Anti-CD47 monoclonal antibody

Abstract

The invention belongs to the field of antibodies, and particularly relates to an anti-CD47 monoclonal antibody, and discloses a highly variable region sequence of the monoclonal antibody, the anti-CD47 monoclonal antibody provided by the invention has good binding activity, good anticoagulation effect and strong SIRP alpha blocking efficacy; the phagocytosis promoting effect is obvious; forteBio is used for detecting the affinity constant of the antibody, the KD value is lower than 1 * 10 <-11 > nM, and the affinity of the antibody is high. The compound has a relatively strong phagocytic function and shows a relatively good anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and antibody drugs; in particular, it relates to an anti-CD47 monoclonal antibody. Background Art

[0002] CD47 antibody, also known as integrin associated protein (IAP), is a member of the immunoglobulin superfamily. Its structure includes an amino-terminal extracellular variable region, a transmembrane region composed of 3 to 5 highly hydrophobic transmembrane segments, and a hydrophilic carboxyl-terminal cytoplasmic tail region. CD47 is widely expressed on the surface of various tissue cells, especially on the surface of tumor cells, including non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), ovarian cancer, glioma, glioblastoma, etc. Weissman et al. of Stanford University systematically studied the expression level of CD47 in various solid tumors. The results showed that CD47 was highly expressed in all human solid tumor cells, and its average expression level was about 3.3 times that of the corresponding normal cells. Moreover, they found that the level of CD47 in patients with solid tumors was negatively correlated with the prognostic index. Furthermore, CD47 has been identified as a cancer stem cell marker for both leukemias and solid tumors (Majeti, et al., 2009 Cell, 138:286-99; Rendtlew et al., 2007 Br J Haematal 138:756-60; Chan et al., 2009 Proc Acad Sci USA, 106:14016-21; Majeti, et al., 2011 Oncogene, 30:1009-19).

[0003] SIRPα (Signal regulatory proteinα), also known as SHPS-1 (Src homology 2domain-containing protein tyrosinephosphatase substeate-1) / BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs) / CD172a, is also a transmembrane glycoprotein with three immunoglobulin-like domains in the extracellular region and a typical immunoreceptor tyrosine inhibitory sequence (ITIM) in the intracellular domain. It has four tyrosine residues and is a potential phosphorylation site.

[0004] CD47 is the ligand of SIRPα, and the two interact through the extracellular domain to form an intercellular communication complex. After CD47 binds to SIRPα, it induces the phosphorylation of SIRPα intracellular ITIM, and the phosphorylation site binds and activates the tyrosine phosphatases SHP-1 and SHP-2 containing the SH2 (Src homology 2) domain, triggering a cascade of signal transduction. SHP-1 is mainly expressed in hematopoietic cells and negatively regulates the function of these cells; while SHP-2 is widely expressed, regulating the small G proteins Ras and Rho, and positively controlling cell growth and proliferation.

[0005] SIRPα is abundantly expressed in neurons and myeloid hematopoietic cells (such as macrophages and dendritic cells), while CD47 is expressed in most cells. The CD47-SIRPα signaling system plays an important role in regulating the phagocytosis of mature blood cells by macrophages. The CD47 molecules on the surface of normal healthy cells (such as red blood cells or platelets) interact with the receptor SIRPα on macrophages to produce inhibitory signals, inhibiting their phagocytic activity, thereby regulating the life cycle of blood cells and their number in the blood. SIRPα on monocytes interacts with CD47 on red blood cells to inhibit Fcγ receptor-dependent phagocytosis through myosin-IIA dephosphorylation. The CD47-SIRPα signaling system inhibits dendritic cell activation, participates in multiple physiological activities such as nervous system development, neutrophil chemotaxis activation, and stromal cell-supported hematopoietic cell production, and also plays a variety of regulatory roles in inducing T cell immune tolerance, activation, and apoptosis.

[0006] In recent years, the role of the CD47-SIRPα signaling system in tumor immune surveillance by regulating macrophages has received attention. The expression level of CD47 is significantly upregulated in many malignant tumors, such as ovarian cancer, acute myeloid leukemia (AML), B-cell lymphoma and solid tumors, and this upregulation is directly related to the poor prognosis of patients with malignant tumors. Expressing mouse CD47 in human myeloid leukemia cells (which express endogenous CD47 at low levels and cannot be transplanted into Rag2-I12rg- mice) can inhibit the phagocytosis of tumor cells by macrophages and promote the successful transplantation of tumor cells. It can be inferred that CD47 on tumor cells interacts with SIRPα on macrophages, inhibiting the clearance of tumor cells by macrophages and promoting the growth and metastasis of tumors in vivo. High expression of CD47 is a common mechanism for tumor cells to escape immune surveillance. Blocking the action of CD47-SIRPα may be a new strategy for tumor immunotherapy.

[0007] Anti-CD47 antibodies, used alone or in combination with other tumor antigen antibodies, have shown good inhibitory effects on tumor growth in mouse transplant models of human acute myeloid leukemia, non-Hodgkin's lymphoma (NHL), and many solid tumors. Humanized anti-CD47 monoclonal antibodies induce macrophages to phagocytose human primary AML cells, completely eliminate human AML cells in vivo, and enable transplanted mice to survive disease-free for a long time; combined with rituximab, it can clear NHL tumors and cure xenograft mice. Moreover, the safety of anti-CD47 antibodies has been confirmed in monkeys. In addition to anti-CD47 antibodies, high-affinity SIRPα mutants (CD172a) can also antagonize CD47 and block the CD47-SIRPα signaling pathway, which can significantly increase the phagocytosis of AML cells by macrophages in AML models and inhibit tumor growth.

[0008] However, the main research bottleneck for antibodies against this target is the development of monoclonal antibody drugs that do not bind to red blood cells or have significant agglutination effects. Summary of the invention

[0009] Based on the deficiencies of the prior art, the present invention provides a human-mouse chimera that specifically binds to CD47, blocks the interaction between CD47 and SIRPα, and has no significant hemagglutination activity.

[0010] The first aspect of the present invention is to provide a human-mouse chimeric antibody that specifically binds to CD47.

[0011] The anti-CD47 antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 sequences.

[0012] wherein the sequence of HCDR1 is SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 10;

[0013] The sequence of HCDR2 is SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 11;

[0014] The sequence of HCDR3 is SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 12;

[0015] The sequence of LCDR1 is SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 20;

[0016] The sequence of LCDR2 is SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 21;

[0017] The sequence of LCDR3 is SEQ ID NO:15 or SEQ ID NO:19 or SEQ ID NO:22.

[0018] CDR (complementarity determining region) usually refers to the region in an antibody that can form complementarity with an antigenic determinant in terms of spatial structure. The variability in an antibody is usually not evenly distributed throughout the variable region of the antibody. The heavy chain variable region and light chain variable region of a monoclonal antibody usually have three hypervariable regions (HVRs), which can usually form complementarity with an antigenic determinant in terms of spatial structure, so the hypervariable region is also called the complementarity determining region (CDR). That is, the heavy chain variable region usually includes three complementarity determining regions, i.e., CDRH1, CDRH2, and CDRH3, and the light chain variable region usually includes three complementarity determining regions, i.e., CDRL1, CDRL2, and CDRL3.

[0019] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:3, HCDR2 with an amino acid sequence as shown in SEQ ID NO:4, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:5.

[0020] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:6, HCDR2 with an amino acid sequence as shown in SEQ ID NO:8, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:9.

[0021] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:7, HCDR2 with an amino acid sequence as shown in SEQ ID NO:8, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:9.

[0022] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:10, HCDR2 with an amino acid sequence as shown in SEQ ID NO:11, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:12.

[0023] In one embodiment, the complementarity determining region of the light chain variable region of the anti-CD47 antibody comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:13, LCDR2 with an amino acid sequence as shown in SEQ ID NO:14, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:15.

[0024] In one embodiment, the complementarity determining region of the light chain variable region of the anti-CD47 antibody comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:16, LCDR2 with an amino acid sequence as shown in SEQ ID NO:17, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:19.

[0025] In one embodiment, the complementarity determining region of the light chain variable region of the anti-CD47 antibody comprises LCDR1 having an amino acid sequence as shown in SEQ ID NO:16, LCDR2 having an amino acid sequence as shown in SEQ ID NO:18, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:19.

[0026] In one embodiment, the complementarity determining region of the light chain variable region of the anti-CD47 antibody comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:20, LCDR2 with an amino acid sequence as shown in SEQ ID NO:21, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:22.

[0027] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody includes HCDR1 with an amino acid sequence as shown in SEQ ID NO: 3, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the complementarity determining region of the light chain variable region includes LCDR1 with an amino acid sequence as shown in SEQ ID NO: 13, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 14, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 15.

[0028] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody includes HCDR1 as shown in SEQ ID NO: 6, HCDR2 as shown in SEQ ID NO: 8, and HCDR3 as shown in SEQ ID NO: 9; the complementarity determining region of the light chain variable region includes LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 19.

[0029] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody includes HCDR1 as shown in SEQ ID NO: 7, HCDR2 as shown in SEQ ID NO: 8, and HCDR3 as shown in SEQ ID NO: 9; the complementarity determining region of the light chain variable region includes LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 18, and LCDR3 as shown in SEQ ID NO: 19.

[0030] In one embodiment, the complementarity determining region of the heavy chain variable region of the anti-CD47 antibody includes HCDR1 with an amino acid sequence as shown in SEQ ID NO: 10, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 12; the complementarity determining region of the light chain variable region includes LCDR1 with an amino acid sequence as shown in SEQ ID NO: 20, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 22.

[0031] In one embodiment, the heavy chain variable region and the light chain variable region of the anti-CD47 antibody further include a framework region, and the framework region may be located between the complementarity determining regions or at both ends of the complementarity determining regions.

[0032] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is as shown in SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 27; or

[0033] The amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 28; or

[0034] The amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29; or

[0035] The amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30.

[0036] In a second aspect of the present invention, an antibody that specifically binds to human CD47 is provided, wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with any one of SEQ ID NO: 23, 24, 25 or 26, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with any one of SEQ ID NO: 27, 28, 29 or 30.

[0037] The method of the present invention for determining the heavy chain and light chain variable region sequences of the anti-CD47 antibody comprises: synthesizing specific primers according to the constant region sequence of the antibody gene, PCR amplifying the heavy chain variable region and the light chain variable region of the monoclonal antibody, recovering the target fragments, cloning them into the PMDA19-T (simple) vector, transforming Escherichia coli E. coli DH5α, picking positive clones, extracting plasmids and sequencing them.

[0038] The antibody of the present invention is a monoclonal antibody, which can bind to and neutralize human CD47, thereby blocking the CD47-SIRPα signaling pathway. In one embodiment, the antibody can promote the phagocytosis of tumor cells by macrophages. In one embodiment, the antibody inhibits the in vivo growth of tumor cells. In one embodiment, the antibody does not increase the phagocytosis of normal blood cells by macrophages. In one embodiment, the antibody does not have significant hemagglutination activity.

[0039] In one embodiment, the antibody is a whole antibody, a Fab fragment, a F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0040] In one embodiment, the antibody is a humanized antibody.

[0041] In one embodiment, the antibody further comprises a heavy chain constant region selected from IgG1 subtype, IgG2 subtype or IgG4 subtype and / or comprises a light chain constant region selected from κ subtype or λ subtype; in a preferred embodiment, the skeleton used for the humanized modification of the antibody of the present invention is IgG1, the heavy chain amino acid sequence is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2.

[0042] In one embodiment, humanized antibodies can be prepared by the following method:

[0043] The heavy chain variable region (VH) and light chain variable region (VL) of the mouse antibody are compared with the human antibody germline gene sequence in the IMGT database, respectively, and the appropriate germline gene sequence is selected to provide the framework region (FR1+FR2+FR3) of the antibody, and the appropriate J region gene sequence is selected to provide the framework region 4 (FR4). This template can be selected according to, for example, the relative total length of the antibody, the size of the CDR, the amino acid residues at the junction between the antibody framework region (FR) and the hypervariable region (CDR), the homology of the entire sequence, etc. The selected template can be a mixture of multiple sequences or a common template, with the purpose of maintaining the appropriate conformation of the parent complementary determining region (CDR) as much as possible. The heavy chain and light chain variable region amino acid sequence of the humanized antibody is finally determined. According to the amino acid sequence of the humanized antibody, the variable region gene is designed and synthesized, and the humanized antibody of IgG1 version, IgG2 version or IgG4 version is prepared.

[0044] The method for determining the subtype of anti-CD47 monoclonal antibody of the present invention is to extract the culture supernatant of hybridoma cells and use IsoStrip TM Mouse monoclonal antibody subtype identification kit (Sino Biological Inc, catalog number SEK003) was used to identify antibody subtypes.

[0045] In one embodiment, the human-mouse chimeric antibody that specifically binds to CD47 has a heavy chain polynucleotide sequence as shown in SEQ ID NO: 31, and a light chain polynucleotide sequence as shown in SEQ ID NO: 35.

[0046] In one embodiment, the human-mouse chimeric antibody that specifically binds to CD47 has a heavy chain polynucleotide sequence as shown in SEQ ID NO: 32, and a light chain polynucleotide sequence as shown in SEQ ID NO: 36.

[0047] In one embodiment, the human-mouse chimeric antibody that specifically binds to CD47 has a heavy chain polynucleotide sequence as shown in SEQ ID NO: 33, and a light chain polynucleotide sequence as shown in SEQ ID NO: 37.

[0048] In one embodiment, the human-mouse chimeric antibody that specifically binds to CD47 has a heavy chain polynucleotide sequence as shown in SEQ ID NO: 34, and a light chain polynucleotide sequence as shown in SEQ ID NO: 38.

[0049] The third aspect of the present invention provides a polynucleotide molecule encoding the anti-CD47 monoclonal antibody as described above.

[0050] In one embodiment, the polynucleotide sequence encoding the heavy chain of the anti-CD47 monoclonal antibody is shown in SEQ ID NO: 31, and the polynucleotide sequence encoding the light chain is shown in SEQ ID NO: 35. In another embodiment, the polynucleotide sequence encoding the heavy chain of the anti-CD47 monoclonal antibody is shown in SEQ ID NO: 32, and the polynucleotide sequence encoding the light chain is shown in SEQ ID NO: 36. In another embodiment, the polynucleotide sequence encoding the heavy chain of the anti-CD47 monoclonal antibody is shown in SEQ ID NO: 33, and the polynucleotide sequence encoding the light chain is shown in SEQ ID NO: 37. In another embodiment, the polynucleotide sequence encoding the heavy chain of the anti-CD47 monoclonal antibody is shown in SEQ ID NO: 34, and the polynucleotide sequence encoding the light chain is shown in SEQ ID NO: 38.

[0051] In a fourth aspect of the present invention, an expression vector is provided, comprising a polynucleotide molecule encoding an anti-CD47 monoclonal antibody provided by the present invention. The expression vector in the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors.

[0052] In a fifth aspect of the present invention, a host cell is provided to transform the expression vector of the present invention. Any cell suitable for expression by the expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Further examples can be CHO cells, BHK cells or HEK293 cells; in a preferred embodiment, the host cell is a CHO cell.

[0053] In a sixth aspect of the present invention, a method for preparing a CD47 monoclonal antibody is provided, the method comprising the following steps: culturing the host cell as described above under conditions suitable for expressing the antibody to thereby express the monoclonal antibody, and purifying and isolating the monoclonal antibody.

[0054] The host cell expresses the anti-human CD47 monoclonal antibody under appropriate expression conditions or obtains hybridoma cells that stably secrete the anti-human CD47 monoclonal antibody through hybridoma technology.

[0055] The host cells used in the present invention are all prior art and can be directly obtained through commercial channels. The culture medium used in the culture is also various conventional culture media. Those skilled in the art can select suitable culture media based on experience and culture them under conditions suitable for the growth of host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time. The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the grouped proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic breaking, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0056] In one experimental scheme, the anti-CD47 antibody can be prepared by the following method: the target gene sequence and the vector pCHO1.0 are both digested with restriction endonucleases AvrII and BstZ17I, and the target fragments are recovered and connected with T4 ligase to construct an expression vector. E. coli DH5α competent cells are transformed respectively, and the plasmids are extracted to obtain recombinant expression plasmids, which are linearized with restriction endonuclease PvuI, transfected into CHO-S cells, and pressure-screened with puromycin and MTX to obtain a cell library expressing the target protein, and after cloning culture, expression level determination and affinity sorting, a monoclonal cell strain expressing the antibody is finally screened; after the antibody protein is prepared, it is further purified, the purity of the antibody is determined, and the affinity, hemagglutination effect, SIRPα blocking efficacy and phagocytosis of the purified antibody are determined.

[0057] In a preferred embodiment, the antibody purification method is affinity chromatography, and the specific steps are: first prepare a protein A affinity column, balance the column with PBS, pass the centrifuged and filtered cell culture supernatant through the column, then wash with PBS until the OD value is close to zero, elute with glycine-hydrochloric acid buffer solution, collect the eluate in the peak area, and use it after dialysis.

[0058] In a preferred embodiment, the method for determining the purity of the antibody is the SDS-PAGE method, and the specific steps are: performing electrophoresis according to the method of Part 4 of the 2015 edition of the Chinese Pharmacopoeia, scanning the electrophoretogram, and identifying its molecular weight and expression level.

[0059] In a preferred embodiment, the method for determining antibody affinity is the ForteBio method, which specifically comprises the following steps: immobilizing the purified CD47 antibody on a ProteinA sensor, binding and dissociating the diluted CD47 protein to the immobilized CD47 ProteinA sensor, respectively obtaining a binding constant and a dissociation constant, and finally obtaining the affinity constant of the CD47 monoclonal antibody.

[0060] In a preferred embodiment, the method for determining whether the antibody blocks the binding of human CD47 to human SIRPα is an ELISA method, monitoring the binding of recombinant SIRPα-his under conditions of increasing amounts of CD47 antibodies, and using an HRP-conjugated anti-his secondary antibody to determine the bound SIRPα.

[0061] In a preferred embodiment, the method for determining the phagocytic effect of antibodies is flow cytometry, and the specific steps are: inoculating macrophages in a cell plate and allowing them to attach for 24 hours, labeling target human cancer cells (Jurkat) with CFSE dye and incubating them with different anti-CD47 monoclonal antibodies or no antibodies, then adding them to macrophage culture medium and incubating them together, washing away unphagocytosed target cells with PBS, collecting macrophages, staining macrophages with anti-human CD14-APC, and analyzing them with a flow cytometer.

[0062] The anti-CD47 monoclonal antibody, conjugate and / or conjugate of the present invention is used in the preparation of a preparation for blocking the binding of CD47 and SIRPα. The EC50 value of the anti-CD47 monoclonal antibody in blocking the binding of CD47 and SIRPα is low, showing a strong SIRPα blocking effect.

[0063] The anti-CD47 monoclonal antibody, conjugate and / or coupling of the present invention is used to promote the phagocytosis of tumor cells by macrophages, and the effect is measured by flow cytometry, and the result is expressed as phagocytosis rate. The present invention provides an antibody with a phagocytosis rate of more than 60% for Jurkat cells.

[0064] In a seventh aspect, the present invention provides use of the anti-CD47 antibody described above in the preparation of an anti-tumor therapeutic drug or a tumor diagnostic drug.

[0065] The tumor therapeutic drug promotes the function of macrophages to phagocytize tumor cells by blocking CD47, thereby killing tumor cells.

[0066] The tumor therapeutic drug can target the CD47 antigen functionally expressed on the surface of tumor cells, bind to or act on the CD47 antigen, and thus treat and / or prevent tumors. The tumors include but are not limited to lung cancer, gastric cancer, cervical cancer, and B lymphoma.

[0067] Advantages and beneficial effects of the present invention:

[0068] The anti-CD47 monoclonal antibody provided by the present invention has an antibody purity of more than 95% after purification; the antibody has good anti-agglutination effect and strong SIRPα blocking effect; the phagocytic effect is obvious; the antibody affinity constant detected by ForteBio has a KD value of less than 1×10 - 11 nM, high antibody affinity. It has strong phagocytic function and shows good anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 It is the electrophoresis pattern of CD47 antibody;

[0070] Figure 2 This is a graph showing the binding activity of CD47 antibodies to cell surface antigens.

[0071] Figure 3A The CD47 antibody dose-dependent blocking curve of the binding of human CD47 to human SIRPa Figure 1 .

[0072] Figure 3B The CD47 antibody dose-dependent blocking curve of the binding of human CD47 to human SIRPa Figure 2 .

[0073] Figure 4 This is a curve chart showing that CD47 antibody promotes phagocytosis of phagocytes.

[0074] Figure 5 This is a graph of the results of a hemagglutination test. DETAILED DESCRIPTION

[0075] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies or recombinant antibodies.

[0076] As used herein, "monoclonal antibody" means an antibody molecule, an antibody preparation having a common heavy chain amino acid sequence and a common light chain amino acid sequence, in contrast to a "polyclonal" antibody preparation containing a mixture of antibodies of different amino acid sequences. The antibodies used in the present invention are derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, rather than the method for producing it. Monoclonal antibodies can be produced by several known techniques, such as phage technology, bacteria, yeast or ribosome display, and the classical methods illustrated by hybridoma-derived antibodies. Therefore, the term (monoclonal) refers to all antibodies derived from a nucleic acid clone.

[0077] Monoclonal antibodies can be obtained by various methods well known to those skilled in the art. For example, monoclonal antibodies can be obtained by the hybridoma method (first proposed by Kohler et al., Nature, 256:495 (1975)) or by recombinant DNA methods (US4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0078] The terms "antibody" and "immunoglobulin" as used herein are heterotetrameric glycoproteins of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. The variable region of each light chain is opposite to the variable region of the heavy chain. Specific amino acids form an interface between the variable regions of the light and heavy chains.

[0079] The antibodies of the present invention include recombinant antibodies. As used herein, "recombinant antibodies" refer to antibodies produced, expressed or separated by a recombinant method, such as antibodies expressed by a recombinant expression vector transfected to a host cell; antibodies separated from a recombinant combinatorial antibody library; antibodies separated from animals (such as mice) because of their human immunoglobulin genes and transgenic; or antibodies produced, expressed, produced or separated in any other manner, wherein a specific immunoglobulin gene sequence (such as a human immunoglobulin gene sequence) is assembled with other DNA sequences. A review of current methods for antibody engineering and improvement can be found in, for example, P. Chsmes, ed., (2012) Antibody Engineering: Methods and Protocals, Second Edition (Methods in Molecular Biology, Book 9070), Humana Press, etc. Recombinant antibodies include, for example, chimeric antibodies and humanized antibodies.

[0080] A "chimeric" protein contains at least one fusion polypeptide that includes regions at positions in the sequence that are different from those that occur in nature. The regions may normally be present in separate proteins but are placed together in the fusion polypeptide; or they may normally be present in the same protein but are placed in a new arrangement in the fusion polypeptide. For example, a chimeric protein can be constructed by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in a desired relationship. As used herein, a "chimeric antibody" refers to an antibody in which the sequence of a variable domain from the germline of a mammalian species (e.g., a mouse) is grafted onto the sequence of a constant domain from the germline of another mammalian species (e.g., a human).

[0081] "Domain" refers to a part of a protein that is physically or functionally distinguished from the rest of the protein or peptide. Physically defined domains include highly hydrophobic or hydrophilic amino acid sequences, such as those that are membrane-bound or cytoplasmic-bound. Domains can also be defined by internal homology, such as caused by gene duplication. Functionally defined domains have different biological functions. For example, the ligand binding domain of a receptor is a domain that binds to a ligand. Antigen binding domain refers to the part of an antigen binding unit and an antibody that binds to an antigen. Functionally defined domains do not need to be encoded by a continuous amino acid sequence. Functionally defined domains can contain one or more physically defined domains. For example, receptors are typically divided into an extracellular ligand binding domain, a transmembrane domain, and an intracellular effector domain.

[0082] The term "variable" as used herein means that some parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a beta-folded configuration, connected by three CDRs that form a continuous loop, and in some cases can form a partial beta-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody. The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0083] "Host cell" includes individual cells or cell cultures that can be or have been recipients of the vector of the present invention.Host cells include the offspring of a single host cell.Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical to the original parent cell (morphologically or in genome or total DNA complementarity).Host cells include cells transfected with the vector of the present invention in vivo. "Host cell" can refer to a prokaryotic cell, a eukaryotic cell, or a cell line cultured as a unicellular entity, which can be used as or has been used as a recipient of a recombinant vector or other transfer polynucleotides, and includes the offspring of the original cell that has been transfected.It should be understood that due to natural, accidental or intentional mutations, the offspring of the unicellular may not necessarily be identical to the original parent in morphology or in genome or total DNA complementarity.

[0084] A "vector" is a nucleic acid molecule, preferably self-replicating, which transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term includes vectors whose primary function is to insert DNA or RNA into a cell, replication vectors whose primary function is to replicate DNA or RNA, and expression vectors whose function is to transcribe and / or translate DNA or RNA. Vectors that provide more than one of the above functions are also included. An "expression vector" refers to a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally refers to a suitable host cell containing an expression vector that can be used to produce a desired expression product.

[0085] The term "treatment" is used herein to refer generally to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely stabilizing or curing a disease and / or adverse reactions attributable to the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal, such as a mouse, rat, rabbit, pig, primate, including humans and other apes, particularly humans, and includes: (a) preventing a disease or symptom from occurring in a subject who may be susceptible to the disease or symptom but has not yet been diagnosed; (b) inhibiting disease symptoms; (c) arresting the development of a disease; (d) alleviating disease symptoms; (e) causing regression of a disease or symptom; or any combination thereof.

[0086] The terms "cancer", "tumor" and "cancer" are used interchangeably in this application and refer to cells that exhibit relatively autonomous growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. Typically, target cells for monitoring or treatment in this application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic and non-metastatic cells.

[0087] A phagocyte refers to a cell that is capable of engulfing. Non-limiting categories of phagocytes include phagocytes, mononuclear cells (eg, histiocytes and monocytes), polymorphonuclear leukocytes (eg, neutrophils), and dendritic cells.

[0088] The following describes the implementation of the present invention through specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific implementations, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0089] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0090] In the numerical range given in the embodiment, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention can also be used to realize the present invention.

[0091] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and conventional techniques in related fields in the art. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial sources.

[0092] Example 1 Obtaining mouse antibodies

[0093] 1. Immunization Procedure

[0094] Five 7-week-old female BALB / c or NZB / W mice were immunized using the following procedure. The mice were injected intraperitoneally with 25 μg of protein antigen per mouse (total volume 125 μL per mouse) every 3 weeks with human CD47 antigen mixed in CpG-ODN adjuvant. Test bleeding was performed by saphenous vein incision 7 days after the second boost. The test bleeding (immune serum) was tested by indirect ELISA assay to determine the best two responding mice for fusion. The mice may need the 3rd and 4th boosts and another test bleeding 7 days after the boost to assess the titer before fusion. When the antibody titer is high enough, the best two responding mice are given a final intravenous boost via the lateral tail vein. Four days after the IV boost, the mice were euthanized for fusion. The spleen was harvested, and the lymphocytes isolated from the spleen were used in the fusion process to produce hybridomas. Hybridoma development isolated lymphocytes and fused with mouse SP2 / 0 myeloma cells according to the standard Roche protocol in the presence of polyethylene glycol (PEG1500). The cells of fusion are cultivated using a single-step cloning method (HAT selection). This method uses the HAT selective culture medium based on semisolid methylcellulose to combine hybridoma selection and cloning into one step. The hybridoma derived from a single cell grows on a semisolid culture medium to form a monoclonal colony. Ten days after the fusion event, 1154 hybridoma clones of the obtained are transferred to a 96-well tissue culture plate and grown in a culture medium containing HT until reaching the logarithmic growth mid-term (5 days).

[0095] 2. Hybridoma Screening

[0096] Tissue culture supernatant from these 1154 hybridomas was tested by indirect ELISA utilizing the antigen (primary screening) screened, and goat anti-IgG / IgM (H&L)-HRP secondary antibodies were used to detect both IgG and IgM antibodies, and developed the color with TMB substrate. In this assay, clones of >0.2OD were taken to carry out the next round of testing. Utilize the antigen screened to test positive cultures again to confirm secretion, utilize irrelevant antigen (human transferrin) to eliminate nonspecific or "sticky" monoclonal antibodies and exclude false positives. All interested clones were subjected to isotype analysis to determine whether they were IgG or IgM isotypes by antibody capture ELISA.

[0097] 3. Hybridoma cell culture

[0098] After transfer to 96-well plates, the hybridoma cell lines of interest are maintained in 24-well culture plates for 32 days. This period is called the stabilization period and the clones are tested for stability and secretion. During this stabilization period, a temporary frozen backup of the cell lines consists of all the clones of interest and is stored at -80°C (can survive for 6 months). During this period, the hybridomas are regularly tested for secretion and specificity.

[0099] 4. Subcloning

[0100] The top hybridoma cell lines (clones) were subcloned to ensure monoclonality. Subcloning was performed by re-plating the parental clone using a single-step cloning system. 24 to 90 subclones were transferred to 96-well culture plates. Subclones were screened by indirect ELISA and antibody capture ELISA. The top subclones of each parent were used for amplification in culture. A second round of subcloning was performed on any parental clones that were <50% cloned.

[0101] Example 2 Identification of subtypes and amplification of variable regions of anti-CD47 mouse monoclonal antibodies

[0102] 1. Antibody subtype identification

[0103] Take the hybridoma cell culture supernatant and use IsoStrip TM Mouse monoclonal antibody subtype identification kit (SinoBiologicallnc, catalog number SEK003) was used to identify antibody subtypes. Monoclonal antibodies CD47-5A10, 5G11, 9C6, and 11F1 were all IgG1 (Kappa) subtypes.

[0104] 2. Antibody variable region amplification

[0105] Candidate hybridoma cells CD47-5A10, 5G11, 9C6, and 11F1 were cultured to a total number of 10 7 The cells were centrifuged at 1000 rpm for 10 min to collect the cells, and total RNA was extracted using a kit (Takara) and reverse transcription kit Primescript TM The first-chain cDNA was synthesized by RT-PCR, and the first-chain cDNA was used as a template for subsequent amplification of the antibody variable region DNA sequence corresponding to the hybridoma cells. According to the subtype identification results, the heavy chain and light chain constant region sequences of the antibody subtype were obtained, involving specific nested PCR primers. The primer sequences used in the amplification reaction were complementary to the first framework region and constant region of the antibody variable region. The heavy chain variable region sequences and light chain variable region sequences of the secreted antibodies of the hybridoma clones CD47-5A10, 5G11, 9C6, and 11F1 are shown in Table 1.

[0106] 1) Cloning of the heavy chain variable regions of mouse antibodies CD47-5A10, 5G11, 9C6, and 11F1

[0107] To design the humanization of mouse antibodies, we first need to obtain DNA fragments containing the coding sequences of the heavy and light chain variable regions of the candidate mouse antibodies CD47-5A10, 5G11, 9C6, and 11F1. We used an mRNA purification kit (Takara) to isolate MRNA from mouse hybridoma cells CD47-5A10, 5G11, 9C6, and 11F1, and prepare cDNA (prime script TM RT-PCR kit, Takara). The heavy chain variable region DNA fragment was isolated from the cDNA by polymerase chain reaction (PCR). The 5'-primer of PCR used 0.4 μm 5'-CCTAGGAGGTSMARCTGCAGSAGTCWGG-3' (primer 1), and the 3'-primer of PCR (primer 2) reacted homologously with the mouse IgG1 heavy chain constant region. The DNA fragment obtained after gel purification was cloned into the PMDA19-T (simple) vector and sequenced to obtain the nucleotide sequence and amino acid sequence of the variable region encoding the heavy chain of the mouse antibodies CD47-5A10, 5G11, 9C6, and 11F1. The nucleotide sequences of the variable regions of the heavy chains of murine antibodies CD47-5A10, 5G11, 9C6, and 11F1 are shown in SEQ ID NOs: 31-34; the amino acid sequences of the variable regions of the heavy chains of murine antibodies CD47-5A10, 5G11, 9C6, and 11F1 are shown in SEQ ID NOs: 23-26.

[0108] 2) Cloning of the light chain variable regions of mouse antibodies CD47-5A10, 5G11, 9C6, and 11F1

[0109] In a similar PCR method, a light chain variable region DNA fragment was isolated from cDNA using a 5' primer 5'-CCTAGGGACATTCAGCTGACCCAGTCTCCA-3' (primer 3) and another 3' primer homologous to the mouse immunoglobulin light chain constant region, i.e., 5'-CATATGGTTAGATCTCCAGCTTGGTCCC-3' (primer 4). These obtained DNA fragments were cloned into the TOPO-TA vector and sequenced to obtain the variable region nucleotide sequence and amino acid sequence encoding the CD47-5A10, 5G11, 9C6, 11F1 mouse hybridoma light chain. The variable region nucleotide sequence of the mouse antibody CD47-5A10, 5G11, 9C6, 11F1 light chain is shown in SEQ ID NO: 35-38; the variable region amino acid sequence of the mouse antibody CD47-5A10, 5G11, 9C6, 11F1 hydrogen chain is shown in SEQ ID NO: 27-30.

[0110] Example 3 Preparation of anti-CD47 human-mouse chimeric antibody

[0111] 1. Preparation of anti-CD47 human-mouse chimeric antibody

[0112] The murine antibodies CD47-5A10, 5G11, 9C6, and 11F1 were humanized to reduce their immunogenicity. The heavy chain variable region (VH) and light chain variable region (VL) of CD47-5A10, 5G11, 9C6, and 11F1 were compared with the human antibody germline gene sequences in the IMGT database, and the appropriate germline gene sequence was selected to provide the antibody framework region (FR1+FR2+FR3), and the appropriate J region gene sequence was selected to provide the framework region 4 (FR4). This template can be selected based on, for example, the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junction between the antibody framework region (FR) and the hypervariable region (CDR), the homology of the entire sequence, etc. The selected template can be a mixture of multiple sequences or a common template, with the aim of maintaining the appropriate conformation of the parent complementary determining region (CDR) as much as possible. The amino acid sequences of the humanized 5A10 heavy chain and light chain variable regions, the humanized 5G11 heavy chain and light chain variable regions, the humanized 9C6 heavy chain and light chain variable regions, and the humanized 11F1 heavy chain and light chain variable regions were finally determined. The variable region genes were designed and synthesized according to the amino acid sequences of the humanized antibodies, and the IgG1 versions of the humanized antibodies CD47-5A10-huIgG1, CD47-5G11-huIgG1, CD47-9C6-huIgG1, and CD47-11F1-huIgG1 were prepared.

[0113] The amino acid sequence of the heavy chain of the chimeric antibody CD47-5A10-huIgG1 (5A10 is underlined) is shown in SEQ ID NO. 39. SEQ ID NO. 39: 5A10-huIgG1-H

[0114] EVKLQESGPELVKPGASVKMSCTASGFTFTNYIIYWVRQEPGQGLEWIAYINPYNDDTEYN

[0115] EKFKGKATLTSDKSSTTVYMELSSLPSEDSAVYYCARGGIRAMDYWGQGTTVTVSS ASTK

[0116] GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

[0117] SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

[0118] KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

[0119] VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL

[0120] TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

[0121] VMHEALHNHYTQKSLSLSPGK

[0122] The amino acid sequence of the chimeric antibody light chain of CD47-5A10-huIgG1 (5A10 is underlined) is shown in SEQ ID NO.40.

[0123] SEQ ID NO.40: 5A10-huIgG1-L

[0124] GLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLAWYLQKPGQSPKL

[0125] LIYKVSNRFSGVPDRFSGSGSGTEFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLE IKR

[0126] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK

[0127] DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0128] The amino acid sequence of the heavy chain of the chimeric antibody of CD47-5G11-huIgG1 (5G11 is underlined) is shown in SEQ ID NO.41. SEQ ID NO.41: 5G11-huIgG1-H

[0129] EVKLQESGPELVKPGASVKISCKASGDSATGYYIHWVKQSPENSLEWIGEINPTSGGTSYSQ

[0130] KFKGKATLSVDKSSSTVYMQLKSLTSEESAVYYCSGGYYAAYWGQGTTVTVSS ASTKGPS

[0131] VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0132] VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0133] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0134] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

[0135] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0136] MHEALHNHYTQKSLSLSPGK

[0137] The amino acid sequence of the light chain of the chimeric antibody of CD47-5G11-huIgG1 (5G11 is underlined) is shown in SEQ ID NO.42. SEQ ID NO.42: 5G11-huIgG1-L

[0138] DIQLTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYSAKTLAAGVPSRF

[0139] SGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPLTIGPGTKLE IKRTVAAPSVFIFPPSDEQLK

[0140] SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0141] EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0142] The amino acid sequence of the heavy chain of the chimeric antibody of CD47-9C6-huIgG1 (9C6 is underlined) is shown in SEQ ID NO. 43. SEQ ID NO. 43: 9C6-huIgG1-H

[0143] EVQLQESGPELVKPGASVKISCKASGDSITGYYIHWVKQSPENSLEWIGEINPTSGGTSYSQ

[0144] KFKGKATLSLDKSSTTVYMQLKSLTSEESAVYYCSGGYYAAYWGQGTTVTVSS ASTKGPS

[0145] VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0146] VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0147] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0148] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

[0149] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0150] MHEALHNHYTQKSLSLSPGK

[0151] The amino acid sequence of the light chain of the chimeric antibody of CD47-9C6-huIgG1 (9C6 is underlined) is shown in SEQ ID NO. 44. SEQ ID NO. 44: 9C6-huIgG1-L

[0152] DIQLTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYSAKTLAEGVPSRF

[0153] SGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPLTIGPGTKLE IKRTVAAPSVFIFPPSDEQLK

[0154] SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0155] EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0156] The amino acid sequence of the heavy chain of the chimeric antibody of CD47-11F1-huIgG1 (11F1 is underlined) is shown in SEQ ID NO. 45. SEQ ID NO. 45: 11F1-huIgG1-H

[0157] EVQLQESGPELVKPGASVKISCKSSDYSFTDYYIHWVKHSHVKSLEWIGRLNPYNGVTIYN

[0158] QNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARSRRYGAMDYWGQGTTVTVSS AS

[0159] TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

[0160] SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

[0161] FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

[0162] VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV

[0163] SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

[0164] CSVMHEALHNHYTQKSLSLSPGK

[0165] The amino acid sequence of the light chain of the chimeric antibody of CD47-11F1-huIgG1 (11F1 is underlined) is shown in SEQ ID NO. 46. SEQ ID NO. 46: 11F1-huIgG1-L

[0166] DIQLTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFS

[0167] GSGSGRDYSFSISNLEPEDIATYYCLHYDNLRTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKS

[0168] GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

[0169] KHKVYACEVTHQGLSSPVTKSFNRGEC

[0170] The amino acid sequences of the heavy and light chain variable regions of humanized antibodies CD47-5A10-huIgG1, CD47-5G11-huIgG1, CD47-9C6-huIgG1, and CD47-11F1-huIgG1 are shown in Table 1, where the CDR regions are defined by the IMGT method.

[0171]

[0172]

[0173]

[0174] 2. Construction, expression and preparation of anti-human CD47 antibody expression vector

[0175] (1) Construction of CD47 antibody expression vector

[0176] According to the heavy chain and light chain sequences obtained above, cDNA encoding CD47-5A10-huIgG1, CD47-5G11-huIgG1, CD47-9C6-huIgG1, and CD47-11F1-huIgG1 were designed and inserted into the pCHO1.0 eukaryotic expression vector to construct a humanized expression vector. The expression vector plasmid contains the cytomegalovirus early promoter factor-enhancer required for high-level expression in mammalian cells. At the same time, the vector plasmid contains a selectable marker gene, thereby conferring kanamycin resistance in bacteria and puromycin resistance in mammalian cells. In addition, the vector plasmid contains a dihydrofolate reductase (DHFR) gene, which can co-amplify antibody genes and DHER genes with methotrexate (MTX) in suitable host cells.

[0177] (2) CD47 antibody expression

[0178] The recombinant expression vector plasmid constructed above is transfected into a mammalian host cell line to express the humanized antibody. In order to stabilize the high level of expression, the preferred host cell line is the DHFR-deficient Chinese hamster ovary (CHO) cell. The preferred transfection method is liposome transfection, and other methods can also be used, including calcium phosphate co-precipitation, electroporation, and protoplasmic fusion. The correctly sequenced plasmid bacterial solution is extracted with the OMEGA kit and linearized with the nuclease PvuI. The linearized plasmid is concentrated to a concentration of more than 1μg / μl and transfected with the transfection reagent Freestyle TM MAX for transfection. 24h before transfection, CHO-S TM Cells were passaged to 0.5×10 6 / ml, during transfection, adjust the cell density to 1×10 6 / ml. 50μg plasmid and 50μl Freestyle MAX were added to OptiPRO-SFM to 1.5ml, and the transfection reagent solution was added to the plasmid solution to mix well. After standing at room temperature for 10min, it was slowly added to the cells to complete the transfection. Two days after transfection, 20ug / ml puromycin and 200nM MTX (Sigma) were added. In order to achieve a higher level of expression, the transfected antibody gene was co-amplified with the DHFR gene inhibited by MTX. The secretion rate of each cell line was determined by limiting dilution subcloning transfectants and ELISA method, and the cell lines with high levels of antibody expression were selected, CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6.

[0179] (3) CD47 antibody purification

[0180] The cell lines CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 obtained in step (2) were expanded and cultured, and the cell culture supernatant was collected. The antibody was purified by Protein A affinity chromatography. First, a Protein A affinity column was prepared. After the column was equilibrated with PBS, the cell culture supernatant that was centrifuged and filtered through a 0.4 μm filter membrane was passed through the column, and then washed with PBS until the OD value was close to zero, eluted with 50 mmol / L pH 7.5 glycine-hydrochloric acid buffer solution, and the eluate in the peak area was collected and dialyzed for later use. The prepared antibodies were named CD47-5A10–huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6.

[0181] Example 4 SDS-PAGE detection of target protein molecular weight and expression level

[0182] SDS-PAGE reduction electrophoresis was used to detect the size and purity of the target protein. Electrophoresis was performed according to the method of Part 4 of the 2015 edition of the Chinese Pharmacopoeia. The results of the electrophoresis scan are shown in Figure 1 , identify the molecular weight and expression level of monoclonal antibodies. According to the electrophoresis results, the light chain is about 25KD, the heavy chain is about 50KD, and the protein purity is greater than 95%.

[0183] Example 5 Determination of Anti-human CD47 Chimeric Antibody Cell Surface Antigen Binding Activity

[0184] CHO-tm-CD47 cells expressing human CD47 transmembrane protein on their cell surface were inoculated in 1640 medium containing 10% FBS and cultured at 37°C and 5.0% CO2. 2×10 6 / ml CHO-tm-CD47 cells in logarithmic growth phase, washed twice with DPBS, 200 μl cells per well of 96-well plate, added 10 μg / ml of the screened anti-CD47 monoclonal antibody, incubated for 1 hour at 37°C, 5.0% CO2 incubator, washed 3 times with PBS, added 10 μg / ml of FITC-labeled fluorescent secondary antibody, incubated for 1 hour at 37°C, 5.0% CO2 incubator, washed 3 times with PBS, and finally added 200 μl PBS per well, and the binding activity of the antibody to CHO-tm-CD47 cells was detected by flow cytometry (Becton-Dickinson, San Jose, CA, US). The results are shown in Tables 2 and Figure 2The positive control antibodies were CC90002 and Hu5F9, and the EC50 results of the antibodies were analyzed using FlowJo software. From the results in Table 2, it can be seen that the EC50 values ​​of the four chimeric antibodies were all smaller than those of the control antibodies CC90002 and Hu5F9.

[0185] Table 2 Antibody EC50 values

[0186] antibody EC50 (ng / ml) CD47-5A10-huIgG1-1B8 25.8 CD47-5G11-huIgG1-2E10 23.34 CD47-9C6-huIgG1-4B6 14.82 CD47-11F1-huIgG1-10G6 11.42 CC90002 31.63 Hu5F9 31.24

[0187] Example 6 Anti-human CD47 chimeric antibody affinity determination

[0188] The affinity determination of the selected anti-human CD47 humanized antibodies CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-hu IgG1-4B6, and CD47-11F1-huIgG1-10G6 was performed using Fortebio technology and according to the instructions of the instrument Fortebio. In the determination test, the affinity of the target protein was measured using Fortebio. The purified CD47 antibody was immobilized on the ProteinA sensor, and the CD47 antibody protein was diluted in 6 times, combined with the immobilized CD47ProteinA sensor, and dissociated to obtain the binding constant and dissociation constant, respectively, and finally the affinity constant of the CD47 monoclonal antibody was obtained. The data obtained were processed and fitted with the experimental data using the 1:1 binding model of Fortebio's analysis software. The fitted data was basically consistent with the experimental data, and the binding and dissociation rate constants Ka and Kd were obtained. The equilibrium dissociation constant KD was obtained using Kd / Ka. The results are shown in Table 3. The KD values ​​of humanized antibodies CD47-5A10, 5G11, 9C6, and 11F1 are less than 1×10 -11 M, retains the affinity and specificity of the parental mouse monoclonal antibody and greatly reduces its immunogenicity.

[0189] Table 3 Chimeric antibody affinity determination

[0190] Antibody KD(M) Kon(1 / Ms) Koff(1 / s) CD47-5A10-huIgG1-1B8 <![CDATA[4.85×10 -12 ]]> <![CDATA[7.85×10 6 ]]> <![CDATA[3.81×10 -5 ]]> CD47-5G11-huIgG1-2E10 <![CDATA[6.22×10 -12 ]]> <![CDATA[3.15×10 6 ]]> <![CDATA[1.96×10 -5 ]]> CD47-9C6-huIgG1-4B6 <![CDATA[1.29×10 -12 ]]> <![CDATA[8.53×10 6 ]]> <![CDATA[1.10×10 -5 ]]> CD47-11F1-huIgG1-10G6 <![CDATA[1.02×10 -12 ]]> <![CDATA[3.57×10 6 ]]> <![CDATA[3.64×10 - 5]]> CC90002 <![CDATA[2.80×10 -9 ]]> <![CDATA[3.75×10 5 ]]> <![CDATA[1.04×10 -3 <!-- 16 -->]]> Hu5F9 <![CDATA[7.16×10 -9 ]]> <![CDATA[6.47×10 4 ]]> <![CDATA[4.63×10 -4 ]]>

[0191] Example 7 Anti-CD47 chimeric antibody dose-dependently blocks the binding of human CD47 to human SIRPα

[0192] Measure the binding of CD47 antibody to SIRPα-His by ELISA. Coating: Human CD47-hFc was diluted to 2 μg / ml with PBS, added to 96-well ELISA plate, 100 μL per well, incubated overnight at 4°C. Blocking: Wash the plate 3 times and block with 1% BSA+PBS, 300 μL per well, incubate at room temperature for 1 hour. Mix the antibody with SIRPα-His: Purified CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 antibodies were diluted to 20 μg / ml with PBST, and diluted 3-fold with PBST solution, with a total of 7 dilution gradients: Human SIRPα-His protein was diluted to 500 ng / ml with PBST, and antibodies and SIRPα-his proteins of different dilution gradients were mixed 1:1, and incubated at room temperature for 30 minutes. Add the mixture of antibody and SIRPα-his protein: 100 μL per well, react at room temperature for 1 hour, and add the mixture of IgG isotype control Rituximab and human SIRP-his protein to the control well. Add secondary antibody: After washing the plate 3 times, add anti-His tag antibody, HRP (1:3000), 100μL per well, and react at room temperature for 1 hour. Color development: After washing the plate 4 times, add TMB color development solution, 100μL per well, and color at room temperature for 30 minutes in the dark. Termination: Directly add stop solution 2.0M H2SO4 to terminate the reaction, 100μL per well. Detection: Immediately after terminating the reaction, place the ELISA plate in the ELISA reader, measure its OD value at 450nm, and save the original data. Data processing: Input the original data into the software SoftMax Pro6.2.1 for data processing. The results are as follows Figure 3A , Figure 3B As shown in Table 4, it can be seen from Table 4 that compared with the commercially available antibody Hu5F9 and the patented antibody CC90002, the antibodies CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 of the present invention all showed enhanced SIRPα blocking efficacy.

[0193] Table 4 Determination of antibody blocking CD47 binding to SIRPα

[0194] Antibody EC50(ng / ml) CD47-5A10-huIgG1-1B8 89.37 CD47-5G11-huIgG1-2E10 109.3 CD47-9C6-huIgG1-4B6 93.28 CD47-11F1-huIgG1-10G6 61.06 CC-90002 155.9 Hu5F9 146.8

[0195] Example 8 Anti-CD47 chimeric antibody promotes phagocytosis of phagocytes

[0196] CD47 is a cell surface receptor that is upregulated on tumor cells and is also thought to promote immune escape by interacting with its natural ligand SIRR-α. Binding of CD47 to SIRPα on macrophages results in decreased phagocytic activity. As described in detail below, it can be determined whether the SIRPα blocking activity of the anti-CD47 antibodies of the present invention and CD47 binding promote tumor cell phagocytosis in the presence of human macrophages.

[0197] PBMCs were isolated from human blood and differentiated into macrophages by culturing in ATM-V medium for 7 days. These monocyte-derived macrophages (MDMs) became adherent and other cells were washed away. MDMs were scraped and re-plated in 12-well plates and allowed to adhere for 24 hours. Jurkat, a human tumor cell line, was selected as the target cell type because of its high CD47 expression. Jurkat cells were labeled with 0.3 μM CFSE at 37 ° C for 15 minutes, then washed and added to MDMs at a ratio of four tumor cells per macrophage, and CD47 antibodies were added at various concentrations. Target cells were subjected to phagocytosis for 3 hours. PBS was then used to wash away target cells that were not engulfed. The remaining macrophages were scraped, stained with antibodies for macrophage marker CD14 coupled to DyLite 649, and analyzed by flow cytometry. Phagocytosis was measured by gating on live cells that were positive for FL4 (CD14+) and then evaluating the percentage of FL1 (CFSE+) positive cells. Taking the background phagocytic rate of macrophages as the starting point, the phagocytic rate of each sample was calculated. The results are shown in Figure 4 and Table 5. As can be seen from Table 5, the anti-CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 antibodies of the present invention all have high phagocytic effects.

[0198] Table 5 Assay for Antibodies to Promote Macrophages to Phagocytose Tumor Cells

[0199]

[0200]

[0201] Example 9 Hemagglutination test

[0202] CD47 is highly expressed on the surface of erythrocytes, so anti-CD47 monoclonal antibodies can specifically bind to CD47 on the surface of erythrocytes. Since each antibody has two antigen binding sites, anti-CD47 monoclonal antibodies may cause erythrocyte agglutination. Monoclonal antibodies that cause erythrocyte agglutination can cause side effects such as anemia and decreased erythrocyte count in the body. Whether anti-CD47 monoclonal antibodies cause erythrocyte agglutination depends mainly on the recognition epitope of the monoclonal antibody on the CD47 molecule. This example uses a classic hemagglutination test to analyze the ability of anti-CD47 monoclonal antibodies to cause erythrocyte agglutination. The CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 antibodies (all 40 μg / ml) obtained in the example were diluted with PBS for a series of multiple dilutions, and then mixed with the prepared 1% erythrocyte suspension in a micro-hemagglutination reaction plate, and placed on a micro-oscillator for 1 min to mix. After the micro-hemagglutination reaction plate is placed at room temperature of 25℃ for 1 hour, the red blood cell agglutination process is recorded by photography, and the micro-hemagglutination reaction plate is tilted at 45° for several minutes, and the red blood cell agglutination degree is further determined by the red blood cell flow rate. The red blood cell agglutination degree can be divided into 4 levels: 4 is agglutinated into a uniform thin layer, and there is no flow when tilted; 3 is agglutinated into a uniform thin layer, and there is slight flow when tilted; 2 is agglutinated into a small amount of thin layer, and the flow is faster when tilted; 1 No thin layer is formed, and the flow rate after tilting is the same as that of the control hole. The results are as follows Figure 5 As shown, human-mouse chimeric antibodies CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 antibodies did not cause or had no significant agglutination.

Claims

1. An anti-CD47 monoclonal antibody, characterized in that: The anti-CD47 antibody comprises a heavy chain variable region having HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region having LCDR1, LCDR2 and LCDR3 sequences; wherein the complementarity determining region of the heavy chain variable region of the antibody comprises HCDR1 having an amino acid sequence as shown in SEQ ID NO: 6, HCDR2 having an amino acid sequence as shown in SEQ ID NO: 8, and HCDR3 having an amino acid sequence as shown in SEQ ID NO: 9; the complementarity determining region of the light chain variable region comprises LCDR1 having an amino acid sequence as shown in SEQ ID NO: 16, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 17, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:

19.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

28.

3. The antibody according to claim 1, characterized in that The polynucleotide sequence of the heavy chain of the antibody is shown in SEQ ID NO: 32; the polynucleotide sequence of the light chain of the antibody is shown in SEQ ID NO:

36.

4. A polynucleotide molecule encoding the anti-CD47 monoclonal antibody according to any one of claims 1 to 3.

5. An expression vector comprising the polynucleotide molecule according to claim 4.

6. A host cell transformed with the expression vector according to claim 5.

7. Use of the CD47 monoclonal antibody according to any one of claims 1 to 3 in the preparation of an anti-tumor therapeutic drug or a drug for the preparation of a tumor diagnosis drug; the tumor is non-Hodgkin's lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, ovarian cancer, glioma, glioblastoma, lung cancer, gastric cancer, cervical cancer or B lymphoma.

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