Anti-cd47 monoclonal antibodies

Abstract

The application 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 application has good binding activity, good anti-aggregation effect, strong SIRP alpha blocking effect, obvious phagocytosis promotion effect, and a low antibody affinity constant KD value (lower than 1x10 ‑11 nM) and high antibody affinity. The antibody has strong phagocytosis and shows good anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and antibody drugs; specifically, it relates to an anti-CD47 monoclonal antibody. Background Technology

[0002] CD47 antibody, also known as integrin-associated protein (IAP), is a member of the immunoglobulin superfamily. Structurally, it consists of an N-terminal extracellular variable region, a transmembrane region composed of 3–5 highly hydrophobic transmembrane segments, and a hydrophilic C-terminal cytoplasmic tail. CD47 is widely expressed on the surface of various tissue cells, especially showing high expression on the surface of tumor cells, including non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), ovarian cancer, glioma, and glioblastoma. Weissman et al. at 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, with an average expression level approximately 3.3 times that of corresponding normal cells. Moreover, they found that the level of CD47 in solid tumor patients was negatively correlated with the prognostic index. In addition, CD47 has been identified as a cancer stem cell marker for both leukemia 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 Protein α), also known as SHPS-1 (Src homology 2 domain-containing protein tyrosine phosphatase substrate-1) / BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs) / CD172a, is a transmembrane glycoprotein with three immunoglobulin-like domains in its extracellular region and a typical immunoreceptor tyrosine inhibitory sequence (ITIM) in its intracellular domain, which has four tyrosine residues and is a potential phosphorylation site.

[0004] CD47 is a ligand for SIRPα, and the two interact through their extracellular domains to form an intercellular communication complex. Upon binding to SIRPα, CD47 induces intracellular ITIM phosphorylation of SIRPα. The phosphorylation site then activates tyrosine phosphatases SHP-1 and SHP-2, which contain SH2 (Src homology 2) domains, triggering a cascade of signal transduction. SHP-1 is primarily expressed in hematopoietic cells, negatively regulating their function; while SHP-2 is widely expressed, regulating the small G proteins Ras and Rho, 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 a crucial role in regulating the phagocytosis of mature blood cells by macrophages. CD47 molecules on the surface of normal healthy cells (such as erythrocytes or platelets) interact with the SIRPα receptor on macrophages to generate an inhibitory signal, suppressing their phagocytic activity and thus regulating the life cycle and quantity of blood cells in the blood. SIRPα on monocytes interacts with CD47 on erythrocytes, inhibiting Fcγ receptor-dependent phagocytosis through myosin-IIA dephosphorylation. The CD47-SIRPα signaling system inhibits dendritic cell activation and participates in various physiological activities such as nervous system development, neutrophil chemotactic activation, and stromal cell-supported hematopoietic cell production. It also plays multiple regulatory roles in inducing T cell immune tolerance, activation, and apoptosis.

[0006] In recent years, the role of the CD47-SIRPα signaling system in regulating macrophage tumor immune surveillance has gained attention. CD47 expression 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 associated with poor prognosis in patients with malignant tumors. Expressing mouse CD47 in human myeloid leukemia cells (which express low levels of endogenous CD47 and cannot be transplanted into Rag2-I12rg- mice) can inhibit macrophage phagocytosis of tumor cells and promote successful tumor cell transplantation. It can be inferred that CD47 on tumor cells interacts with SIRPα on macrophages, inhibiting macrophage clearance of tumor cells and promoting tumor growth and metastasis in vivo. High expression of CD47 is a common mechanism for tumor cells to escape immune surveillance, and blocking CD47-SIRPα may be a novel strategy for tumor immunotherapy.

[0007] Anti-CD47 antibodies, used alone or in combination with other tumor antigen antibodies, have shown excellent inhibitory effects on tumor growth in mouse transplantation models of human acute myeloid leukemia, non-Hodgkin's lymphoma (NHL), and many solid tumors. Humanized anti-CD47 monoclonal antibodies induce macrophage phagocytosis of primary human AML cells, completely eliminating human AML cells in vivo and resulting in long-term disease-free survival in transplanted mice. Combined with rituximab, they can clear NHL tumors and cure xenograft mice. Furthermore, the safety of anti-CD47 antibodies has been confirmed in monkeys. In addition to anti-CD47 antibodies, the high-affinity SIRPα mutant (CD172a) can also antagonize CD47, thereby blocking the CD47-SIRPα signaling pathway. In AML models, it significantly increases macrophage phagocytosis of AML cells and inhibits tumor growth.

[0008] However, the main bottleneck in antibody research targeting 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 shortcomings of the prior art, this invention provides a human-mouse chimeric compound that specifically binds to CD47, blocks the interaction between CD47 and SIRPα, and does not have significant hemagglutination activity.

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

[0011] The anti-CD47 antibody contains heavy chain variable regions of HCDR1, HCDR2, and HCDR3 sequences and light chain variable regions containing LCDR1, LCDR2, and LCDR3 sequences.

[0012] 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] A CDR (complementarity determining region) generally refers to a region in an antibody that is spatially complementary to the antigenic determinant. The variability in an antibody is usually not uniformly distributed throughout its variable regions. Monoclonal antibodies typically have three hypervariable regions (HVRs) in both the heavy and light chain variable regions. These regions are often spatially complementary to the antigenic determinant, hence the term complementarity determining region (CDR). Specifically, the heavy chain variable region usually includes three CDRs: CDRH1, CDRH2, and CDRH3, while the light chain variable region typically includes three CDRs: CDRL1, CDRL2, and CDRL3.

[0019] 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.

[0020] 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: 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 includes 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 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.

[0023] In one embodiment, the complementarity-determining region of the light chain variable region of the anti-CD47 antibody 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.

[0024] In one embodiment, the complementarity-determining region of the light chain variable region of the anti-CD47 antibody includes 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 includes 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: 18, and LCDR3 with 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 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.

[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 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; the complementarity-determining region of the light chain variable region includes 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.

[0029] 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: 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; the complementarity-determining region of the light chain variable region includes 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: 18, and LCDR3 with an amino acid sequence 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 as shown in SEQ ID NO: 10, HCDR2 as shown in SEQ ID NO: 11, and HCDR3 as shown in SEQ ID NO: 12; the complementarity-determining region of the light chain variable region includes LCDR1 as shown in SEQ ID NO: 20, LCDR2 as shown in SEQ ID NO: 21, and LCDR3 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, which may be located between 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, the present invention provides an antibody that specifically binds to human CD47, wherein the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to 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 is at least 90% identical to any one of SEQ ID NO: 27, 28, 29 or 30.

[0037] The method for determining the heavy and light chain variable region sequences of the anti-CD47 antibody in this invention is as follows: specific primers are synthesized based on the constant region sequence of the antibody gene, the heavy and light chain variable regions of the monoclonal antibody are amplified by PCR, the target fragment is recovered, cloned into the PMDA19-T (simple) vector, transformed into Escherichia coli DH5α, positive clones are selected, plasmids are extracted and sequenced.

[0038] The antibody described in this invention is a monoclonal antibody that binds to and neutralizes human CD47, thereby blocking the CD47-SIRPα signaling pathway. In one embodiment, the antibody promotes 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, an 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, IgG2 or IgG4 subtypes and / or a light chain constant region selected from κ or λ subtypes; in a preferred embodiment, the backbone used for humanization 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 implementation, the humanized antibody can be prepared using the following method:

[0043] The heavy chain variable region (VH) and light chain variable region (VL) of the murine antibody were compared with human antibody germline gene sequences in the IMGT database. Suitable germline gene sequences were selected to provide the frame region (FR1+FR2+FR3) of the antibody, and a suitable J region gene sequence was selected to provide frame region 4 (FR4). This template can be selected based on factors such as the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junction between the antibody frame region (FR) and the hypervariable region (CDR), and the overall sequence homology. The selected template can be a mixture of multiple sequences or a shared template, with the aim of maintaining the appropriate conformation of the parental complementarity-determining region (CDR) as much as possible. The amino acid sequences of the heavy and light chain variable regions of the humanized antibody were finally determined. Based on the amino acid sequence of the humanized antibody, variable region genes were designed and synthesized, and IgG1, IgG2, or IgG4 versions of the humanized antibody were prepared.

[0044] The method of this invention for determining the anti-CD47 monoclonal antibody subtype is to extract the supernatant of hybridoma cell culture and use IsoStripping... TM The Mouse Monoclonal Antibody Subtype Identification Kit (Sino Biological Inc., catalog number SEK003) identifies 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] In a third aspect, the present invention provides a polynucleotide molecule encoding an anti-CD47 monoclonal antibody as described above.

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

[0051] A fourth aspect of the invention provides an expression vector comprising a polynucleotide molecule encoding an anti-CD47 monoclonal antibody provided by the invention. The expression vector in this 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, the invention provides a host cell for transforming the expression vector described herein. Any cell suitable for expression in the expression vector can serve as a host cell; for example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Further examples include CHO cells, BHK cells, or HEK293 cells; in a preferred embodiment, the host cell is a CHO cell.

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

[0054] The host cells express the anti-human CD47 monoclonal antibody under appropriate expression conditions, or hybridoma cells that stably secrete the anti-human CD47 monoclonal antibody are obtained through hybridoma technology.

[0055] The host cells used in this invention are all existing technologies and can be obtained directly through commercial channels. The culture media used in the culture are also various conventional culture media. Those skilled in the art can select suitable culture media based on experience and culture the cells under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using appropriate methods (such as temperature change or chemical induction), and the cells are cultured for a further period of time. The recombinant polypeptides in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the grouped proteins can be separated and purified using various separation methods based on 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0056] In one experimental protocol, the anti-CD47 antibody can be prepared by the following method: The target gene sequence and vector pCHO1.0 are digested with restriction endonucleases AvrII and BstZ17I, and the target fragment is recovered and ligated with T4 ligase to construct an expression vector. The vector is then transformed into E. coli DH5α competent cells, and the plasmid is extracted to obtain a recombinant expression plasmid. This plasmid is linearized with restriction endonuI, transfected into CHO-S cells, and screened under pressure with puromycin and MTX to obtain a cell bank expressing the target protein. After clonal culture, expression level determination, and affinity sequencing, a monoclonal cell line expressing the antibody is finally screened. The antibody protein is then further purified, and its purity, affinity, hemagglutination effect, SIRPα blocking efficacy, and phagocytic effect are measured.

[0057] In a preferred embodiment, the antibody purification method is affinity chromatography, and the specific steps are as follows: first, a protein A affinity column is prepared, the column is equilibrated with PBS, the centrifuged and filtered cell culture supernatant is passed through the column, then washed with PBS until the OD value is close to zero, eluted with glycine-hydrochloric acid buffer solution, the eluent in the peak region is collected, and then dialyzed for later use.

[0058] In a preferred embodiment, the method for determining antibody purity is SDS-PAGE, specifically involving electrophoresis according to the method in Part IV of the 2015 edition of the Chinese Pharmacopoeia, scanning the electrophoretic image, and identifying its molecular weight and expression level.

[0059] In a preferred embodiment, the method for determining antibody affinity is the ForteBio method, which includes the following steps: the purified CD47 antibody is immobilized by the ProteinA sensor; the diluted CD47 protein is bound to the immobilized CD47 ProteinA sensor; the protein is then dissociated; the binding constant and dissociation constant are obtained respectively; and finally, the affinity constant of the CD47 monoclonal antibody is determined.

[0060] In a preferred embodiment, the method for determining the antibody blocking the binding of human CD47 to human SIRPα is an ELISA method, which monitors the binding of recombinant SIRPα-his under conditions of increased CD47 antibody levels. The binding of SIRPα is determined using HRP-conjugated anti-his secondary antibody.

[0061] In a preferred embodiment, the method for determining the antibody-induced phagocytosis is flow cytometry, specifically comprising the following steps: seeding macrophages in a cell plate and allowing them to attach for 24 hours; labeling target human cancer cells (Jurkat) with CFSE dye and co-incubating with different anti-CD47 monoclonal antibodies or without antibodies; then adding them to macrophage culture medium for co-incubation; washing away unphagocytosed target cells with PBS; collecting macrophages; staining macrophages with anti-human CD14-APC; and analyzing the results using flow cytometry.

[0062] The application of the anti-CD47 monoclonal antibody, conjugate and / or conjugate described in this invention in the preparation of formulations that block the binding of CD47 to SIRPα shows that the anti-CD47 monoclonal antibody has a low EC50 value in blocking the binding of CD47 to SIRPα, indicating a strong SIRPα blocking efficacy.

[0063] The application of the anti-CD47 monoclonal antibody, conjugate, and / or conjugate described in this invention in promoting the phagocytosis of tumor cells by macrophages, the effect of which was determined by flow cytometry and the results were expressed as phagocytosis rate. This invention provides an antibody with a phagocytosis rate of over 60% on Jurakat cells.

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

[0065] The tumor treatment drug kills tumor cells by blocking CD47, thereby promoting the function of macrophages to engulf tumor cells.

[0066] The aforementioned tumor treatment drug is a drug that targets the CD47 antigen, which is functionally expressed on the surface of tumor cells, and binds to or acts on the CD47 antigen to treat and / or prevent tumors. The tumors include, but are not limited to, lung cancer, gastric cancer, cervical cancer, and B-cell lymphoma.

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

[0068] The anti-CD47 monoclonal antibody provided by this invention has a purity of over 95% after purification; it exhibits good anti-agglutination effect, strong SIRPα blocking efficacy, and significant phagocytosis promotion; the antibody affinity constant detected by ForteBio shows a KD value below 1×10⁻⁶. - 11 It contains nM molecules and has high antibody affinity. It exhibits strong phagocytic function and demonstrates good anti-tumor effects. Attached Figure Description

[0069] Figure 1 This is the electrophoretic pattern of the CD47 antibody;

[0070] Figure 2 This is a curve showing the binding activity of CD47 antibody with cell surface antigen.

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

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

[0073] Figure 4 This is a graph showing the effect of CD47 antibody on phagocytosis by phagocytes.

[0074] Figure 5 This is a graph showing the results of a blood cell agglutination test. Detailed Implementation

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

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

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

[0078] As used herein, the terms "antibody" and "immunoglobulin" refer to heterotetraglycoproteins of approximately 150,000 Daltons with the same structural characteristics, composed 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, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. The variable regions of each light chain are opposite to those of the heavy chain. Specific amino acids form interfaces between the variable regions of the light and heavy chains.

[0079] The antibodies of this invention include recombinant antibodies. As used herein, “recombinant antibody” means an antibody produced, expressed, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into host cells; antibodies isolated from a library of recombinant combined antibodies; antibodies isolated from animals (e.g., mice) that are transgenic due to their human immunoglobulin gene; or antibodies produced, expressed, generated, or isolated in any other manner in which a specific immunoglobulin gene sequence (e.g., the human immunoglobulin gene sequence) is assembled with other DNA sequences. Reviews of current methods for antibody engineering and improvement can be found, for example, in P. Chsmes, ed., (2012) Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology, Book 9070), HumanaPress, etc. Recombinant antibodies include, for example, chimeric antibodies and humanized antibodies.

[0080] A "chimeric" protein contains at least one fusion polypeptide that contains a region in its sequence at a location different from its natural counterpart. This region, which would normally exist in a single protein, is placed together in the fusion polypeptide; or they would normally exist in the same protein, but are arranged in a new configuration within the fusion polypeptide. For example, chimeric proteins can be constructed through chemical synthesis or by creating and translating polynucleotides in which peptide regions are encoded in a desired relationship. As used herein, a "chimeric antibody" refers to an antibody in which a sequence of a variable domain from a mammalian species (e.g., mouse) is grafted onto a sequence of a constant domain from another mammalian species (e.g., human).

[0081] A "domain" is a portion of a protein that is physically or functionally distinct from the rest of that protein or peptide. Physically defined domains include highly hydrophobic or hydrophilic amino acid sequences, such as those that are membrane-bound or cytoplasm-bound. Domains can also be defined by, for example, internal homology caused by gene replication. Functionally defined domains have different biological functions. For example, the ligand-binding domain of a receptor is the domain that binds to a ligand. An antigen-binding domain is the portion of 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 may contain one or more physically defined domains. For example, receptors are typically classified into extracellular ligand-binding domains, transmembrane domains, and intracellular effector domains.

[0082] The term "variable" as used herein refers to the fact that certain portions of the variable region in an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called architecture regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally p-sheet oriented and linked by three CDRs forming a connecting loop, and in some cases, partially p-sheet structures. The CDRs in each chain are tightly packed together through the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

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

[0084] A "vector" is a nucleic acid molecule, preferably self-replicating, that transfers an inserted nucleic acid molecule into a host cell and / or between host cells. This genus 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. It also includes vectors that provide more than one of the above functions. An "expression vector" refers to a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell containing an expression vector capable of producing a desired expression product.

[0085] The term “treatment” is used herein to refer broadly to achieving the desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete stabilization or cure of the disease and / or adverse reactions attributable to the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals such as mice, rats, rabbits, pigs, primates, including humans and other apes, particularly humans, and the term includes: (a) preventing the occurrence of a disease or symptoms in subjects who may be susceptible to the disease or symptoms but have not yet been diagnosed; (b) suppressing disease symptoms; (c) halting the development of the disease; (d) alleviating disease symptoms; (e) causing the remission of the disease or symptoms; or any combination thereof.

[0086] The terms “cancer,” “tumor,” and “carcinoma” are used interchangeably in this application to refer to cells that exhibit relatively autonomous growth, resulting in an abnormal growth phenotype characterized by significantly uncontrolled cell proliferation. Typically, target cells used for monitoring or treatment in this application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells.

[0087] Phagocytes are cells that can engulf other cells. Non-restrictive categories of phagocytes include phagocytes, mononuclear cells (e.g., tissue cells and monocytes), polymorphonuclear leukocytes (e.g., neutrophils), and dendritic cells.

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

[0089] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated herein, the singular forms “a,” “an,” and “this” include the plural forms.

[0090] Within the numerical ranges given in the embodiments, it should be understood that, unless otherwise stated in the present invention, any value between the two endpoints of each numerical range and any value in between may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of the present invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of the present invention may be used to implement the present invention.

[0091] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise stated, the materials and reagents used in the following examples are commercially available.

[0092] Example 1: Obtaining murine antibodies

[0093] 1. Immunization program

[0094] Five 7-week-old female BALB / c or NZB / W mice were immunized using the following procedure. Every 3 weeks, mice were intraperitoneally injected with 25 μg of protein antigen per mouse (total volume 125 μL per mouse) mixed with human CD47 antigen in CpG-ODN adjuvant. Test bleeding was performed 7 days after the second booster via a saphenous vein incision. This test bleeding (immune serum) was tested by an indirect ELISA assay to determine the two best-responding mice for fusion. The mouse may require a third and fourth booster and another test bleeding 7 days after the booster to assess the pre-fusion titer. When the antibody titer was sufficiently high, the two best-responding mice were given a final intravenous booster via the lateral tail vein. Four days after the fourth booster, the mice were euthanized for fusion. The spleen was harvested, and lymphocytes isolated from the spleen were used to generate hybridomas during the fusion process. Hybridoma development involved isolating lymphocytes and fusing them with mouse SP2 / 0 myeloma cells in the presence of polyethylene glycol (PEG1500) according to the standard Roche protocol. Fusion cells were cultured using a single-step cloning method (HAT selection). This method uses a HAT-selective medium based on semi-solid methylcellulose to combine hybridoma selection and cloning in one step. Single-cell-derived hybridomas were grown on the semi-solid medium to form monoclonal colonies. Ten days after the fusion event, 1154 resulting hybridoma clones were transferred to 96-well tissue culture plates and grown in HT-containing medium until reaching mid-log growth (5 days).

[0095] 2. Hybridoma screening

[0096] Tissue culture supernatants from these 1154 hybridomas were tested using an indirect ELISA with a selected antigen (primary screening). Both IgG and IgM antibodies were detected using goat anti-IgG / IgM (H&L)-HRP secondary antibody, and color development was performed using a TMB substrate. Clones with >0.2 OD were used for the next round of testing. Positive cultures were retested with the selected antigen to confirm secretion, and an unrelated antigen (human transferrin) was used to eliminate nonspecific or “sticky” monoclonal antibodies and rule out false positives. All clones of interest were subjected to isotype analysis using an antibody capture ELISA to determine whether they were IgG or IgM isotypes.

[0097] 3. Hybridoma cell culture

[0098] After transfer to 96-well plates, the hybridoma cell lines of interest were cultured in 24-well plates for 32 days. This period, known as the stationary phase, was used to test whether the clones remained stable and secreted. During this stationary phase, a temporary frozen backup of the cell lines, consisting of all clones of interest, was stored at -80°C (surviving for up to 6 months). During this period, the secretion and specificity of the hybridomas were tested periodically.

[0099] 4. Subcloning

[0100] Subcloning of top-performing hybridoma cell lines (clones) was performed to ensure monoclonal integrity. Parental clones were re-platened using a single-step cloning system. 24 to 90 subclones were transferred to 96-well plates. Subclones were screened by indirect ELISA and antibody capture ELISA. Top-performing subclones from each parent were used for amplification in culture. A second round of subcloning was performed on any parental clones with <50% clone content.

[0101] Example 2: Identification of anti-CD47 murine monoclonal antibody subtypes and amplification of variable regions

[0102] 1. Antibody subtype identification

[0103] Hybridoma cell culture supernatant was collected and processed using IsoStrip. TM The Mouse Monoclonal Antibody Isotype Identification Kit (SinoBiologicallnc, catalog number SEK003) identifies antibody isotypes. The monoclonal antibodies CD47-5A10, 5G11, 9C6, and 11F1 are all isotypes of IgG1 (Kappa).

[0104] 2. Amplification of antibody variable regions

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

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

[0107] To design and humanize murine antibodies, it is first necessary to obtain a DNA fragment containing the coding sequences for the variable regions of the heavy and light chains of the candidate murine antibody CD47-5A10, 5G11, 9C6, and 11F1. mRNA was isolated from mouse hybridoma cells CD47-5A10, 5G11, 9C6, and 11F1 using an mRNA purification kit (Takara), and cDNA was prepared accordingly. TM RT-PCR kit (Takara). Heavy chain variable region DNA fragments were isolated from cDNA using polymerase chain reaction (PCR). The 5' primer for PCR was 0.4 μm 5'-CCTAGGAGGTSMARCTGCAGSAGTCWGG-3' (primer 1), and the 3' primer (primer 2) reacted homologously with the constant region of the mouse IgG1 heavy chain. The purified DNA fragment was cloned into the PMDA19-T (simple) vector and sequenced to obtain the nucleotide and amino acid sequences encoding the variable regions of the mouse antibody CD47-5A10, 5G11, 9C6, and 11F1 heavy chains. The variable region nucleotide sequences of the heavy chains CD47-5A10, 5G11, 9C6, and 11F1 of the murine antibody are shown in SEQ ID NO: 31-34; the variable region amino acid sequences of the heavy chains CD47-5A10, 5G11, 9C6, and 11F1 of the murine antibody are shown in SEQ ID NO: 23-26.

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

[0109] Using a similar PCR method, primer 5'-CCTAGGGACATTCAGCTGACCCAGTCTCCA-3' (primer 3) and another 3' primer homologous to and antisense the constant region of the mouse immunoglobulin light chain, namely 5'-CATATGGTTAGATCTCCAGCTTGGTCCC-3' (primer 4), were used to isolate the variable region DNA fragments of the light chain from cDNA. These obtained DNA fragments were cloned into the TOPO-TA vector and sequenced to obtain the nucleotide and amino acid sequences of the variable regions encoding the CD47-5A10, 5G11, 9C6, and 11F1 mouse hybridoma light chains. The nucleotide sequences of the variable regions of the mouse antibody CD47-5A10, 5G11, 9C6, and 11F1 light chains are shown in SEQ ID NO: 35-38; the amino acid sequences of the variable regions of the hydrogen chains of the mouse antibody CD47-5A10, 5G11, 9C6, and 11F1 are 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] Humanization studies were conducted on murine antibodies CD47-5A10, 5G11, 9C6, and 11F1 to reduce their immunogenicity. The heavy chain variable regions (VH) and light chain variable regions (VL) of CD47-5A10, 5G11, 9C6, and 11F1 were compared with human antibody germline gene sequences in the IMGT database. Suitable germline gene sequences were selected to provide the frame regions (FR1+FR2+FR3) of the antibody, and suitable J region gene sequences were selected to provide frame region 4 (FR4). This template was selected based on factors such as the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junctions between the antibody frame regions (FR) and hypervariable regions (CDR), and the overall sequence homology. The selected template can be a mixture of multiple sequences or a shared template, with the aim of maintaining the appropriate conformation of the parental complementarity-determining regions (CDRs) as much as possible. The amino acid sequences of the humanized 5A10 heavy chain, light chain variable region, humanized 5G11 heavy chain, light chain variable region, humanized 9C6 heavy chain, light chain variable region, and humanized 11F1 heavy chain and light chain variable region were finally determined. Based on the amino acid sequences of the humanized antibodies, variable region genes were designed and synthesized, and IgG1 versions of the humanized antibodies CD47-5A10-huIgG1, CD47-5G11-huIgG1, CD47-9C6-huIgG1, and CD47-11F1-huIgG1 were prepared.

[0113] The heavy chain amino acid sequence of the chimeric antibody CD47-5A10-huIgG1 (5A10 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 light chain amino acid sequence of the chimeric antibody CD47-5A10-huIgG1 (5A10 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 heavy chain amino acid sequence of the chimeric antibody CD47-5G11-huIgG1 (5G11 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 light chain amino acid sequence of the chimeric antibody CD47-5G11-huIgG1 (5G11 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 heavy chain amino acid sequence of the chimeric antibody CD47-9C6-huIgG1 (9C6 underlined) is shown in SEQ ID NO.43. SEQ ID NO.43: 9C6-huIgG1-H

[0143] [[ID=十六]]EVQLQESGPELVKPGASVKISCKASGDSITGYYIHWVKQSPENSLEWIGEINPTSGGTSYSQ

[0144] [[ID=十七]]KFKGKATLSLDKSSTTVYMQLKSLTSEESAVYYCSGGYYAAYWGQGTTVTVSS ASTKGPS

[0145] VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0146] VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0147] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0148] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

[0149] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0150] MHEALHNHYTQKSLSLSPGK

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

[0152] [[ID=十八]]DIQLTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYSAKTLAEGVPSRF

[0153] [[ID=十九]]SGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPLTIGPGTKLE IKRTVAAPSVFIFPPSDEQLK

[0154] SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0155] EKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0157] [[ID=二十]]EVQLQESGPELVKPGASVKISCKSSDYSFTDYYIHWVKHSHVKSLEWIGRLNPYNGVTIYN

[0158] [[ID=二十一]]QNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARSRRYGAMDYWGQGTTVTVSS AS

[0159] TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

[0160] SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

[0161] FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

[0162] VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV

[0163] SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

[0164] CSVMHEALHNHYTQKSLSLSPGK

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

[0166] [[ID=二十二]]DIQLTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFS

[0167] [[ID=二十三]]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 region is 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] Based on the obtained heavy and light chain sequences, cDNA encoding CD47-5A10-huIgG1, CD47-5G11-huIgG1, CD47-9C6-huIgG1, and CD47-11F1-huIgG1 was designed and inserted into the pCHO1.0 eukaryotic expression vector to construct a humanized expression vector. This expression vector plasmid contains the cytomegalovirus early initiation factor-enhancer, required for high-level expression in mammalian cells. Simultaneously, the vector plasmid contains selectable marker genes, thereby conferring kanamycin resistance in bacteria and puromycin resistance in mammalian cells. Furthermore, the vector plasmid contains the dihydrofolate reductase (DHFR) gene, which, in suitable host cells, can co-amplify the antibody gene and the DHER gene with methotrexate (MTX).

[0177] (2) CD47 antibody expression

[0178] The recombinant expression vector plasmid constructed above was transfected into a mammalian host cell line to express the humanized antibody. For stable, high-level expression, the preferred host cell line was DHFR-deficient Chinese hamster ovary (CHO) cells. The preferred transfection method was liposome transfection, but other methods, including calcium phosphate co-precipitation, electroporation, and protoplasmic fusion, could also be used. Plasmids were extracted from correctly sequenced bacterial cultures using an OMEGA kit and linearized with the restriction endonuclease PvuI. The linearized plasmid was concentrated to a concentration above 1 μg / μl and transfected using the Freestyle transfection reagent. TM MAX was used for transfection. CHO-S was added 24 hours before transfection. TM Cells were passaged to 0.5 × 10⁻⁶ cells. 6 / ml, during transfection, adjust cell density to 1×10⁶. 6 / ml. Add 50μg of plasmid and 50μl of Freestyle MAX to OptiPRO-SFM to a final volume of 1.5ml. Add the transfection reagent solution to the plasmid solution and mix well. After standing at room temperature for 10 min, slowly add the solution to the cells to complete transfection. Two days after transfection, add cells containing 20ug / ml puromycin and 200nM MTX (Sigma). To achieve higher levels of expression, the transfected antibody gene was co-amplified using the DHFR gene, which is inhibited by MTX. The secretion rate of each cell line was determined by limiting dilution subclonal transfectants and ELISA. Cell lines expressing high levels of antibody 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 equilibrating the column with PBS, the cell culture supernatant, which had been centrifuged and filtered through a 0.4 μm filter membrane, was passed through the column. Then, it was washed with PBS until the OD value was close to zero. Elution was performed with 50 mmol / L glycine-hydrochloric acid buffer at pH 7.5. The eluent in the peak region 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, respectively.

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

[0182] SDS-PAGE reduction electrophoresis was used to determine the size and purity of the target protein. Electrophoresis was performed according to the methods described in Part IV of the 2015 edition of the Chinese Pharmacopoeia. The electrophoresis results are shown below. [[ID=二十四]]Figure 1 The molecular weight and expression level of the monoclonal antibodies were identified. According to the electrophoresis results, the light chain was approximately 25 kDa, the heavy chain approximately 50 kDa, and the protein purity was greater than 95%.

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

[0184] CHO-tm-CD47 cells expressing human CD47 transmembrane protein were seeded in 1640 medium containing 10% FBS and cultured at 37°C with 5.0% CO2. Two × 10⁶ cells were collected. 6 / ml of CHO-tm-CD47 cells in logarithmic growth phase were washed twice with DPBS. 200 μl of cells were added to each well of a 96-well plate, followed by 10 μg / ml of the selected anti-CD47 monoclonal antibody. The cells were incubated at 37°C for 1 hour in a 5.0% CO2 incubator. After washing three times with PBS, 10 μg / ml of FITC-labeled fluorescent secondary antibody was added, and the cells were incubated at 37°C for 1 hour in a 5.0% CO2 incubator. After washing three times with PBS, 200 μl of PBS was added to each well. The binding activity of the antibody to CHO-tm-CD47 cells was detected by flow cytometry (Becton-Dickinson, San Jose, CA, US). Results are shown in Table 2 and [Table data would be inserted here]. [[ID=二十五]]Figure 2The positive control antibodies were CC90002 and Hu5F9. The EC50 values ​​of the antibodies were analyzed using FlowJo software. As shown in Table 2, the EC50 values ​​of all four chimeric antibodies were lower than those of the control antibodies CC90002 and Hu5F9.

[0185] Table 2 Antibody EC50 values

[0186] [[ID=二十六]]Antibody [[ID=二十七]]EC50 (ng / ml) [[ID=二十八]]CD47-5A10-huIgG1-1B8 25.8 [[ID=二十九]]CD47-5G11-huIgG1-2E10 23.34 [[ID=三十]]CD47-9C6-huIgG1-4B6 14.82 CD47-11F1-huIgG1-10G6 11.42 CC90002 31.63 Hu5F9 31.24

[0187] Example 6: Affinity determination of anti-human CD47 chimeric antibody

[0188] The affinity assays for the selected anti-human CD47 humanized antibodies CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 were performed using Fortebio technology, following the Fortebio instrument's instruction manual. In the assay, the affinity of the target protein was measured using Fortebio. The purified CD47 antibody was immobilized by the ProteinA sensor. The CD47 antibody protein was serially diluted six times and bound to the immobilized CD47 ProteinA sensor. After dissociation, the binding and dissociation constants were obtained, and finally, the affinity constant of the CD47 monoclonal antibody was determined. The processed data were fitted to the experimental data using a 1:1 model in Fortebio's analysis software. The fitted data were largely consistent with the experimental data, yielding the binding and dissociation rate constants Ka and Kd. 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 1×10⁻⁶. -11 M retains the affinity and specificity of the parent mouse monoclonal antibody while significantly reducing its immunogenicity.

[0189] Table 3. Affinity assay of chimeric antibodies

[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 SIRPa.

[0192] The binding of CD47 antibody to SIRPα-His was measured by ELISA. Coating: Human CD47-hFc was diluted to 2 μg / ml with PBS and added to 100 μL per well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. Blocking: After washing the plate three times, 300 μL of 1% BSA + PBS was added to each well and incubated at room temperature for 1 hour. The antibody was mixed 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 serially diluted 3-fold with PBST solution, resulting in a total of 7 dilutions. Human SIRPα-His protein was diluted to 500 ng / ml with PBST. The antibodies and SIRPα-his protein of different dilutions were mixed 1:1 and incubated at room temperature for 30 min. The antibody-SIRPα-his protein mixture was added: 100 μL per well, and the reaction was carried out at room temperature for 1 h. A mixture of IgG isotype control Rituximab and human SIRP-his protein was added to the control wells. Secondary antibody addition: After washing the plate three times, add 100 μL of anti-His tag antibody and HRP (1:3000) per well, and react at room temperature for 1 hour. Color development: After washing the plate four times, add 100 μL of TMB chromogenic buffer per well, and incubate at room temperature in the dark for 30 minutes. Termination: Directly add 100 μL of 2.0 M H2SO4 stop solution per well to terminate the reaction. Detection: Immediately after terminating the reaction, place the plate in a microplate reader and measure its OD value at 450 nm. Save the raw data. Data processing: Input the raw data into SoftMax Pro 6.2.1 software for data processing. Results are as follows: Figure 3A , Figure 3B As shown in Table 4, 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 show enhanced SIRPα blocking efficacy.

[0193] Table 4. Assay for antibody blocking of CD47-SIRPα binding.

[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 by phagocytes

[0196] CD47 is a cell surface receptor that is upregulated on tumor cells and is also believed to promote immune escape through its interaction with its natural ligand, SIRR-α. Binding of CD47 to SIRR-α on macrophages leads to decreased phagocytic activity. As detailed below, it can be determined whether the SIRR-α blocking activity of the anti-CD47 antibody described in this invention and the presence of CD47 binding promote tumor cell phagocytosis in the presence of human macrophages.

[0197] PBMCs were isolated from human blood and cultured in ATM-V medium for 7 days to differentiate monocytes into macrophages. These monocyte-derived macrophages (MDMs) became adhesive, and other cells were washed away. MDMs were scraped off and re-coated into 12-well plates, allowing them to adhere for 24 hours. The human tumor cell line Jurkat was selected as the target cell type due to 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, with CD47 antibody added at various concentrations. Target cells underwent phagocytosis for 3 hours. Unphagocytosed target cells were then washed away with PBS. The remaining macrophages were scraped off, stained with an antibody against CD14-conjugated macrophages to DyLite 649, and analyzed by flow cytometry. Phagocytosis was measured by gate selection of FL4-positive (CD14+) viable cells followed by assessment of the percentage of FL1 (CFSE+) positive cells. Starting with the baseline phagocytic rate of macrophages, the phagocytic rate of each sample was calculated, and the results are shown in [the table below]. 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 antibody-induced macrophage phagocytosis of tumor cells

[0199]

[0200]

[0201] Example 9: Blood Cell Agglutination Test

[0202] CD47 is highly expressed on the surface of red blood cells, therefore anti-CD47 monoclonal antibodies can specifically bind to CD47 on the surface of red blood cells. Since each antibody has two antigen-binding sites, anti-CD47 monoclonal antibodies may cause red blood cell agglutination. Monoclonal antibodies that cause red blood cell agglutination can cause side effects such as anemia and decreased red blood cell count in vivo. Whether anti-CD47 monoclonal antibodies cause red blood cell agglutination depends primarily on the recognition epitope of the monoclonal antibody on the CD47 molecule. This example analyzes the ability of anti-CD47 monoclonal antibodies to cause red blood cell agglutination using a classic hemagglutination assay. The antibodies CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 (all 40 μg / ml) obtained in the example were serially diluted with PBS, then mixed with the prepared 1% red blood cell suspension in a micro-hemagglutination reaction plate, and vortexed for 1 min on a micro-shaker to mix thoroughly. After incubating the micro-coagulation reaction plate at 25°C for 1 hour, the erythrocyte aggregation process was recorded photographically. The plate was then tilted at a 45° angle for several minutes, and the degree of erythrocyte aggregation was further determined by the rate of erythrocyte flow. The degree of erythrocyte aggregation was classified into four levels: 4. A uniform thin layer formed, with no flow upon tilting; 3. A uniform thin layer formed, with slight flow upon tilting; 2. A small thin layer formed, with relatively fast flow upon tilting; 1. No thin layer formed, and the flow rate was the same as the control well after tilting. Results are as follows: Figure 5 As shown, the human-mouse chimeric antibodies CD47-5A10-huIgG1-1B8, CD47-5G11-huIgG1-2E10, CD47-9C6-huIgG1-4B6, and CD47-11F1-huIgG1-10G6 did not induce or exhibit significant agglutination.

Claims

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

19.

2. The antibody as described in 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 as described in claim 1, characterized in that, The polynucleotide sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 32; the polynucleotide sequence of its light chain variable region is shown in SEQ ID NO:

36.

4. A polynucleotide molecule encoding the anti-CD47 monoclonal antibody as claimed in any one of claims 1-3.

5. An expression vector comprising the polynucleotide molecule of claim 4.

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

7. The use of the CD47 monoclonal antibody as described in any one of claims 1-3 in the preparation of antitumor therapeutic drugs or in the preparation of diagnostic tumor drugs; wherein the tumor is non-Hodgkin's lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, ovarian cancer, glioma, lung cancer, gastric cancer, cervical cancer or B-cell lymphoma.

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