and/or treatment of a CD 47-associated disease; preferably, the drug is an antibody conjugated drug.

14. The use of claim 13, wherein the disease is a tumor;

Preferably, the tumor is selected from one or more of the following: leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.

15. A detection reagent or kit comprising the antibody or antigen-binding portion thereof of any one of claims 1-8.