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CN111518208B - anti-CD 47 antibodies and uses thereof - Google Patents

anti-CD 47 antibodies and uses thereof Download PDF

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CN111518208B
CN111518208B CN201910104772.2A CN201910104772A CN111518208B CN 111518208 B CN111518208 B CN 111518208B CN 201910104772 A CN201910104772 A CN 201910104772A CN 111518208 B CN111518208 B CN 111518208B
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CN111518208A (en
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王岩
郑倩倩
高新
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Qichen Baitai Beijing Pharmaceutical Technology Co ltd
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Qichen Baitai Beijing Pharmaceutical Technology Co ltd
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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
Technical Field
The present application relates to the field of antibodies, more specifically, 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 present 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 present 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 present application provides an expression vector comprising a nucleic acid molecule according to the second aspect.
In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.
In a fifth aspect, the present 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 as described in the first aspect.
In other aspects, the present 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.
An antibody or antigen binding portion thereof that specifically binds CD47 of the present application is 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 or antigen binding portions thereof of the present application bind to CD47 expressed on the surface of a cell membrane, blocking the 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. CDR sequences of VH or VL are defined in two common ways, namely Kabat definition and 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); 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 embodiments of the present application, 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 half maximal effect concentration (concentration for 50%of maximal effect,EC50), meaning a concentration that causes 50% 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 (except for the 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 present 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 disclosed herein 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 present 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 present 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
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.
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.
The present 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 present application. For example, HEK293E cells, CHO cells, and the like are suitable for use in the present invention. 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. Using FreeStyle TM 293F cells were transiently expressed in Freestole medium, followed by purification of the fusion protein using nickel column (Ni) to finally obtain 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 with myeloma cells (p3x63ag8.653) prepared in advance in a ratio of 1:2, 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 then subjected to liquid exchange, and after liquid exchange, ELISA screening of hybridoma lines was performed, and the obtained positive lines were obtained in hybridomas containing 20% fbs Subcloning was performed in medium using 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 with ELISA and FACS detected as double positive are transferred into 24 holes from 96 holes for culture, and transferred into 25cm after full growth 2 The culture flask was expanded.
(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 present 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 containing an antibody or antigen-binding portion thereof of the present application is administered to a patient that is 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 present 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 particular aspects, embodiments or examples of the present application may be applied to any other aspects, embodiments or examples 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 should not be construed as limiting the present 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 having ordinary skill in the art and are described in numerous publications, see for example Sambrook, j., fritsch, e.f. and maniis, 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. FreeStyle was expressed in FreeStyle medium with recombinant expression vector pcDNA3.3-hCD47-ECD-6His TM Transient transfection of 293F cellsAnd (5) expression. 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. FreeStyle was isolated in FreeStyle medium using recombinant expression vector pcDNA3.3-P02 TM 293F cells were transiently transfected for expression. P02 was then purified using a Protein A column (Protein purification liquid chromatography system/AKTA Purifier 10, GE) according to the protocol provided by the manufacturer to give 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: the cells were mixed at a ratio of P3X63Ag8.653=1:2, the supernatant was centrifuged at 2000rpm/5min, and the cell pellet was shaken off at 400. Mu.l/1X 10 8 Adding 1mg/ml Pronase (Pronase) into B lymphocyte, incubating for 15 seconds, adding 10ml fetal bovine serum to stop reaction, adding electrotransfer solution (ECF), centrifuging, removing supernatant, re-suspending with ECF, performing viable cell count, and regulating B lymphocyte density to 2×10 6 Individual 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. According to 1X 10 6 The density of individual B cells/plate, 200. Mu.l/well, the cell suspension was added to a 96-well cell culture plate 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 which are detected as positive by ELISA and FACS are transferred into a 24-well plate from a 96-well plate for culture, and transferred into 25cm after full growth 2 Is cultured in a flask.
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 placed at 37℃in 5% CO 2 Culturing in an 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 25cm after full growth 2 Is cultured in a flask.
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.
The cells in the flask were resuspended, after cell counting, the supernatant was discarded after centrifugation at 1000rpm for 5min, and the suspension was blown with fetal bovine serum containing 10% DMSO at 5X 10 6 The density of individual cells/tubes was frozen in a cryopreservation cassette, 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 st SrancDNA synthesis kit, followed by G treatment of the 5 'end of the synthesized cDNA template, and PCR was performed by 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 are set forth in SEQ ID NOs: 63-72; encoding the amino acid sequence shown in SEQ ID NOs:54-62 are shown in SEQ ID NOs: 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 developed with 2M H 2 SO 4 (50. Mu.l/well) the color development was stopped. 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
Culturing Jurkat cells expressing human CD47 protein on cell membrane surface to total cell amount required for experiment, diluting the cells to 1×10 with 1% BSA/PBS solution 6 Individual cells/ml, and at 1X 10 5 The individual cells/100. Mu.l/well were plated onto 96 well U-shaped bottom plates and centrifuged at 1500rpm for 4 minutes, and the supernatant 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 anti-human CD47 monoclonal antibodies and cell membrane surface expressed human CD47 eggs were measured on a BD flow cytometer White binding. 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
Culturing Jurkat cells expressing human CD47 protein on the surface of cell membrane to the total amount of cells required for experiment, diluting the cells to 1×10 with 1% BSA/PBS solution 6 Individual cells/ml, and at 1X 10 5 The individual cells/100. Mu.l/well were plated onto 96 well U-shaped bottom plates and centrifuged at 1500rpm for 4 minutes, and the supernatant 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
Respectively culturingCT26-hCD47 cells, CT26-mCD47 cells and CT26-cCD47 cells expressing human, mouse and monkey CD47 proteins on the cell membrane surface until the total cell amount reaches the experimental requirement, diluting the cells to 1×10 with 1% BSA/PBS solution 6 Each cell/ml was used in an amount of 100. Mu.l (1X 10) 5 Cell/well density plating, centrifugation at 1500rpm for 4 min, and supernatant 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 μl 1% 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 μl 1% 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
Culturing CT26-hCD47 cells expressing human CD47 protein on the surface of cell membrane until the total amount of the cells reaches the experimental requirement, diluting the cells to 1×10 with 1% BSA/PBS solution 6 Each cell/ml was used in an amount of 100. Mu.l (1X 10) 5 Cell/well density plating, centrifugation at 1500rpm for 4 min, and supernatant discarded. The initial concentration of the anti-human CD47 monoclonal antibody (A1-A6, positive control antibody P02) was 10. Mu.g/ml, and the total of 11 spots were diluted in a 2-fold gradient, and 100. Mu.l/well of the monoclonal antibody weight was addedCells were suspended, two duplicate wells were set for each concentration of antibody, and incubated at 4℃for 1 hour. 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. Carefully withdraw the needle, pour the liquid into a centrifuge tube, centrifuge at 1000rpm for 3 minutes at 4 ℃, remove supernatant, add 2ml of DMEM medium (DMEM+10% FBS+1% PS), count cells, centrifuge at 1000rpm for 3 minutes, remove supernatant, add 2ml of DMEM medium, place at 37 ℃,5% CO 2 Culturing overnight in incubator, changing liquid the next day, digesting with pancreatin two days later, performing cell count, inoculating 96-well plate, and 4×10 each well 4 Individual cells/well (100 μl), 5% CO at 37deg.C 2 Incubation in incubator 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 at 2X 10 4 Individual cells/wells were mixed into macrophages and anti-human CD47 monoclonal antibody at 37 ℃,5% co 2 Incubate in incubator 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 in 100 μl, 1×10 density 6 Raji cells of individual cells/ml, mice were observed for neoplasia, and on day 6 after tumor loading (tumor volume about 50-150mm 3 ) Administration is started. 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-administered group was calculated, and the results are shown in Table 8 and FIG. 4, which show that the same dose was administeredThe tumor inhibition rate of the anti-human CD47 monoclonal antibody to be detected is equivalent to that of a positive control antibody.
TABLE 8 tumor inhibition of anti-human CD47 monoclonal antibodies
It should be understood that while the application describes the invention in terms of the specific forms described above, the invention is not limited to the specific details described in these specific forms. It will be apparent to those skilled in the art that various equivalent changes can be made to the technical features contained in the invention as described herein without departing from the spirit of the invention as described herein, and such changes should be considered as falling within the scope of the invention.
Sequence listing
<110> Qimen Baitai (Beijing) pharmaceutical technology Co., ltd
<120> anti-CD 47 antibodies and uses thereof
<160> 84
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Asn Tyr Val Leu His
1 5
<210> 2
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Gly Tyr Phe Met Asn
1 5
<210> 3
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Asn Tyr Trp Met Asn
1 5
<210> 4
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Tyr Tyr Val Met His
1 5
<210> 5
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Gly Tyr Thr Met His
1 5
<210> 6
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Asp Ser Glu Met His
1 5
<210> 7
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Ser Tyr Trp Met Asn
1 5
<210> 8
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Asn Tyr Val Met His
1 5
<210> 9
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Tyr Phe Asn Pro Tyr Asn Asp Asp Ser Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 10
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 11
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Asn Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 12
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 13
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Leu Ile Asn Pro Tyr Thr Gly Gly Thr Asn Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 14
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Val Ile Asp Pro Glu Thr Gly Asn Ser Ala Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 15
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Leu Ile Asn Pro Tyr Thr Asp Gly Thr Asn Tyr Asn Gln Asn Phe Lys
1 5 10 15
Gly
<210> 16
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Asn Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Arg
1 5 10 15
Asp
<210> 17
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Tyr Ile Asn Pro Tyr Asn Asn Asp Ile Lys Ser Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 18
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Gly Gly Tyr Tyr Ser Met Asp Tyr
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Gly Gly Tyr Gly Gly Met Asp Tyr
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Trp Arg Gly Tyr Ala Leu Asp Tyr
1 5
<210> 21
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Gly Gly Thr Thr Ala Pro Asp Tyr
1 5
<210> 22
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Met Gly Leu Ala Trp Phe Ala Tyr
1 5
<210> 23
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Gly Gly Val Phe Gly Tyr Pro Phe Asp Tyr
1 5 10
<210> 24
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Trp Arg Gly Tyr Ala Met Asp Tyr
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Gly Gly Leu Phe Ser Met Asp Tyr
1 5
<210> 26
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Arg Ala Ser Gln Ser Leu Ala His Ser Asn Gly Tyr Thr Tyr Leu Gln
1 5 10 15
<210> 27
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu
1 5 10 15
<210> 28
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Arg Ala Ser Lys Ser Ile Ser Lys Phe Leu Ala
1 5 10
<210> 29
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Arg Tyr Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu
1 5 10 15
<210> 30
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Phe His
1 5 10 15
<210> 31
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Ser Ala Thr Ser Ser Val Arg Ser Ser Tyr Leu His
1 5 10
<210> 32
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 33
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Arg Ser Ser Gln Ser Ile Val His Ile Lys Gly Asn Thr Tyr Leu Glu
1 5 10 15
<210> 34
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 34
Lys Val Ser Lys Arg Phe Ser
1 5
<210> 35
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 35
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 36
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 36
Ser Gly Ser Thr Leu Gln Ser
1 5
<210> 37
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 37
Lys Val Ser Ile Arg Phe Ser
1 5
<210> 38
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 38
Arg Thr Ser Asn Leu Ala Ser
1 5
<210> 39
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 39
Ser Gln Ser Thr His Val Pro Tyr Thr
1 5
<210> 40
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 40
Phe Gln Gly Ser His Val Pro Tyr Thr
1 5
<210> 41
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 41
Gln Gln His Asn Glu Tyr Pro Trp Thr
1 5
<210> 42
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Phe Gln Gly Ser His Val Pro Trp Thr
1 5
<210> 43
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 43
Gln Gln Trp Arg Gly Tyr Pro Tyr Thr
1 5
<210> 44
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 44
Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Val Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Phe Asn Pro Tyr Asn Asp Asp Ser Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Tyr Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 45
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
Glu Val Gln Val Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Gly Tyr Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 46
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 46
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly His Thr Phe Thr Asn Tyr
20 25 30
Trp Met Asn Trp Ile Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Val Gly Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Trp Arg Gly Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 47
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 47
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Tyr
20 25 30
Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Thr Thr Ala Pro Asp Tyr Trp Gly Gln Ala Thr Thr
100 105 110
Leu Thr Val Ser Ser
115
<210> 48
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 48
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Arg Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Thr Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Thr Gly Gly Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met Gly Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 49
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Ser
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Val Tyr Gly Leu Glu Trp Thr
35 40 45
Gly Val Ile Asp Pro Glu Thr Gly Asn Ser Ala Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Gly Val Phe Gly Tyr Pro Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 50
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Asn Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Thr Asp Gly Thr Asn Tyr Asn Gln Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met Gly Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 51
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 51
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe
50 55 60
Arg Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Trp Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 52
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 52
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asn Asp Ile Lys Ser Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala Phe
65 70 75 80
Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Gly Gly Leu Phe Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 53
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 53
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Arg Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Thr Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Thr Gly Gly Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met Gly Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 54
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 54
Asp Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp
1 5 10 15
Gln Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Ala His Ser Asn
20 25 30
Gly Tyr Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro
35 40 45
Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
65 70 75 80
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr
85 90 95
His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 55
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 55
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 56
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 56
Cys Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro
1 5 10 15
Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys
20 25 30
Phe Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Asn Leu Leu
35 40 45
Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro
85 90 95
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 57
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 57
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Arg Arg Tyr Ser Gln Ser Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Ile Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 58
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 58
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Phe His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
100 105 110
<210> 59
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 59
Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ala Ala
1 5 10 15
Ser Leu Gly Gln Lys Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val
20 25 30
Arg Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro
35 40 45
Lys Pro Leu Ile His Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
65 70 75 80
Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Arg
85 90 95
Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 60
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 60
Asp Phe Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 61
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 61
Cys Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro
1 5 10 15
Gly Glu Pro Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys
20 25 30
Phe Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn His Leu Leu
35 40 45
Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro
85 90 95
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 62
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 62
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ile
20 25 30
Lys Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 63
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
gagttccagc tgcagcagag cggaccagaa ctggtgaagc caggagccag cgtgaagatg 60
tcttgcaagg ccagcggcta caccttcacc aactacgtgc tgcattgggt gaagcagaag 120
ccaggacagg gactcgagtg gatcggatac ttcaacccct acaacgacga cagcaagtac 180
aacgagaagt tcaagggcaa ggccaccctg accagcgata agagcagcag caccgcctac 240
atggagctgt ctagcctgac cagcgaggac tcagccgtgt actattgcgc cagaggcgga 300
tactacagca tggactattg gggccaggga acaacagtga cagtgtccag c 351
<210> 64
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
gaggttcagg tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60
tcctgcaagg cttctggtta ctcatttact ggctacttta tgaactgggt gaagcagagc 120
catggaaaga gccttgagtg gattggacgt attaatcctt acaatggtga tactttctac 180
aaccagaagt tcaagggcaa ggccacattg actgtagaca aatcctctag cacagcccac 240
atggagctcc tgagcctgac atctgaggac tctgcagtct attattgtgg aagggggggc 300
tacggaggaa tggactactg gggtcaagga acctcagtca ccgtctcctc a 351
<210> 65
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60
tcctgcaagg cttctggcca cacgttcacc aactactgga tgaactggat taaacagagg 120
cctgagcaag gccttgagtg gattggaaat attgatcctt acgatagtga aactcactac 180
aatcaaaagt tcaaggacaa ggccatattg actgtcggca aatcctccag cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aatatggaga 300
ggctatgctt tggactactg gggtcaagga acctcagtca ccgtctcctc a 351
<210> 66
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60
tcctgcaagg cttctggata cacattcact tactatgtta tgcactgggt gaagcagaag 120
cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaagtac 180
aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240
atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagagggggt 300
actacggccc ccgactactg gggccaagcc accactctca cagtctcctc a 351
<210> 67
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggagcttc aatgaggatg 60
tcctgcaagg cttctggtta ctcgttcact ggctacacca tgcactgggt gaagcagagc 120
cacggaacga accttgagtg gattggactt attaatcctt acactggtgg tactaactac 180
aaccagaagt tcaagggcaa ggccacatta actttagaca agtcatccag cacagcctac 240
atggagctcc tcagtctgac atctgaggac tttgcagtct attactgtgc aagaatgggc 300
ctggcctggt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 351
<210> 68
<211> 357
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
caggttcaac tgcagcagtc tggggctgaa ctggtgaggc ctggggcttc agtgacgctg 60
tcctgcaagg cttcgggcta ctcatttact gactctgaaa tgcactgggt gaagcagaca 120
cctgtgtatg gcctggaatg gactggagtt attgatcctg aaactggaaa tagtgcctac 180
aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccaa cacagcctac 240
atggaactcc gcagcctgac atctgaggac tctgccgtct attactgtac aagaggaggg 300
gttttcggct acccctttga ctactggggc caaggcacca ctctcacagt ctcctca 357
<210> 69
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggagcttc aatgaagatc 60
tcctgcaagg cttctggtta ctcattcact ggctacacca tgcactgggt gaaacagagc 120
catggaaaca accttgagtg gattggactt attaatcctt acactgatgg tactaactac 180
aaccagaact tcaagggcaa ggccacatta actttagaca agtcatccag cacagcctac 240
atggagctcc tcagtctgac atctgaggac tctgcagtct attactgtgc aagaatgggc 300
ctggcctggt ttgcttactg gggccaaggg actctggtca ctgtctcagc a 351
<210> 70
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60
tcctgcaagg cttctggcta cacgttcacc agctactgga tgaactgggt taagcagagg 120
cctgagcaag gccttgagtg gattggaaat attgatcctt acgatagtga aactcactac 180
aatcaaaagt tcagggacaa ggccatattg actgtcgaca aatcctccag cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aatatggaga 300
ggctatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351
<210> 71
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60
tcctgcaagg cttctggata cacattcact aactatgtta tgcactgggt gaagcagaag 120
cctgggcagg gccttgagtg gattggatat attaatcctt acaataatga tattaagtcc 180
aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccac cacagccttc 240
atggacctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aaaagggggg 300
ctattctcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351
<210> 72
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggagcttc aatgaggatg 60
tcctgcaagg cttctggtta ctcgttcact ggctacacca tgcactgggt gaagcagagc 120
catggaacga accttgagtg gattggactt attaatcctt acactggtgg tactaactac 180
aaccagaagt tcaagggcaa ggccacatta actttagaca agtcatccag cacagcctac 240
atggagctcc tcagtctgac atctgaggac tctgcagtct attactgtgc aagaatgggc 300
ctggcctggt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 351
<210> 73
<211> 333
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
gacgtgatga cccagacccc tctgtctctg ccagtgtctc tgggagacca ggcctctatc 60
tcttgcagag ccagccagtc tctggcccat agcaacggct acacctacct ccagtggtat 120
ctgcagaagc caggccagag ccctaaactg ctgatctaca aggtgtccaa gcggttcagc 180
ggcgtgccag atagattcag cggaagcgga agcggcaccg acttcaccct gaagatcagc 240
agagtggagg ccgaggatct gggagtgtac ttctgcagcc agagcaccca cgtgccttac 300
acattcggcc agggcaccaa ggtggagatc aag 333
<210> 74
<211> 336
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300
tacacgttcg gaggggggac caagctggaa ataaaa 336
<210> 75
<211> 324
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
tgtgatgtcc agataaccca gtctccatct tatcttgctg catctcctgg agaaaccatt 60
actattaatt gcagggcaag taagagcatt agcaaatttt tagcctggta tcaagagaaa 120
cctgggaaaa ctaataacct tcttatctac tctggatcca ctttgcaatc tggaattcca 180
tcaaggttca gtggcagtgg atctggtaca gatttcactc tcaccatcag tagcctggag 240
cctgaagatt ttgcaatgta ttactgtcaa cagcataatg aatacccgtg gacgtttggt 300
ggaggcacca agctggaaat caaa 324
<210> 76
<211> 336
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctctcgca gatatagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgattt acaaagtttc catccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300
tggacgttcg gtggaggcac caagctggaa atcaaa 336
<210> 77
<211> 336
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta ttttcattgg 120
tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300
tacacgttcg gaggggggac caaactggaa atgaaa 336
<210> 78
<211> 333
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
tccagaggag aaaatgtgct cacccagtct ccagcaataa tggctgcctc tctggggcag 60
aaggtcacca tgacctgcag tgccacctca agtgtaaggt ccagttactt gcactggtac 120
cagcagaagt caggcgcttc ccccaaaccc ttgattcata ggacatccaa cctggcttct 180
ggagtcccag ctcgcttcag tggcagtggg tctgggacct cttactctct cacaatcagc 240
agcgtggagg ctgaagatga tgcaacttat tactgccagc agtggagagg ttacccgtac 300
acgttcggag gggggaccaa gctggaaata aaa 333
<210> 79
<211> 336
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
gattttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggcga tcaagcctcc 60
atctcttgca gatctagtca gagccttgta cacagtaatg gcaacaccta tttacattgg 120
tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300
tacacgttcg gaggggggac caagctggaa ataaaa 336
<210> 80
<211> 324
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
tgtgatgtcc agataaccca gtctccatct tatcttgctg catctcctgg agaacccatt 60
actattaatt gcagggcaag taagagcatt agcaaatttt tagcctggta tcaagagaaa 120
cctgggaaaa ctaatcacct tcttatctac tctggatcca ctttgcaatc tggaattcca 180
tcaaggttca gtggcagtgg atctggtaca gatttcactc tcaccatcag tagcctggag 240
cctgaagatt ttgcaatgta ttactgtcaa cagcataatg aatacccgtg gacgttcggt 300
ggaggcacca agctggaaat caaa 324
<210> 81
<211> 336
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gagcattgta catattaagg gaaacaccta tttagaatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agtagagtgg aggctgagga tctgggagtt tattattgtt ttcaaggttc acatgttccg 300
tggacgttcg gtggaggcac caagctggaa atcaaa 336
<210> 82
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
cccccccccc cccccccc 18
<210> 83
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
ctcagggaar tarccyttga c 21
<210> 84
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
tcactgccat caatcttcca c 21

Claims (20)

1. An antibody or antigen-binding portion thereof that specifically binds CD47, comprising HCDR1, HCDR2 and HCDR3 of a heavy chain variable region, and LCDR1, LCDR2 and LCDR3 of a light chain variable region, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No. 5; the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 13; the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 22; and the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 30; the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 35; and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 39.
2. The antibody or antigen-binding portion thereof of 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 48 and 53.
3. The antibody or antigen-binding portion thereof of claim 1, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 58.
4. The antibody or antigen-binding portion thereof of any one of claims 1-3, wherein the antibody or antigen-binding portion thereof is capable of specifically binding to human CD 47.
5. The antibody or antigen-binding portion thereof of any one of claims 1-3, wherein the antibody is a full-length antibody, a single chain antibody, or a bispecific antibody.
6. The antibody or antigen-binding portion thereof of any one of claims 1-3, wherein 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 1-3, wherein the antibody is a monoclonal antibody.
8. The antibody or antigen-binding portion thereof of any one of claims 1-3, wherein the antibody is murine or humanized.
9. The antibody or antigen binding portion thereof of claim 1, 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.
10. The antibody or antigen-binding portion thereof of claim 9, wherein 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.
11. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-10.
12. An expression vector comprising the nucleic acid molecule of claim 11.
13. A host cell comprising the nucleic acid molecule of claim 11 or the expression vector of claim 12.
14. The host cell of claim 13, which is a prokaryotic cell, yeast, insect cell, or mammalian cell.
15. The host cell of claim 14, wherein the prokaryotic cell is e. And/or the mammalian cell is a HEK293E cell or CHO cell.
16. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-10 and a pharmaceutically acceptable carrier.
17. The pharmaceutical composition of claim 16, further comprising one or more additional active ingredients.
18. Use of an antibody or antigen-binding portion thereof according to any one of claims 1-10, or a pharmaceutical composition according to claim 16 or 17, in the manufacture of a medicament for the treatment of lymphoma.
19. The use of claim 18, wherein the medicament is an antibody conjugated medicament.
20. A detection reagent or kit comprising the antibody or antigen-binding portion thereof of any one of claims 1-10.
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CN106084052A (en) * 2016-06-17 2016-11-09 长春金赛药业有限责任公司 Anti-CD47 monoclonal antibody and application thereof
WO2018095428A1 (en) * 2016-11-28 2018-05-31 江苏恒瑞医药股份有限公司 Cd47 antibody, antigen-binding fragment and medical use thereof

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WO2018095428A1 (en) * 2016-11-28 2018-05-31 江苏恒瑞医药股份有限公司 Cd47 antibody, antigen-binding fragment and medical use thereof

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