Anti-human CD47 antibody and antigen-binding fragment thereof, preparation method and application

Abstract

The present invention provides an anti-human CD47 antibody or an antigen-binding fragment thereof, a preparation method and use. The present invention also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof and a vector comprising the isolated polynucleotide of the present invention. The present invention also provides the use of the antibody or antigen-binding fragment of the present invention in the preparation of a medicament for treating and / or preventing a disease that benefits from enhancing an immune response, wherein the disease is cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biological immunology technology, and specifically relates to an anti-human CD47 antibody and an antigen-binding fragment thereof that can specifically bind to human CD47 protein and block its binding to SIRPα, thereby blocking the inhibitory effect of CD47 on macrophages expressing SIRPα. The present invention also relates to a preparation method and use of the antibody and the antigen-binding fragment thereof. Background Art

[0002] CD47 is a 45-50kD transmembrane glycoprotein that belongs to the immunoglobulin (Ig) superfamily. Studies have found that CD47, which is highly expressed on tumor cells, binds to the ligand SIRPα on macrophages, causing SIRPα tyrosine phosphorylation, thereby sending inhibitory regulatory signals and inhibiting the phagocytosis of macrophages. Correspondingly, blocking this pathway can relieve the inhibitory effect of macrophages on the phagocytosis of tumor cells, enhance the body's immune response to tumor cells, and provide a new approach for tumor immunotherapy.

[0003] In tumor therapy targeting the CD47-SIRPα axis, the most important mechanism is the activation of macrophages, thereby enhancing the phagocytic effect of macrophages on tumor cells. Secondly, blocking CD47 may further recruit macrophages into tumor tissues, as well as cytokines and chemokines that recruit additional immune cells to tumor tissues, such as monocyte chemoattractant protein 3 (MCP-3). The secretion of these cytokines contributes to the efficacy of CD47 blockade therapy. Thirdly, therapy targeting CD47-SIRPα may also change the polarization state of macrophages in tumors. Macrophages can be divided into M1 (type I) and M2 (type II) according to their phenotype and functional activity. M1 macrophages can produce a large number of proinflammatory cytokines, mediate resistance to intracellular parasites and inhibit tumor growth; M2 macrophages produce fewer proinflammatory molecules, participate in tissue damage repair, angiogenesis and promote tumor growth. Studies have found that blocking CD47-SIRPα can significantly increase the number of M1 macrophages in the mouse microenvironment, while there is no significant increase in mouse M2 macrophages. Finally, other immune cells may also respond to CD47 blockade therapy. SIRPα is highly expressed on myeloid immune cells, so it may be a key regulator of the myeloid lineage. In mice, CD47 regulates antigen uptake by SIRPα+ dendritic cells, and using a syngeneic immune-competent tumor model, it was found that the therapeutic effect of CD47 blockade depends on dendritic cells. By stimulating antigen presentation by macrophages or dendritic cells, therapies targeting the CD47-SIRPα axis can promote adaptive immune responses in tumors.

[0004] CD47 is widely expressed on a variety of cells, especially on newly born red blood cells. Therefore, therapeutic antibodies targeting CD47 may cause anemia. On the other hand, the concentration of free anti-human CD47 antibodies in the body will also be greatly reduced due to the adsorption of anti-human CD47 antibodies by red blood cells.

[0005] Therefore, developing new therapeutic anti-human CD47 antibodies that have stronger selectivity for binding to CD47 protein on the surface of tumor cells and weaker binding to CD47 protein on the surface of red blood cells has become one of the goals of new therapeutic anti-human CD47 antibodies. Summary of the invention

[0006] The main purpose of the present invention is to provide an efficient and safe anti-human CD47 antibody. The present invention also provides a preparation method and use of the antibody. The anti-human CD47 antibody of the present invention has a stronger binding selectivity with the CD47 protein on the surface of tumor cells and a weaker binding with the CD47 protein on the surface of red blood cells, and treats various cancers by regulating human immune function.

[0007] In one aspect, the present invention provides an anti-human CD47 antibody or an antigen-binding fragment thereof, comprising one or more heavy chain complementary determining regions selected from the following:

[0008] VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1;

[0009] VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; and

[0010] VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:3.

[0011] According to the antibody or antigen-binding fragment thereof of the present invention, the anti-human CD47 antibody or antigen-binding fragment thereof further comprises one or more light chain complementary determining regions selected from the following:

[0012] VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:4;

[0013] VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:5; and

[0014] VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0015] According to the antibody or antigen-binding fragment thereof of the present invention, wherein the anti-human CD47 antibody or antigen-binding fragment thereof is a camelized single domain antibody, scFv, scFv dimer, BsFv, dsFv, dsFv2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds bifunctional antibody, nanobody, domain antibody or bivalent domain antibody;

[0016] Preferably, the anti-human CD47 antibody or its antigen-binding fragment further comprises a human heavy chain constant region and / or a human light chain constant region; more preferably, the human heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the human light chain constant region is selected from the light chain constant region of human IgG1, IgG2, IgG3 or IgG4; further preferably, the human heavy chain constant region is the heavy chain constant region of human IgG1, and the human light chain constant region is a κ chain.

[0017] In one embodiment according to the present invention, the heavy chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.

[0018] In one embodiment of the present invention, the light chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.

[0019] In one embodiment of the present invention, the heavy chain of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.

[0020] In one embodiment of the present invention, the light chain of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.

[0021] In one embodiment of the present invention, the heavy chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:13.

[0022] In one embodiment of the present invention, the light chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:14.

[0023] In one embodiment of the present invention, the heavy chain of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:15.

[0024] In one embodiment of the present invention, the light chain of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:16.

[0025] In another embodiment of the present invention, the heavy chain and light chain of the anti-human CD47 antibody or its antigen-binding fragment antibody are connected by a self-cleavage sequence, and the self-cleavage sequence is selected from a 2A peptide segment. The self-cleavage sequence 2A peptide segment is derived from foot-and-mouth disease viruses (FMDV). The 2A peptide segment or its similar peptide segment is composed of 18 to 22 amino acids, and the C-terminal sequence is highly conserved: -D X E X NPGP-. When the ribosome translates to this sequence, it "jumps" to achieve automatic cleavage (self-cleavage) without the need for protease. Self-cleavage occurs between the C-terminal GP residues. (See "Research Progress in Construction of Multi-Gene Expression Vectors Based on 2A Peptide Strategy", China Biotechnology, 2013, 33(1): 104-108). Preferably, the 2A peptide segment is selected from P2A, T2A or E2A; more preferably, the self-cleavage sequence is P2A.

[0026] In another aspect, the present invention also provides an isolated polynucleotide encoding the above-mentioned antibody or antigen-binding fragment thereof;

[0027] Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 11;

[0028] Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 12;

[0029] Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 17;

[0030] Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:18.

[0031] In yet another aspect, the present invention also provides a vector comprising the isolated polynucleotide of the present invention.

[0032] In yet another aspect, the present invention further provides a host cell comprising the vector of the present invention. Preferably, the host cell is a mammalian cell, more preferably a cell of a human, mouse, sheep, horse, dog or cat, and even more preferably a Chinese hamster ovary cell.

[0033] Furthermore, the present invention also provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell under conditions capable of expressing the polynucleotide of the present invention.

[0034] Furthermore, the present invention also provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention.

[0035] Furthermore, the present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0036] Furthermore, the present invention also provides use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicament or a cell for cell therapy, wherein the medicament or the cell is used to prevent and / or treat a disease that benefits from enhancing an immune response.

[0037] In one embodiment according to the present invention, the disease is cancer, preferably, the disease is lymphoma with high CD47 expression, such as acute myeloid leukemia and myelodysplastic syndrome.

[0038] Compared with the prior art, the anti-human CD47 antibody of the present invention has stronger binding selectivity to the CD47 protein on the surface of tumor cells, weaker binding to the CD47 protein on the surface of red blood cells, and has no agglutination effect on red blood cells, which is one of the goals of the new therapeutic anti-human CD47 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0040] Figure 1 The figure shows the detection of the binding between the anti-human CD47 antibody of the present invention and the CD47 protein using the ELISA method.

[0041] FIG2 shows the binding of the anti-human CD47 antibody of the present invention to human / mouse tumor cells that highly express human / mouse CD47. Figure 2A MKN-45, A2780, and SW620 are CD47-positive cells; Figure 2B It was shown that the antibodies UM03-L4 and UM03-C4 of the present invention strongly bind to MKN-45, A2780 and SW620; Figure 2C The anti-human CD47 antibody of the present invention was shown to bind to the mouse tumor cell MC-38 which highly expresses mouse CD47, and it was found that UM03-L4 only weakly bound to mouse CD47.

[0042] Figure 3 This indicates that the anti-human CD47 antibody of the present invention can inhibit the binding of CHO-CD47 cells to SIRPα protein.

[0043] FIG4 shows the binding of the anti-human CD47 antibody of the present invention to human erythrocytes and the agglutination effect on human erythrocytes. Figure 4A It shows that the anti-human CD47 antibodies UM03-L4, UM03-C4 and the positive control antibody of the present invention bind to human erythrocytes; Figure 4BThis indicates that the anti-human CD47 antibodies UM03-L4 and UM03-C4 of the present invention have almost no agglutination effect on human erythrocytes, while the positive control antibody has a stronger agglutination effect on human erythrocytes.

[0044] FIG5 shows the binding of the anti-human CD47 chimeric antibody of the present invention to monkey erythrocytes and the agglutination effect on monkey erythrocytes. Figure 5A It shows that the anti-human CD47 antibody UM03-L4 of the present invention and the positive control antibody bind to monkey red blood cells; Figure 5B This indicates that the anti-human CD47 antibody UM03-L4 of the present invention has almost no agglutination effect on monkey erythrocytes, while the positive control antibody has a stronger agglutination effect on monkey erythrocytes.

[0045] Figure 6 This is the antigen-antibody affinity test of the anti-human CD47 antibody of the present invention.

[0046] Figure 7 The anti-tumor effect of the anti-human CD47 antibody of the present invention in mice.

[0047] Figure 8 This is a drug metabolism test of the anti-human CD47 antibody of the present invention in cynomolgus monkeys. DETAILED DESCRIPTION

[0048] The following description of the application is only for illustrating the various embodiments of the application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive a variety of equivalents, variations and modifications without departing from the scope of the application, and it should be understood that such equivalent embodiments are included within the scope of the invention. All documents cited in this application, including publications, patents and patent applications, are incorporated by reference in their entirety.

[0049] definition

[0050] The term "antibody" in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that can bind to a specific antigen. A natural complete antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and the first, second and third constant regions; each light chain consists of a variable region and a constant region. The heavy chains of mammals can be divided into α, δ, ε, γ and μ, and the light chains of mammals can be divided into λ or κ. The antibody is "Y" shaped, and the neck of the Y-shaped structure is composed of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y"-shaped structure includes the variable region and the first constant region of one of the heavy chains, which is combined with the variable region and constant region of one light chain. The variable regions of the light chain and the heavy chain determine the binding of the antigen. The variable region of each chain contains three hypervariable regions, called complementary determining regions (CDRs). The CDR of the light chain (L) comprises LCDR1, LCDR2, LCDR3, and the CDR of the heavy chain (H) comprises HCDR1, HCDR2, HCDR3. The CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature. (AI-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273 (4): 927 (1997); Chothia, C. et al., J. Mol. Biol., 186 (3): 651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196: 901 (1987); Chothia, C. et al., Nature, 342 (6252): 877-83 (1989); Kabat, EA et al., National Institutes of Health, Bethesda, Md. (1991)). Among them, the three CDRs are separated by flanking continuous portions called framework regions (FRs), which are more highly conserved than CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not related to antigen binding, but have a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region. Depending on whether they contain α, δ, ε, γ and μ heavy chains, antibodies can be divided into five major categories or isoforms: IgA, IgD, IgE, IgG and IgM. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0051] The term "antigen binding fragment" as used herein refers to an antibody fragment formed by an antibody portion containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have a complete antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bivalent single-chain antibodies (BsFv), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen binding fragment may bind to the same antigen as the parent antibody. In certain embodiments, an antigen binding fragment may contain one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0052] The "Fab" fragment of an antibody refers to the portion of the antibody molecule that is composed of a light chain (including the variable region and the constant region) and the variable region and part of the constant region of a heavy chain linked together by disulfide bonds.

[0053] "Fab'" fragment refers to the Fab fragment that includes part of the hinge region.

[0054] "F(ab')2" refers to a dimer of Fab.

[0055] The Fc region of an antibody is responsible for a variety of different effector functions such as ADCC and CDC, but does not participate in antigen binding.

[0056] The "Fv" segment of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site. The Fv fragment consists of the variable region of one light chain and one heavy chain.

[0057] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region directly linked to a heavy chain variable region or connected through a peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85: 5879 (1988)).

[0058] "Single-chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc portion of an antibody.

[0059] "Camelized single domain antibody", "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" all refer to antibodies containing two VH domains but no light chain (Riechmann L. and Muyldermans S., J Immunol Methods. 231 (1-2): 25-38 (1999); Muyldermans S., J Biotechnol. 74 (4): 277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries and llamas). Despite the lack of light chains, camelized antibodies have confirmed full antigen binding function (Hamers Casterman C. et al., Nature 363(6428): 446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae: a case of evolutionary innovation, Immunogenetics. 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. 109(1): 93-101 (2003)). The variable region (VH domain) of the heavy chain antibody is the smallest known antigen binding unit produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. 21(13): 3490-8. Epub (2007)).

[0060] "Nanobody" refers to an antibody fragment that consists of a VH domain from a heavy chain antibody and two constant regions CH2 and CH3.

[0061] "Diabodies" include small antibody fragments with two antigen binding sites, wherein the fragment contains a VH domain and a VL domain connected on the same polypeptide chain (see, Holliger P. et al., Proc Natl Acad Sci USA. 90 (14): 6444-8 (1993); EP404097; WO93 / 11161). The linker between the two domains is very short, so that the two domains on the same chain cannot pair with each other, thereby forcing the two domains to pair with the complementary domains of another chain to form two antibody binding sites. The two antibody binding sites can target and bind to the same or different antigens (or antigenic epitopes).

[0062] "Domain antibodies" refer to antibody fragments that contain only one heavy chain variable region or one light chain variable region. In some cases, two or more VH domains are covalently linked by a polypeptide linker to form a bivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0063] In certain embodiments, "(dsFv)2" contains three peptide chains: two VH genes are connected by a polypeptide linker and are bound to two VL groups by disulfide bonds.

[0064] In certain embodiments, a "bispecific ds bifunctional antibody" contains VL1-VH2 (connected by a polypeptide linker) and VH1-VL2 (also connected by a polypeptide linker), and the two are linked by a disulfide bond between VH1 and VL1.

[0065] "Bispeci?c dsFv" or "dsFv-dsFv" contains three polypeptide chains: a VH1-VH2 group, wherein the heavy chains of the two are connected by a polypeptide linker (e.g., a long flexible linker) and are bound to the VL1 and VL2 groups, respectively, by disulfide bonds, and each pair of heavy and light chains paired by disulfide bonds has different antigenic specificity.

[0066] In some embodiments, the "scFv dimer" is a bivalent bifunctional antibody or a bivalent single-chain antibody (BsFv), containing two dimerized VH-VL (connected by a polypeptide linker) groups, wherein the VH of the two groups cooperates with the VL of the other group to form two binding sites, and the two binding sites can target and bind to the same antigen (or antigenic epitope) or different antigens (or antigenic epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody, containing VH-VL groups connected to each other. L1 -V H2 (connected by a polypeptide linker) and V H1 -V L2 (connected by a polypeptide linker), wherein V H1 and V L1 Collaboration, V H2 and V L2 Collaborative, and each collaborating pair has a different antigenic specificity.

[0067] The term "fully human" as used in this application, when used for an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human immune cell, or derived from a non-human source such as a transgenic non-human animal using a human antibody library, or other sequences encoding human antibodies. In certain embodiments, a fully human antibody does not contain amino acid residues (particularly antigen-binding residues) derived from a non-human antibody.

[0068] The term "humanization" used in this application, when used for antibodies or antigen-binding fragments, refers to antibodies or antigen-binding fragments including CDRs derived from non-human animals, FR regions derived from humans, and constant regions (when applicable) derived from humans. Because humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as therapeutic agents for people in certain embodiments. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment is substantially entirely composed of human sequences except that the CDR sequence is non-human. In some embodiments, the FR region derived from humans may include the same amino acid sequence as the human antibody from which it comes, or it may include some amino acid changes, for example, no more than 10,9,8,7,6,5,4,3,2 or 1 amino acid changes. In some embodiments, the amino acid changes may be present only in the heavy chain FR region, only in the light chain FR region, or simultaneously in two chains. In some preferred embodiments, the humanized antibody includes human FR1-3 and human JH and JK.

[0069] The term "chimeric" used in this application refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or a light chain derived from one species, and the remainder of the heavy chain and / or the light chain derived from a different species. In an illustrative example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal such as a mouse.

[0070] The term "CD47" refers to a ligand that binds to SIRPα. Others are named IAP, MER6, or OA3, etc. It has a molecular weight of 35.2 kDa and is stored in SwissProt with accession number Q08722.

[0071] "Specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application specifically bind to human and / or monkey CD47, and their binding affinity (K D )≤10 -6 M. K in this application D It is the ratio of the dissociation rate to the association rate (k off / k on ), can be determined by surface plasmon resonance methods, for example using an instrument such as Biacore.

[0072] "UM03-L4" used in the present application refers to a human-mouse chimeric antibody having a heavy chain as shown in SEQ ID NO: 9, a light chain as shown in SEQ ID NO: 10, and heavy and light chain constant regions of human IgG4 and kappa chains, respectively.

[0073] "UM03-C4" used in the present application refers to a humanized antibody having a heavy chain as shown in SEQ ID NO: 15, a light chain as shown in SEQ ID NO: 16, and heavy and light chain constant regions of human IgG4 and κ chain, respectively.

[0074] In the present application, when "conservative substitution" is used for an amino acid sequence, it means that an amino acid residue is substituted with another amino acid residue having a side chain of similar physicochemical properties. For example, conservative substitution can be performed between hydrophobic side chain amino acid residues (e.g., Met, Ala, VaL, Leu and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys and Arg) or between aromatic side chain residues (e.g., Trp, Tyr and Phe). It is known in the art that conservative substitution usually does not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.

[0075] When "percent sequence identity" is used for amino acid sequences (or nucleic acid sequences), it means that after performing sequence alignment and introducing intervals when necessary to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence, the amino acid (or nucleic acid) residues identical to the reference sequence account for the percentage of amino acid (or nucleic acid) residues in the candidate sequence. Conservative substitutions of the amino acid residues may or may not be considered to be identical residues. Sequences may be aligned by tools disclosed in the art to determine the percentage sequence identity of amino acid (or nucleic acid) sequences. Those skilled in the art may use the default parameters of the tools or appropriately adjust the parameters according to the needs of the alignment, for example, by selecting a suitable algorithm.

[0076] The “T cells” used in the present application include CD4+T cells, CD8+T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

[0077] As used herein, "effector function" refers to the biological activity of the Fc region of an antibody in combination with its effector, such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of an antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the Fc region of an antibody to Fc receptors on effector cells, and phagocytosis.

[0078] "Cancer" or "cancerous condition" in this application refers to any medical condition mediated by tumor or malignant cell growth, proliferation or metastasis, and causing solid tumors and non-solid tumors such as leukemia. "Tumor" in the present invention refers to the solid material of tumor and / or malignant cells.

[0079] "Treatment" or "therapy" of a condition includes preventing or alleviating the condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing full or partial reversal of a condition, curing a condition, or some combination thereof. "Treatment" or "therapy" for cancer may refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination thereof. "Treatment" or "therapy" for tumors includes eliminating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0080] "Isolated" substances have been artificially altered from their natural state. If an "isolated" substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not isolated, but it can be considered "isolated" if the substance with which it coexists in nature is sufficiently separated and present in a sufficiently pure state. In certain embodiments, the purity of antibodies and antigen-binding fragments is at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, as determined by electrophoresis methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (such as ion exchange chromatography or reversed-phase HPLC).

[0081] In the present invention, "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted and the protein can be expressed. The vector can be used to transform, transduce or transfect a host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40)). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including but not limited to viral particles, liposomes or protein shells.

[0082] In the present invention, "host cell" refers to a cell into which an exogenous polynucleotide and / or vector is introduced.

[0083] The term "therapeutically effective amount" or "effective dose" in the present invention refers to the dose or concentration of a drug that is effective in treating a disease. For example, for the use of the antibody or antigen-binding fragment thereof disclosed in the present invention, the therapeutically effective amount is the dose or concentration at which the antibody or antigen-binding compound can eliminate all or part of a tumor, inhibit or slow tumor growth, inhibit tumor cell metastasis, alleviate any symptoms or markers associated with a tumor or cancer condition, prevent or delay the development of a tumor or cancer condition, inhibit or eliminate viruses or virus-infected cells, or some combination thereof.

[0084] "Pharmaceutically acceptable" means that the referenced carriers, solvents, diluents, excipients and / or salts are generally chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0085] "Cell therapy" refers to a treatment method in which biologically active cell materials are transplanted into a patient's body to produce the desired therapeutic effect. In a specific embodiment, the cell therapy is performed by modifying specific cells so that the cells express antibodies or antigen-binding fragments thereof, with the purpose of increasing the therapeutic effect of the cells or enhancing their ability to specifically act on specific tissue sites.

[0086] Regarding the anti-human CD47 antibody of the present invention

[0087] In certain embodiments, the present application provides an exemplary anti-human CD47 antibody UM03-C4.

[0088] Those skilled in the art will appreciate that the aforementioned CDR sequences can be modified to include substitutions of one or more amino acids, thereby obtaining improved biological activity, such as improved binding affinity to human CD47. For example, a library of antibody variants (e.g., Fab or FcFv variants) can be produced and expressed using phage display technology, followed by screening for antibodies with affinity to human CD47. In another example, computer software can be used to simulate the binding of the antibody to human CD47 and identify the amino acid residues on the antibody that form the binding interface. Substitution of these residues can be avoided to prevent reduced binding affinity, or these residues can be targeted for substitution to form a stronger binding. In certain embodiments, at least one (or all) substitutions in the CDR sequence are conservative substitutions.

[0089] In certain embodiments, the antibody or antigen-binding fragment comprises one or more CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences of SEQ ID NO: 1-6, and at the same time retains a binding affinity to human CD47 that is similar to or even higher than that of its parent antibody. The parent antibody has substantially the same sequence, but its corresponding CDR sequence has 100% sequence identity to the sequences listed in SEQ ID NO: 1-6.

[0090] In some embodiments, the antibodies or antigen-binding fragments described herein can be used at ≤10 -7 The binding affinity (K D ) binds specifically to human CD47 as measured by surface plasmon resonance. Binding affinity values ​​can be expressed as K D The value is expressed by the ratio of the dissociation rate to the association rate when the binding of the antigen and the antigen-binding molecule reaches equilibrium (k off / k on ) is calculated. The antigen binding affinity (e.g., K D ) may be suitably determined by any suitable method known in the art, including, for example, plasmon resonance binding using an instrument such as Biacore.

[0091] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to human CD47 at an EC50 (i.e., half binding concentration) of 10 ng / mL-10 μg / mL. The binding of the antibody or antigen-binding fragment to human CD47 can be determined by methods known in the art, such as sandwich methods such as ELISA, Western blotting, FACS or other binding assays. In an illustrative example, the antibody to be tested (i.e., the primary antibody) is bound to immobilized human CD47 or cells expressing human CD47, and then the unbound antibody is washed away, and a labeled secondary antibody is introduced, which can bind to the primary antibody and thus detect the bound secondary antibody. When immobilized CD47 is used, the detection can be performed on an ELISA plate, or when cells expressing human CD47 are used, the detection can be performed using FACS analysis.

[0092] The antibody is specific for human CD47. In certain embodiments, the antibody does not bind to murine CD47, but binds with similar binding affinity to monkey CD47 and to human CD47.

[0093] In some embodiments, the antibodies have a constant region of the IgG4 isotype, which has reduced or eliminated effector functions. Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing CD47. Some normal cells can express CD47. In order to avoid potential undesirable toxicity to these normal cells, certain embodiments of the antibodies of the present invention have reduced or even eliminated effector functions. There are many known tests for evaluating ADCC or CDC activity, such as Fc receptor binding assays, complement Clq binding assays, and cell lysis assays, which can be easily selected by those skilled in the art. Without wishing to be bound by theory, it is believed that antibodies with reduced or eliminated effector functions such as ADCC and CDC will not cause or minimize cytotoxicity to cells expressing CD47 (such as those normal cells), thereby avoiding undesirable side effects.

[0094] In some embodiments, the antibodies or antigen-binding fragments described herein have reduced side effects. For example, the anti-anti-human CD47 antibodies and antigen-binding fragments thereof may have fully human IgG sequences, so their immunogenicity is lower than that of humanized antibodies. For another example, the antibodies and antigen-binding fragments thereof may have IgG2 or IgG4 forms to eliminate ADCC and CDC.

[0095] In some embodiments, the advantage of the antibodies described herein is that they can be used in combination with immunogenic substances, such as tumor cells, purified tumor antigens, cells transfected with encoded immunostimulatory factors, and tumor vaccines. In addition, the anti-human CD47 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemotherapy and radiotherapy, target-based small molecule therapies, and other emerging immune checkpoint regulator therapies. In some embodiments, the antibodies and antigen-binding fragments thereof can be used as the basic molecules of antibody-drug conjugates, bispecific or multivalent antibodies.

[0096] In some embodiments, the antibodies and antigen-binding fragments thereof described in the present application are camelized single chain domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragments, Fab, Fab', F(ab')2, dsdiabodies, nanobodies, domain antibodies or bivalent domain antibodies.

[0097] In some embodiments, the antibodies described herein include an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region includes a heavy chain and / or light chain constant region. The heavy chain constant region includes a CH1, CH1-CH2 or CH1-CH3 region. In some embodiments, the immunoglobulin constant region may further include one or more modifications to obtain the desired properties. For example, the constant region may be modified to reduce or eliminate one or more effector functions to enhance FcRn receptor binding or introduce one or more cysteine ​​residues.

[0098] In certain embodiments, the antibody and antigen-binding fragment thereof further comprise a conjugate. It is conceivable that the antibody or antigen-binding fragment thereof in the present invention can be connected to a variety of conjugates (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York (1989)). These conjugates can be connected to the antibody or antigen-binding substance by other means such as covalent binding, affinity binding, embedding, coordinate binding, complexation, binding, mixing or addition. In certain embodiments, the antibodies and antigen-binding fragments disclosed in the present invention can be engineered to contain specific sites other than the epitope binding portion, which can be used to bind one or more conjugates. For example, such sites can contain one or more reactive amino acid residues, such as cysteine ​​residues and histidine residues, for assisting covalent connection with the conjugate. In certain embodiments, the antibody can be indirectly connected to the conjugate, or connected through another conjugate. For example, the antibody or antigen-binding fragment thereof can be bound to biotin and then indirectly bound to a second conjugate, which is connected to avidin. The conjugate can be a detectable label, a pharmacokinetic modification part, a purification part or a cytotoxic part. The example of detectable label can include fluorescent label (such as fluorescein, rhodamine, dansyl, phycoerythrin or Texas Red), enzyme substrate marker (such as horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase or beta-D galactosidase), stable isotope or radioisotope, chromophore part, digoxin, biotin / avidin, DNA molecule or gold to detect. In some embodiments, the conjugate can be a pharmacokinetic modification part such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include such as carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol copolymers, etc. In some embodiments, the conjugate can be a purification part such as magnetic beads. "Cytotoxic part" can be any reagent that is harmful to cells or may damage or kill cells.Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenipogan, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycinolone, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-mercaptoguanine, cytarabine, 5-fluorouracil, 1,2-dopamine ... uracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and ampicillin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0099] Polynucleotides and recombinant methods

[0100] Using genetic engineering techniques known in the art, the amino acid sequences of the antibodies and antigen-binding fragments thereof described in the present application can be converted into corresponding DNA coding sequences. Due to the degeneracy of the genetic code, the converted DNA sequences can be completely consistent, while the encoded protein sequence remains unchanged.

[0101] Using recombinant techniques known in the art, a vector comprising a polynucleotide encoding the antibody and its antigen-binding fragment can be introduced into a host cell for cloning (amplifying DNA) or gene expression. In another embodiment, the antibody and its antigen-binding fragment can be prepared by homologous recombination methods known in the art. A variety of vectors are available. Vector components generally include, but are not limited to, two or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancing sequence, a promoter (e.g., SV40, CMV, EF-1a), and a transcription termination sequence.

[0102] In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and will include plasmids such as but not limited to pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2 and other vectors available from the laboratory or commercially available. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0103] The vector comprising the polynucleotide encoding the antibody and its antigen-binding fragment can be introduced into the host cell for cloning or gene expression. The host cell suitable for cloning or expressing the DNA in the vector in the present invention is a prokaryotic cell, yeast or the above-mentioned higher eukaryotic cell. Prokaryotic cells suitable for the purposes of the present invention include true bacteria such as, Gram-negative bacteria or Gram-positive bacteria, for example, Enterobacteriaceae, such as, Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as, Salmonella typhimurium, Serratia, such as, Serratia marcescens, and Shigella, and Bacillus such as, Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as, Peptidobacterium chlororaphis and Streptomyces.

[0104] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cells to clone or express vectors encoding antibodies or antigen-binding fragments thereof. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly used and suitable for use in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, Kluyveromyces lactis, Kluyveromyces fragilis (ATCC12424), Kluyveromyces bulgaricus (ATCC16045), Kluyveromyces weigeni (ATCC24178), Kluyveromyces krusonii (ATCC56500), Kluyveromyces drosophila (ATCC36906), Kluyveromyces thermotolerans and Kluyveromyces marxianus: Yarrowia lipolytica (EP402226); Pichia pastoris (EP183,070); Candida: Trichoderma reesei (EP244234); Neurospora; western Schwann yeast, such as western Schwann yeast; and filamentous fungi, such as Neurospora, Penicillium, Tolypoxetine and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.

[0105] The host cells suitable for expressing glycosylated antibodies or antigen-binding fragments thereof provided in the present invention are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. A variety of baculoviral strains and variants thereof and corresponding permissive insect host cells have been found, from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the Bm-5 variant of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0106] However, of greatest interest are spinal cells, and their cultivation (tissue culture) has become routine. Examples of mammalian host cells that can be used include SV40-transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al., Gen Virol. 36: 59 (1977)); baby hamster kidney cells (B blood, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse testicular supporting cells (TM4, Ma ther, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (HepG2). In certain preferred embodiments, the host cell is a 293F cell.

[0107] Host cells are transformed with the above-mentioned expression or cloning vectors capable of producing the antibodies and antigen-binding fragments thereof, and cultured in a conventional nutrient medium which has been modified to be suitable for inducing promoters, selecting transformed cells or amplifying genes encoding target sequences.

[0108] The host cells used to produce the antibodies and antigen-binding fragments thereof in the present invention can be cultured in a variety of culture media known in the art. The culture media may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the culture media, such as temperature, pH value and the like, are the conditions previously used for selecting host cells for expression and are well known to those of ordinary skill in the art.

[0109] When recombinant techniques are used, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, the particle debris of the host cell or cleavage fragments is first removed, for example, by centrifugation or ultrasound. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is dissolved in the presence of uranyl acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes or more. Cell debris is removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is usually first concentrated using a commercially available protein concentration filter, such as an lAmicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF can be added in any of the aforementioned steps to inhibit protein degradation, as well as antibiotics to prevent the growth of accidental contaminants.

[0110] The antibodies produced from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography columns, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. The type of antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein A is suitable as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is suitable for all murine isoforms and human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986)). Agarose is the most commonly used matrix for attachment of affinity ligands, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrene)benzene can achieve faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.TM resin (JT Baker, Phillipsburg, NJ). Other protein purification techniques may also be determined based on the desired antibody, such as fractionation in an ion exchange column, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resin (e.g., polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0111] Following any preliminary purification steps, the mixture containing the antibody of interest and impurities can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0 to 0.25 M salt concentration).

[0112] Reagent test kit

[0113] The present application provides a kit comprising the antibody or antigen-binding fragment thereof. In some embodiments, the kit is used to detect the presence or level of CD47 in a biological sample. The biological sample may include cells or tissues.

[0114] In some embodiments, the kit includes an antibody or antigen-binding fragment thereof conjugated to a detectable label. In some embodiments, the kit includes an unlabeled antibody and further includes a secondary antibody capable of binding to the unlabeled antibody with a label. The kit may further include instructions for use and packaging that separates each component in the kit.

[0115] In some embodiments, the anti-human CD47 antibody or antigen-binding fragment thereof is connected to a substrate or instrument for sandwich assays such as ELISA or immunochromatographic assays. Applicable substrates or instruments may be, for example, microtiter plates and test strips.

[0116] Pharmaceutical compositions and methods of treatment

[0117] The present application further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

[0118] The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in the present application may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending / dispersing agents, integrators, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components well known in the art, or various combinations thereof.

[0119] Applicable components may include, for example, antioxidants, fillers, adhesives, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Applicable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl gallate. Including one or more antioxidants such as methionine in a composition containing an antibody disclosed in the present invention or its antigen-binding fragment can reduce the oxidation of the antibody or its antigen-binding fragment. The reduction of oxidation can prevent or reduce the reduction of binding affinity, thereby improving antibody stability and extending shelf life.

[0120] Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous media such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinyl pyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), integrating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents as carriers may be added to the pharmaceutical composition in multiple dose containers and include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, glucose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

[0121] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, saccharin sodium, cellulose, magnesium carbonate, etc.

[0122] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, a liquid solvent, a suspending agent, an emulsifier, or a solid form suitable for producing a liquid solvent, a suspending agent, or an emulsifier. Injectable formulations can include sterile and / or pyrogen-free solutions, sterile dried solubles that are combined with solvents before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions for injection, sterile dried insoluble products that are combined with media before use, and sterile and / or pyrogen-free emulsions. The solvent can be an aqueous phase or a non-aqueous phase.

[0123] In certain embodiments, the unit dose of the injection preparation is packaged in an ampoule, a tube or a syringe with a needle. It is well known in the art that all preparations for injection should be sterile and pyrogen-free.

[0124] In certain embodiments, a sterile lyophilized powder can be prepared by dissolving the antibody or antigen-binding fragment thereof disclosed in the present application in a suitable solvent. The solvent may contain a component that can improve the stability of the powder or a reconstituted solution obtained from the powder, or improve other pharmacological components of the powder or the reconstituted solution. Applicable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, brown sugar or other applicable substances. The solvent may contain a buffer, such as a citrate buffer, a sodium phosphate or potassium phosphate buffer or other buffer known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. The solution is subsequently filtered and sterilized under standard conditions known in the art, and then lyophilized to obtain an ideal preparation. In one embodiment, the resulting solvent is dispensed into small tubes and lyophilized. Each small tube can accommodate a single dose or multiple doses of the anti-human CD47 antibody or its antigen-binding fragment or its composition. Each vial may be filled with slightly more than required for each dose or multiple doses (eg, 10% overdose) to ensure accurate sampling and dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0125] The lyophilized powder is reconstituted with water for injection to obtain a preparation for injection. In one embodiment, the lyophilized powder can be added to sterile pyrogen-free water or other applicable liquid carriers for reconstitution. The exact amount is determined by the selected therapy and can be determined based on empirical values.

[0126] Also provided are methods of treatment comprising administering a therapeutically effective amount of an antibody described herein to a subject in need thereof.

[0127] The therapeutically effective dose of the antibodies provided in this application depends on a variety of factors known in the art, such as body weight, age, past medical history, current treatment, the health status of the subject and the potential for cross infection, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. Those skilled in the art (e.g., doctors or veterinarians) may reduce or increase the dose in proportion to these or other conditions or requirements.

[0128] In certain embodiments, the antibodies provided by the present invention can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the antibody is administered at a dosage of about 50 mg / kg or less, and in certain embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less or 0.1 mg / kg or less. A certain specific dose can be administered at multiple intervals, such as once a day, twice a day or more, twice a month or more, once a week, once every two weeks, once every three weeks, once a month or once every two months or more. In certain embodiments, the dosage can vary with the course of treatment. For example, in certain embodiments, the initial dosage can be higher than the subsequent dosage. In certain embodiments, the dosage is adjusted during the course of treatment according to the response of the subject being administered.

[0129] Dosage regimens may be adjusted to achieve the optimum response (eg, therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over a period of time.

[0130] The antibodies disclosed in the present invention can be administered by administration methods known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection) or non-injection administration (e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration).

[0131] In certain embodiments, the antibodies can be used to treat disorders related to their molecular mechanisms, including tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycoses, fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

[0132] How to use

[0133] The present application further provides methods of using the antibodies.

[0134] In some embodiments, the present application provides a method for treating a condition or disorder associated with the antibody mechanism in an individual, comprising administering a therapeutically effective amount of the antibody described herein.

[0135] The antibodies disclosed in the present invention can be administered alone or in combination with one or more other treatments or substances. For example, the antibodies or antigen-binding fragments thereof disclosed in the present invention can be used in combination with chemotherapy, radiotherapy, cancer treatment surgery (such as tumor resection), antiviral drugs, one or more antiemetics or other chemotherapy-induced complications, or any other therapeutic substances for cancer or viruses. In some such embodiments, when the antibodies disclosed in the present invention are used in combination with one or more therapeutic substances, they can be administered simultaneously with the one or more therapeutic substances. In some such embodiments, the antibodies can be administered simultaneously as part of the same pharmaceutical composition. However, antibodies "used in combination" with other therapeutic substances do not need to be administered simultaneously or in the same composition as the therapeutic substance. The meaning of "used in combination" in the present invention also includes that antibodies administered before or after another therapeutic substance are also considered to be "used in combination" with the therapeutic substance, even if the antibody and the second substance are administered by different administration methods. Where possible, other therapeutic substances used in combination with the antibodies disclosed herein can be administered according to the methods in the product instructions of the other therapeutic substances, or according to Physicians' Desk Reference, 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or according to other methods known in the art.

[0136] In some embodiments, the therapeutic substance can induce or enhance an immune response to cancer. For example, tumor vaccines can be used to induce an immune response to certain tumors or cancers. Cytokine therapy can be used to improve the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon α, β and γ, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF, interleukins such as 1L-L, 1L-1a, 1L-2, 1L3, 1L-4, 1L-5, 1L-6, 1L-7, 1L-8, 1L-9, 1L-10, 1L-11 and 1L-12, and tumor necrosis factors such as TNF-α and TNF-β. Reagents that inactivate immunosuppressive targets can also be used, such as PD-1 antibodies, TGF-β inhibitors, IL-10 inhibitors and Fas ligand inhibitors. Another group of agents includes those that activate an immune response against tumor or cancer cells, for example, those that enhance T cell activation (such as T cell co-stimulatory signaling pathways such as CTLA-4, ICOS, OX40, 4-1BB, etc. pathways), and those that enhance dendritic cell function and antigen presentation.

[0137] The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All of the following specific compositions, materials and methods, in whole or in part, are within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the present invention, but are only intended to illustrate that specific embodiments are within the scope of the present invention. Those skilled in the art may develop equivalent compositions, materials and methods without adding creativity and without departing from the scope of the present invention. It should be understood that the various modifications made to the method of the present invention may still be included within the scope of the present invention. The inventor intends to include such changes within the scope of the present invention.

[0138] Example 1: Obtaining mouse anti-human CD47 monoclonal antibody

[0139] The inventors of the present application constructed a CHO cell line that overexpressed human CD47 protein and used it to immunize mice. The spleen cells of the immunized mice were taken and hybridoma cells were fused with SP2 / 0-AG14 cells, and an appropriate amount of the fused cells were plated on a 96-well plate. The supernatant of each well was taken on the 10th day after fusion, and the binding activity of the mouse antibody secreted by the hybridoma cells to human CD47 (see Example 4 for the method) and its inhibitory activity on SIRPα / CD47 binding (see Example 6 for the method) were detected by ELISA, and a series of hybridoma cells with high activity were obtained. The hybridoma cell with the best activity was selected, and the heavy chain variable region cDNA sequence and light chain variable region cDNA sequence corresponding to the secreted antibody were obtained by sequencing. The amino acid sequence of the heavy chain variable region encoded by it is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region encoded by it is shown in SEQ ID NO: 8. The heavy chain variable region and light chain variable region of the mouse antibody were connected to the constant region of the human IgG4 heavy chain and the constant region of the κ chain, respectively, to obtain the human-mouse chimeric antibody UM03-L4, whose heavy chain sequence is shown in SEQ ID NO: 9 and the light chain sequence is shown in SEQ ID NO: 10.

[0140] The amino acid sequence of the heavy chain variable region of the hybridoma cell is shown below;

[0141] SEQ ID NO:7:

[0142] EVKLVESGGDLVQPGGSRKLSCAASGFTFSDYGMAWIRQAPGKGPEWIAFITNLASSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARAGDYRSFPYWGQGTPVTVSA

[0143] The amino acid sequence of the light chain variable region of the hybridoma cell is shown below;

[0144] SEQ ID NO:8:

[0145] EILLTQSPAIIAASPGEKVTITCSASSSVNYVNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSTFPPYTFGGGTKLEIK

[0146] The heavy chain amino acid sequence of the human-mouse chimeric antibody UM03-L4 is shown below;

[0147] SEQ ID NO:9:

[0148] EVKLVESGGDLVQPGGSRKLSCAASGFTFSDYGMAWIRQAPGKGPEWIAFITNLASSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARAGDYRSFPYWGQG TPVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0149] The light chain amino acid sequence of the human-mouse chimeric antibody UM03-L4 is shown below;

[0150] SEQ ID NO:10:

[0151] EILLTQSPAIIAASPGEKVTITCSASSSVNYVNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSTFPPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0152] Example 2: Humanization of antibodies

[0153] The sequences of the heavy chain variable region and light chain variable region of the mouse antibody obtained in Example 1 were analyzed, and the heavy chain complementary determining region CDR was obtained to be the following sequence: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the light chain complementary determining region was obtained to be the following sequence: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0154] SEQ ID NO: 1GFTFSDYGMA

[0155] SEQ ID NO:2FITNLASSIYYADTVTG

[0156] SEQ ID NO: 3ARAGDYRSFPY

[0157] SEQ ID NO: 4SASSSVNYVN

[0158] SEQ ID NO: 5GISNLAS SEQ ID NO: 6QQRSTFPP

[0159] The human germline antibody sequence database (IGMT) was searched to obtain human germline antibody sequences with high homology to the heavy / light chain variable regions of mouse antibodies, and their framework regions were combined with the CDRs of the above-mentioned mouse antibodies, i.e., the so-called CDR grafting. At the same time, some amino acids in the framework region were back mutated, and finally the humanized antibody UM03-C4 was obtained. Its heavy chain sequence is shown in SEQ ID NO: 15, and its light chain sequence is shown in SEQ ID NO: 16.

[0160] The heavy chain variable region sequence of the humanized antibody UM03-C4 is as follows:

[0161] SEQ ID NO:13

[0162] QVQLVESGGGVVQPGGSLRLSCAASGFTFSDYGMAWIRQAPGKGPEWIAFITNLASSIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGDYRSFPYWGQGTLVTVSA

[0163] The light chain variable region sequence of the humanized antibody UM03-C4 is as follows:

[0164] SEQ ID NO:14

[0165] EIVLTQSPATLSLSPGERATLSCSASSSVNYVNWYQQKPGQAPRILIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSTFPPYTFGQGTKLEIK

[0166] The heavy chain sequence of the humanized antibody UM03-C4 is as follows:

[0167] SEQ ID NO:15

[0168] QVQLVESGGGVVQPGGSLRLSCAASGFTFSDYGMAWIRQAPGKGPEWIAFITNLASSIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGDYRSFPYWGQG TLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0169] The light chain sequence of the humanized antibody UM03-C4 is as follows:

[0170] SEQ ID NO:16

[0171] EIVLTQSPATLSLSPGERATLSCSASSSVNYVNWYQQKPGQAPRILIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSTFPPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0172] Example 3: Preparation of Antibodies

[0173] The cDNA sequences encoding the heavy chain and light chain of UM03-L4 are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the cDNA sequences encoding the heavy chain and light chain of UM03-C4 are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0174] SEQ ID NO: 11

[0175]

[0176] SEQ ID NO:12

[0177] GAGATCCTGCTGACCCAGAGCCCCGCCATCATCGCCGCCAGCCCTGGAGAGAAGGTGACCATCACATGTTCCGCCAGCAGCAGCGTGAATTACGTGAACTGGTATCAACAGAAGCCTGGCAGCAGCCCTAAGATCTGGATCTACGGCATCTCCAACCTGGCCTCCGGCGTGCCTGCCAGGTTCAGCGGAAGCGGCAGCGGCACCAGCTTCAGCTTCACAATCAATAGCATGGAGGCCGAGGATGTGGCCACATACTACTGTCAGCAGAGATCCACATTCCCTCCCTACACATTCGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTSEQ ID NO:17

[0178]

[0179] SEQ ID NO: 18

[0180] GAGATTGTGCTGACCCAGAGCCCCGCCACACTGAGTCTGAGTCCCGGCGAGAGAGCAACACTGAGTTGTAGCGCCAGCAGTAGTGTGAACTACGTGAACTGGTATCAGCAGAAGCCTGGACAGGCTCCCAGAATCCTGATCTACGGCATCTCCAACCTGG CCAGCGGAGTGCCCGCCAGATTCAGCGGAAGTGGCAGCGGGACAGACTTCACCCTGACCATCAGCAGCCTGGAACCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAGAAGCACCTTCCCCCCCTATACATTTGCCAGGGAACCAAGCTGGAAATCAAA CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0181] The cDNA sequence was connected to the sequence encoding the signal peptide and cloned into the mammalian cell expression vector pcDNA3.4. The heavy chain expression plasmid and the light chain expression plasmid were transfected into HEK293 cells at a molar ratio of 2:1 using Lipofectamine 2000 transfection reagent (Invitrogen) and cultured for 7 days at 37°C and 5% carbon dioxide. The culture supernatant was collected and the antibody in the supernatant was purified by Protein A affinity chromatography. The purified antibody was dialyzed with PBS solution and concentrated by freeze drying and stored at -20°C.

[0182] Example 4: Binding of Antibodies to CD47 Protein

[0183] Coat a 96-well high affinity plate with 100 μL / well of human CD47 protein solution at a concentration of 1 μg / mL at 4°C and shake overnight. The next day, wash three times with 300 μL PBST (Tween20: 0.5‰), then block with 100 μL / well of 5% BSA / PBS for 2 hours and shake at room temperature. Wash three times with 300 μL PBST. Prepare a gradient dilution solution of the antibody sample with PBS. Add 100 μL / well to a 96-well plate and shake at room temperature for 1 hour. Wash three times with 300 μL PBST. Prepare a secondary antibody goat anti-mouse IgG HRP or goat anti-human IgG HRP solution and add 100 μL / well to a 96-well plate and shake at room temperature for 1 hour. Wash four times with 300 μL PBST. Add 100 μL / well TMB and develop color for 20 minutes. Add 100 μL / well 0.6N H2SO4 to stop color development and measure OD 450 nm.

[0184] After testing, the results are as follows Figure 1 As shown, the EC50 of the chimeric antibody UM03-L4 binding to human CD47 is 0.2499 μg / mL, and the EC50 of the humanized antibody UM03-C4 binding to human CD47 is 0.1687 μg / mL.

[0185] Example 5: Binding of Antibodies to Human Tumor Cells Highly Expressing Human CD47

[0186] Gastric cancer cell line MKN-45, ovarian cancer cell line A2780 and colon cancer cell line SW620 (ATCC) are human tumor cells that highly express CD47. First, the inventors of the present application used flow cytometry to verify the expression of CD47 on the above three cell lines. Use PBS to prepare concentration gradient solutions of anti-human CD47 detection antibody (eBioscience, 17-0479-42) and APC isotype (eBioscience, 17-4714-41), anti-huIgG 633 (life technologies, A21091) to prepare a final concentration of 2× working solution. Collect the above three tumor cells separately, wash them with PBS once, count them separately, and dilute them into 4*10 6 / ml cell suspension; 50μL of antibody working solution was added to 50μL of cell suspension, and incubated at 4℃ in the dark for 30min; after washing twice with PBS, the corresponding fluorescent labeled secondary antibody (anti-huIgG 633) was added, and incubated at 4℃ in the dark for 30min. After washing twice with PBS, the cells were suspended with 400μL FACS buffer, and the binding of antibodies to cells was detected by flow cytometry. The results showed that all three cell lines were CD47 positive cells ( Figure 2A )

[0187] The inventors of the present application tested the binding of chimeric antibody UM03-L4 and humanized antibody UM03-C4 to the above three tumor cell lines. The method was the same as above. Figure 2B As shown, the results showed that the chimeric antibody UM03-L4 and the humanized antibody UM03-C4 all bound to three human tumor cells that highly expressed human CD47. The test results are summarized in Table 1.

[0188] Similarly, the inventors of the present application detected the binding of antibody UM03-L4 to mouse tumor cells MC-38 that highly expressed mouse CD47, and found that UM03-L4 did not bind to mouse CD47. Figure 2C As shown, the isotype antibody control is APC isotype (Biolegend, 400511), and the CD47 detection antibody is anti-mouse CD47 detection antibody (Biolegend, 127513).

[0189] Table 1 EC50 (μg / ml) of humanized antibody UM03-C4 binding to human tumor cells

[0190] UM03-L4 UM03-C4 MKN-45 0.3811 0.3544 A2780 0.142 0.1191 SW620 0.2163 0.1262

[0191] Example 6: Antibodies inhibit the binding of CHO-CD47 cells to SIRPα protein

[0192] First, construct a stable CHO cell line overexpressing human CD47. Prepare a concentration gradient solution of the antibody with PBS to a final concentration of 2× working solution. Collect CHO-CD47 cells, centrifuge and resuspend in culture medium, and adjust the density to 4*10 6 / ml, take 50μL of antibody working solution and add it to 50μL cell suspension, incubate at 4℃ in the dark for 30min; wash twice with PBS, add 100μL 4μg / ml SIRPa-mFc (AcroBiosystems) or PBS, incubate at 4℃ for 30min; wash twice with PBS, add the corresponding fluorescent labeled secondary antibody, incubate at 4℃ in the dark for 30min, wash twice with PBS, suspend with 400μL FACS buffer, and detect the binding of antibody to cells by flow cytometer.

[0193] like Figure 3 As shown, SIRPα protein can bind to CHO-CD47 cells, and the binding can be inhibited by chimeric antibody UM03-L4 or humanized antibody UM03-C4, with IC50 of 3.383 μg / mL and 2.398 μg / mL, respectively.

[0194] Example 7: Binding of Antibodies to Human Red Blood Cells and Agglutination of Human Red Blood Cells

[0195] The inventors of the present application detected the binding of chimeric antibody UM03-L4 and humanized antibody UM03-C4 and positive control antibody Hu5F9 (US Pat. No. US 9017675B2 and PLoS ONE 2015, 10(9): e0137345) to human erythrocytes and their agglutination effect on human erythrocytes. First, erythrocytes were separated from the peripheral blood of volunteers and suspended in saline to form a 2% concentration erythrocyte suspension. PBS was used to prepare a concentration gradient solution of the antibody to prepare a 2× working solution at a final concentration. 50 μL of the antibody working solution was added to 50 μL of the erythrocyte suspension and incubated at 4°C in the dark for 60 minutes; after washing twice with PBS, the corresponding fluorescent labeled secondary antibody was added and incubated at 4°C in the dark for 30 minutes. After washing twice with PBS, the antibody was suspended in 400 μL FACS buffer and the binding of the antibody to erythrocytes was detected by flow cytometry. For the red blood cell agglutination test, 50 μL of antibody working solution was added to 50 μL of red blood cell suspension, placed in a 96-well round-bottom plate, and left to stand at room temperature for 2 h before observation and photography.

[0196] like Figure 4A As shown, the EC50 of chimeric antibody UM03-L4 and humanized antibody UM03-C4 binding to human erythrocytes were 1.21 μg / ml and 0.93 μg / ml, and the upper plateaus of binding were 586 and 473, respectively. The EC50 and upper plateau of positive control antibody Hu5F9 binding to human erythrocytes were 1.954 μg / ml and 892, respectively. Figure 4B As shown, the chimeric antibody UM03-L4 and the humanized antibody UM03-C4 had almost no agglutination effect on human erythrocytes, while the positive control antibody had a stronger agglutination effect on human erythrocytes.

[0197] Example 8: Binding of Antibodies to Monkey Red Blood Cells and Agglutination of Monkey Red Blood Cells

[0198] Similarly, the inventors of the present application detected the binding of the chimeric antibody UM03-L4 and the positive control antibody Hu5F9 to monkey erythrocytes and the agglutination of monkey erythrocytes, and the detection method was the same as that of human erythrocytes. Figure 5A As shown, the EC50 of the chimeric antibody UM03-L4 and the positive control antibody binding to monkey erythrocytes were 0.9070 μg / ml and 0.5860 μg / ml, and the upper binding plateaus were 186 and 392, respectively; similarly, Figure 5B The results showed that the chimeric antibody UM03-L4 had almost no agglutination effect on monkey erythrocytes, while the positive control antibody had a stronger agglutination effect on monkey erythrocytes.

[0199] Example 9: Antigen-antibody affinity detection

[0200] The affinity of antigen-antibody binding was detected using SPR technology (Surface Plasmon Resonance). Briefly, UM03-C4 at a concentration of 4 μg / mL was incubated with protein A sensor chip (GE, Cat#29127556) for 30 seconds for antibody capture. In the antigen binding stage, gradiently diluted CD47 protein was used as the mobile phase to bind to the UM03-C4 antibody captured on the sensor chip for 120 seconds. In the dissociation stage, HBS-EP buffer was used for continuous elution for 360 seconds. The binding of CD47 to the antibody on the sensor chip was quantitatively detected using Biacore T200 (GE Healthcare). The test results are shown in Tables 2 and Figure 6 As shown, the affinity of UM03-C4 is 0.4 nM.

[0201] Table 2 Affinity and kinetic data of UM03-C4 and CD47

[0202] Receptors Ligand ka(1 / Ms) kd(1 / s) KD(M) CD47 UM03-C4 1.02E+06 1.933E-04 1.90E-10

[0203] Example 10: Mouse tumor efficacy model

[0204] Daudi cells (ATCC, human lymphoma cells) are tumor cells with high expression of CD47. 6 Daudi cells were inoculated subcutaneously into female NCG mice. When the tumor grew to about 150 mm 3 Afterwards, UM03-C4 antibody 2.5 mg / kg, UM03-C4 antibody 5 mg / kg, UM03-C4 antibody 10 mg / kg, positive antibody Hu5F9 (5 mg / kg) and IgG1 isotype control antibody (5 mg / kg) were intravenously administered, with 6 mice in each group. The administration was once every 3 days for a total of 4 times.

[0205] like Figure 7 The results showed that since this model is an advanced tumor model, the mice in the positive antibody Hu5F9 group failed to show a longer survival time compared with the control group mice, while the UM03-C4 antibody treatment group showed clear efficacy at all doses.

[0206] Example 11: Drug Metabolism in Cynomolgus Monkeys

[0207] Cynomolgus monkeys (n=2) were intravenously injected with UM03-C4 at a dose of 15 mg / kg. Blood was collected at 0, 0.25, 4, 8, 24, 48, 72, 96, 144, 192, 240 and 336 hours before and after administration. The blood drug concentration was detected by ELISA and a drug-time curve was drawn. The results are shown in Figure 8The pharmacokinetic parameters were calculated at the same time, and the results are shown in Table 1. In this experiment, moderate to mild anemia was observed in cynomolgus monkeys, and no obvious thrombocytopenia was observed.

[0208] Table 3 Pharmacokinetic parameters of UM03-C4 in cynomolgus monkeys (15 mg / kg, n=2)

[0209]

[0210] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention. Sequence Listing <110> Suzhou Zelgen Biopharmaceutical Co., Ltd. <120> Anti-human CD47 antibody and antigen-binding fragment thereof, preparation method and application <130> DIC19110084 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Gly Phe Thr Phe Ser Asp Tyr Gly Met Ala 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <400> 2 Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val Thr 1 5 10 15 Gly <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <400> 3 Ala Arg Ala Gly Asp Tyr Arg Ser Phe Pro Tyr 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <400> 4 Ser Ala Ser Ser Ser Val Asn Tyr Val Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <400> 5 Gly Ile Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <400> 6 Gln Gln Arg Ser Thr Phe Pro Pro 1 5 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <400> 7 Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Pro Val Thr Val Ser Ala 115 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <400> 8 Glu Ile Leu Leu Thr Gln Ser Pro Ala Ile Ile Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Val 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ile Trp Ile Tyr 35 40 45 Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Phe Ser Phe Thr Ile Asn Ser Met Glu Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 445 <212> PRT <213> Artificial Sequence <400> 9 Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Pro Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 10 <211> 214 <212> PRT <213> Artificial Sequence <400> 10 Glu Ile Leu Leu Thr Gln Ser Pro Ala Ile Ile Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Val 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ile Trp Ile Tyr 35 40 45 Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Phe Ser Phe Thr Ile Asn Ser Met Glu Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 11 <211> 1335 <212> DNA <213> Artificial Sequence <400> 11 gaggtgaagc tggtggagag cggcggcgac ctggtgcagc caggaggatc cagaaagctg 60 agctgtgccg ccagcggctt cacattcagc gactacggca tggcctggat caggcaggcc 120 cctggcaagg gccccgagtg gatcgccttc atcaccaatc tggcctcctc catctactac 180 gccgacacag tgaccggcag gttcaccatc agcagagaga acgccaagaa cacactgtac 240 ctggagatgt cctccctgag aagcgaggac acagccatgt actactgtgc cagagccggc 300 gactacagga gcttccccta ctggggccag ggcacacctg tgaccgtgtc cgccgctagc 360 acaaagggcc ccagcgtgtt tcctctggcc ccctgcagca gaagcaccag cgagtccacc 420 gccgccctgg gatgcctggt gaaggactac ttccctgagc ccgtgacagt gagctggaat 480 agcggcgccc tgacaagcgg cgtgcacacc tttcctgccg tgctgcagtc cagcggcctg 540 tacagcctga gcagcgtggt gacagtgcct agctcctccc tgggcacaaa gacatacacc 600 tgcaatgtgg accacaagcc cagcaacaca aaggtggaca agagagtgga gagcaagtac 660 ggccctcctt gtcccccttg tcctgcccct gagtttctgg gcggcccctc cgtgtttctg 720 tttcctccca agcctaagga taccctgatg atctccagaa cccccgaggt gacctgtgtg 780 gtggtggatg tgagccagga ggaccccgag gtgcagttca actggtacgt ggacggcgtg 840 900. gaggtgcaca atgccaagac caagcccagg gaggagcagt ttaactccac atacagggtg gtgtccgtgc tgaccgtgct gcaccaggac tggctgaatg gcaaggagta caagtgtaag 960 gtgagcaaca agggcctgcc tagctccatc gagagacca tctccaaggc caagggccag cctagggagc cccaggtgta cacactgcct cccagccagg aggagatgac aaagaaccag 1080 gtgagcctga catgcctggt gaaaggcttc tacccttccg acatcgccgt ggagtggggag tccaacggcc agcctgagaa caattacaag acaacccccc ccgtgctgga ttccgacggc agctttttcc tgtactccag actgaccgtg gacaagagca gatggcagga gggcaatgtg tttagctgtt ccgtgatgca cgaggccctg cacaatcact acacacaga gtccctgagc ctgagcctgg gcaaa 1335 <210> 12 <211> 642 <212> DNA <213> Artificial Sequence <400> 12 gagatcctgc tgacccagag ccccgccatc atcgccgcca gccctggaga gaaggtgacc 60 atcacatgtt ccgccagcag cagcgtgaat tacgtgaact ggtatcaaca gaagcctggc 120 agcagcccta agatctggat ctacggcatc tccaacctgg cctccggcgt gcctgccagg 180 ttcagcggaa gcggcagcgg caccagcttc agcttcacaa tcaatagcat ggaggccgag 240 gatgtggcca catactactg tcagcagaga tccacattcc ctccctacac attcggcggc 300 ggcaccaagc tggagatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagttcgc ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <400> 13 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Asn Tyr Val 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ile Leu Ile Tyr 35 40 45 Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 445 <212> PRT <213> Artificial Sequence <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 16 <211> 214 <212> PRT <213> Artificial Sequence <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Asn Tyr Val 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ile Leu Ile Tyr 35 40 45 Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 17 <211> 1335 <212> DNA <213> Artificial Sequence <400> 17 caggtccagc tggtcgagtc aggcggcggc gtcgtgcagc ctggaggatc actgagactg 60 agctgcgcag ccagcggctt cacattcagc gactacggca tggcctggat cagacaggca 120 cccggcaaag ggccagagtg gattgctttc atcaccaacc tggccagcag catctactac 180 gccgacaccg tgacaggcag attcaccatc agcagagaca acagcaagaa cacactgtac 240 ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc cagggccgga 300 gactacagat cctttcccta ctgggggcag ggaaccctgg tgaccgtcag cgccgctagc 360 accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca 420 gccgccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa gacctacacc 600 tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agagagttga gtccaaatat 660 ggtcccccat gcccaccatg cccagcacct gagttcctgg ggggaccatc agtcttcctg 720 ttccccccaa aacccaagga cactctcatg atctcccgga cccctgaggt cacgtgcgtg 780 gtggtggacg tgagccagga agaccccgag gtccagttca actggtacgt ggatggcgtg 840 gaggtgcata atgccaagac aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg gcaaggagta caagtgcaag 960 gtctccaaca aaggcctccc gtcctccatc gagaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacacaga gagcctctcc ctgtctctgg gtaaa <210> 18 <211> 642 <212> DNA <213> Artificial Sequence <400> 18 gagattgtgc tgacccagag ccccgccaca ctgagtctga gtcccggcga gagagcaaca 60 ctgagttgta gcgccagcag tagtgtgaac tacgtgaact ggtatcagca gaagcctgga 120 caggctccca gaatcctgat ctacggcatc tccaacctgg ccagcggagt gcccgccaga 180 ttcagcggaa gtggcagcgg gacagacttc accctgacca tcagcagcct ggaacccgag 240 gatttcgccg tgtactactg ccagcagaga agcaccttcc ccccctatac atttggccag 300 ggaaccaagc tggaaatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagttcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

Claims

1. An anti-human CD47 antibody or an antigen-binding fragment thereof, comprising the following heavy chain complementary determining region: VH CDR1, the amino acid sequence of which is shown in SEQ ID NO: 1; VH CDR2, the amino acid sequence of which is shown in SEQ ID NO:2; and VH CDR3, the amino acid sequence of which is shown in SEQ ID NO:3; Furthermore, the anti-human CD47 antibody or antigen-binding fragment thereof further comprises the following light chain complementary determining region: VL CDR1, the amino acid sequence of which is shown in SEQ ID NO:4; VL CDR2, the amino acid sequence of which is shown in SEQ ID NO:5; and VL CDR3, the amino acid sequence of which is shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-human CD47 antibody or antigen-binding fragment thereof is scFv, scFv dimer, dsFv, dsFv2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, or ds bifunctional antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-human CD47 antibody or antigen-binding fragment thereof further comprises a human heavy chain constant region and / or a human light chain constant region.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein The human heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the human light chain constant region is selected from the light chain constant region of human IgG1, IgG2, IgG3 or IgG4.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein The human heavy chain constant region is the heavy chain constant region of human IgG1, and the human light chain constant region is a κ chain.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The amino acid sequence of the heavy chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 7; The amino acid sequence of the light chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

8.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The heavy chain of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9; The light chain of the anti-human CD47 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

10.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The amino acid sequence of the heavy chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 13; The amino acid sequence of the light chain variable region of the anti-human CD47 antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

14.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The amino acid sequence of the heavy chain of the anti-human CD47 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 15; The amino acid sequence of the light chain of the anti-human CD47 antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

16.

10. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. The polynucleotide according to claim 10, wherein The polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 11; Furthermore, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:

12.

12. The polynucleotide according to claim 10, wherein The polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 17; Furthermore, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:

18.

13. A vector comprising the isolated polynucleotide according to any one of claims 10-12.

14. A host cell comprising the vector according to claim 13; The host cell is a mammalian cell.

15. The host cell according to claim 14, wherein The host cell is a cell of a human, mouse, sheep, horse, dog or cat.

16. The host cell according to claim 14, wherein The host cell is a Chinese hamster ovary cell.

17. A method for preparing the anti-human CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing the host cell according to claim 14 under conditions capable of expressing the isolated polynucleotide according to claim 10.

18. A kit comprising the anti-human CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

19. A pharmaceutical composition comprising the anti-human CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a medicament for treating a condition that benefits from enhancing an immune response; in, The disorder is a lymphoma with high CD47 expression.

21. The use according to claim 20, wherein the disorder is acute myeloid leukemia and myelodysplastic syndrome.

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