CD47 antibody specifically activated by tumor microenvironment

Abstract

The invention provides a CD47 antibody specifically activated by a tumor microenvironment. Specifically, the invention provides a bispecific antibody which is connected with a substrate and a masking body which are highly expressed in a tumor environment and can recognize cleavage in a chemical coupling manner. The antibody activity of the bispecific antibody is hindered in normal tissue, and the antibody activity is activated in a tumor environment. In addition, the bispecific antibody also has significantly reduced toxic and side effects.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a CD47 antibody specifically activated by a tumor microenvironment. Background Art

[0002] CD47 is a membrane protein involved in the increase in intracellular calcium concentration during cell adhesion to the extracellular matrix. The encoded protein is also a receptor for the C-terminal cell binding domain of coagulase and may play a role in membrane trafficking and signal transduction. The CD47-SIRPα (signal regulatory protein α) pathway, also known as the "don't eat me" signal, was first discovered in 1990. SIRPα is the first protein member of the SIRP family to be discovered and is present in myeloid cells and includes all forms of macrophages.

[0003] SIRPα has an extracellular immunoglobulin functional region that binds to a ligand and an intracellular inhibitory motif containing an ITIM (immunoreceptor tyrosine-based inhibitory motif), which can bind to SHP-1 (containing an SH2 motif), Protein Tyrosine Phosphatase 1) or SHP2 (also known as PTPN11) to transmit information.

[0004] It has been reported that tumor heterogeneity and the tumor microenvironment (TME) of ovarian cancer affect the efficacy of immunotherapy and patient prognosis. The TME of ovarian cancer is intrinsically heterogeneous. CD47 plays an important role in cell functional behaviors and immune homeostasis associated with cancer prognosis.

[0005] CD47 has a long N-terminal extracellular domain, five transmembrane domains, and a short intracellular domain. CD47 binds to SIRPα and induces SHP1 and SHP2 activation by tyrosine phosphorylation of SIRPαITIM, leading to myosin IIA dephosphorylation and inhibition of cytoskeletal reorganization, a step required for macrophage phagocytosis of target cells.

[0006] The role of CD47 in phagocytosis was discovered in CD47-deficient mice. If the red blood cells of CD47-deficient mice were taken out and given to wild-type mice, the red blood cells lacking CD47 would be quickly cleared, and inhibiting macrophages would reverse this result, indicating that macrophages play a very important role in this process.

[0007] In addition to regulating normal tissue homeostasis, CD47 has also been found to be abundantly expressed on tumor cells (both hematological and non-hematological cells). CD47 is not directly involved in the regulation of tumor cell proliferation and survival. Antibody inhibition of CD47 can increase the phagocytosis of tumor cells even in the absence of T cells, B cells, and NK cells, indicating that tumor cells can evade phagocytosis of macrophages and thus avoid being cleared by macrophages.

[0008] However, the anti-CD47 antibodies in the prior art still have many deficiencies, and the art still needs to provide an anti-CD47 antibody with low toxicity and high safety to the human body. Summary of the invention

[0009] The purpose of the present invention is to provide a CD47 antibody that specifically activates the tumor microenvironment.

[0010] In the first aspect of the present invention, a mutant CD47 antibody is provided, wherein the mutant CD47 antibody introduces one or more mutations in the complementarity determining region (CDR region) of the heavy chain variable region and / or the light chain variable region relative to the wild-type CD47 antibody sequence, and the mutation is selected from the following group:

[0011] The Y33, R55, Y61, W65, Y105 or L107 position of the heavy chain variable region; and / or the Q27, Y49, T53, F92, Y93, Y96 or N98 position of the light chain variable region comprises a mutation in which one or more amino acids are replaced with cysteine ​​(Cys);

[0012] Wherein, the affinity of the wild-type CD47 antibody binding to CD47 is E0, the affinity of the mutant CD47 antibody binding to CD47 is E1, and E1 / E0≤5.5 (preferably E1 / E0≤3, preferably ≤1.5, and more preferably E1 / E0≤1);

[0013] Wherein, the wild-type CD47 antibody has a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises three heavy chain variable regions as shown below:

[0014] HCDR1 SGYDMC (SEQ ID NO. 18),

[0015] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.19),

[0016] HCDR3 GRSSYRLNL (SEQ ID NO. 20), and

[0017] The light chain variable region comprises three light chain variable regions as shown below:

[0018] LCDR1 QASQSISNELS(SEQ ID NO.21),

[0019] LCDR2 RASTLES(SEQ ID NO.22),

[0020] LCDR3 QSTFYGDYGNA(SEQ ID NO.23);

[0021] Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain CD47 binding affinity.

[0022] In another preferred embodiment, the amino acid sequence of any of the above CDRs contains a derivative CDR sequence that has been added, deleted, modified and / or substituted with 1, 2 or 3 amino acids, and the derivative antibody composed of VH and VL containing the derivative CDR sequence can retain the affinity for binding to CD47.

[0023] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0024] In another preferred embodiment, the derivative sequence that has been subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain CD47 binding affinity is an amino acid sequence with a homology or sequence identity of at least 85%.

[0025] In another preferred embodiment, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0026] In another preferred embodiment, the wild-type CD47 antibody includes: a single-chain antibody, a double-chain antibody, a monoclonal antibody, a chimeric antibody (such as a human-mouse chimeric antibody), a mouse antibody, or a humanized antibody.

[0027] In another preferred embodiment, the heavy chain variable region of the mutant CD47 antibody comprises three heavy chain variable regions as shown below:

[0028] HCDR1 SGYDMC (SEQ ID NO. 18),

[0029] HCDR2 CIYTRSSGGTYCASWAKG (SEQ ID NO.24),

[0030] HCDR3 GRSSYRLNL (SEQ ID NO. 20), and

[0031] The light chain variable region comprises three light chain variable regions as shown below:

[0032] LCDR1 QASQSISNELS(SEQ ID NO.21),

[0033] LCDR2 RASTLES(SEQ ID NO.22),

[0034] LCDR3 QSTFYGDYGNA(SEQ ID NO.23)

[0035] Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain CD47 binding affinity.

[0036] In another preferred embodiment, the mutant CD47 antibody includes a PEGylated CD47 antibody.

[0037] In another preferred embodiment, the PEGylation is carried out using PEG with a molecular weight of 5-100 kD (preferably 10-50 kD).

[0038] In another preferred embodiment, the PEGylation includes site-specific PEGylation or non-site-specific PEGylation.

[0039] In another preferred example, the affinity for binding to CD47 is the EC50 value for binding to CD47.

[0040] In another preferred example, the ratio of the affinity E1 of the mutant CD47 antibody binding to CD47 to the affinity E0 of the corresponding wild-type CD47 antibody binding to CD47 (E1 / E0) is 0.4-5.5, preferably 0.5-2, more preferably 0.7-1.5, and more preferably 0.8-1.2.

[0041] In another preferred embodiment, the wild-type CD47 antibody is a CD47 mouse antibody or a CD47 humanized antibody.

[0042] In another preferred example, the wild-type CD47 antibody comprises a heavy chain variable region as shown in SEQ ID NO.7, and a light chain variable region as shown in SEQ ID NO.8.

[0043] In another preferred example, the amino acid sequence of the heavy chain variable region of the wild-type CD47 antibody has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the heavy chain variable region of SEQ ID NO.7.

[0044] In another preferred example, the amino acid sequence of the light chain variable region of the wild-type CD47 antibody has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the light chain variable region of SEQ ID NO.8.

[0045] In another preferred embodiment, the humanized CD47 antibody comprises a heavy chain variable region as shown in SEQ ID NO.11, 12, 13 or 14, and a light chain variable region as shown in SEQ ID NO.15, 16 or 17; or comprises

[0046] An amino acid sequence of a heavy chain variable region that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO. 11, 12, 13 or 14; and

[0047] A light chain variable region amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO.15, 16 or 17.

[0048] In another preferred embodiment, the mutant CD47 antibody comprises one or more mutations in the complementarity determining region (CDR) of the heavy chain variable region and / or light chain variable region of the wild-type CD47 antibody; preferably 1, 2, 3, 4 or 5 amino acid mutations.

[0049] In another preferred example, the mutant CD47 antibody comprises a mutation in which one or more amino acids are replaced with cysteine ​​(Cys) in the complementarity determining region (CDR) of the heavy chain variable region relative to the wild-type CD47 antibody; and / or comprises a mutation in which one or more amino acids are replaced with cysteine ​​(Cys) in the complementarity determining region (CDR) of the light chain variable region relative to the wild-type CD47 antibody.

[0050] In another preferred embodiment, the mutant CD47 antibody comprises a mutation selected from the following group in the complementary determining region of the heavy chain variable region corresponding to the wild-type CD47 antibody: Y33C, R55C, Y61C, W65C, Y105C or L107C, or a combination thereof, preferably comprising a mutation of Y61C; and / or

[0051] The complementarity determining region corresponding to the light chain variable region of the wild-type CD47 antibody comprises a mutation selected from the group consisting of Q27C, Y49C, T53C, F92C, Y93C, Y96C or N98C or a combination thereof, preferably comprising a mutation of Y49C.

[0052] In another preferred embodiment, all position numbers of amino acids mentioned for amino acid residues of an antibody are based on the Kabat numbering.

[0053] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the mutant CD47 antibody has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the heavy chain variable region of SEQ ID NO.7, 11, 12, 13 or 14; and

[0054] The amino acid sequence of the light chain variable region of the mutant CD47 antibody has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the light chain variable region of SEQ ID NO.8, 15, 16 or 17.

[0055] In another preferred embodiment, no interchain disulfide bond or intrachain disulfide bond is formed in the complementarity determining region (CDR) of the variable region of the antibody.

[0056] In another preferred embodiment, the mutant CD47 antibody comprises: (i) a Fab fragment; (ii) a F(ab')2 fragment; (iii) a Fd fragment; (iv) a Fv fragment; (v) a single-chain Fv (scFv) molecule; or (vi) a dAb fragment.

[0057] In another preferred embodiment, the mutant CD47 antibody is a bispecific, trispecific or multispecific antibody.

[0058] In another preferred embodiment, the mutant CD47 antibody includes a modified mutant CD47 antibody.

[0059] In another preferred embodiment, the modified mutant CD47 antibody is a tumor microenvironment-specific activated antibody (TMEAbody), that is, a masked antibody.

[0060] In another preferred embodiment, the modified mutant CD47 antibody includes, but is not limited to, connecting a blocker to the cysteine ​​at the mutation site of the mutant CD47 antibody via a cleavable connecting peptide, wherein the mutation site is located in the complementarity determining region (CDR region) of the heavy chain variable region and / or the light chain variable region of the mutant CD47 antibody.

[0061] In another preferred embodiment, the mutant CD47 antibody includes a modified mutant CD47 antibody having the structure shown below:

[0062] Ab-(P-R1)n

[0063] in,

[0064] Ab is a mutant CD47 antibody;

[0065] R1 is each independently a blocker;

[0066] P is a cleavable connecting peptide;

[0067] n is a positive integer from 1 to 5;

[0068] “-” is a chemical bond, a linker or a connector.

[0069] In another preferred embodiment, the masking body refers to a group that prevents Ab from binding to its ligand or receptor.

[0070] In another preferred embodiment, the masking body is selected from polyethylene glycol-C 1-5 -Alkylcarbonyl group:

[0071]

[0072] wherein each R is independently a C1-4 alkyl group; each n is independently an integer in the range of 1 to 30,000, such as an integer of 1 to 15,000, 1 to 5,000, 1 to 2,000, 1 to 300, 1 to 150, 1 to 50, 1 to 20 or 3 to 12; polyethylene glycol or Peg m represents polyethylene glycol with a molecular weight of 44 to 132,000, such as polyethylene glycol with a molecular weight of 1,000 to 50,000 or 10,000 to 30,000; m represents the molecular weight of the polyethylene glycol; and the wavy line represents the position where R2 is connected.

[0073] In another preferred embodiment, the masking body is selected from the following group: PEG5K, PEG10K, PEG20K, or PEG40K.

[0074] In another preferred embodiment, the R1 is wherein n is independently an integer from 1 to 2000, 1 to 300, 1 to 150, 1 to 50 or 1 to 20.

[0075] In another preferred embodiment, the P comprises the following structure:

[0076] R2-R3-R4, among which,

[0077] R2 is absent or is a cleavable linker that can be activated by one or more proteolytic enzymes or is a chemical bond that can be activated by acid in a pathological microenvironment;

[0078] R3 does not exist, or is a linker arm that automatically falls off after R2 is cut or R2 is activated by acid in a pathological microenvironment; when R2 does not exist, R3 is a chemical bond that is activated by acid in a pathological microenvironment;

[0079] R4 is a group that can covalently bind to the sulfur atom of the cysteine ​​residue contained in the mutant CD47 antibody.

[0080] In another preferred embodiment, the R1 is connected to R2 in P through a chemical bond.

[0081] In another preferred embodiment, R2 is a peptide that can be activated or cleaved by one or more proteolytic enzymes, proteases or peptidases, wherein the protease is selected from the following group: asparagine endopeptidase, granzyme, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, astrin, factor Xa, chymosin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidin protease, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, peptidase, interstitial protease, plasma protease, Nepenthes protease, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-113 converting enzyme, thrombin, FAP (FAP-a), transmembrane peptidase (meprin), dipeptidyl peptidase and dipeptidyl peptidase-4 (DPPIV / CD26), preferably asparagine endopeptidase.

[0082] In another preferred embodiment, R2 is a connecting peptide that can be cleaved under the acidic conditions of the pathological microenvironment; preferably, R2 is a tripeptide, wherein the first amino acid residue of the tripeptide connected to R1 is selected from Ala, Thr, Val and Ile, the second amino acid residue in the middle is selected from Ala, Thr, Val and Asn, and the acid residue of the third amino acid connected to Ab is selected from Asn and Asp; wherein R2 is connected to R1 through the amino group of the first amino acid residue in the form of an amide, ester, carbamate, urea or hydrazone bond, and is connected to R3 through the carboxyl group of the third amino acid residue in the form of an amide, ester, carbamate, urea or hydrazone bond; preferably, the tripeptide is selected from Ala-Ala-Asn and Ala-Ala-Asp.

[0083] In another preferred embodiment, Ala-Ala-Asn can be recognized and cleaved by asparagine endopeptidase, and Ala-Ala-Asp can be recognized and cleaved by granzyme.

[0084] In another preferred embodiment, R2 is a connecting peptide that can be cleaved under the acidic conditions of the pathological microenvironment, and is selected from amide, ester, carbamate, urea and hydrazone bonds; preferably, the structure of R1-R2-R3-R4 is represented by the following formula:

[0085]

[0086] Wherein X and Y are independently NR' or O, Z is H or C1-10 alkyl, R' is H or C1-4 alkyl; R3 is connected to R1 and R4 through X and Y in the form of amide, ester, carbamate, urea or hydrazone bond.

[0087] In another preferred embodiment, R4 is

[0088]

[0089] In another preferred embodiment, the structure of P-R1 is as follows:

[0090]

[0091] Wherein R1 is PEG20K, and the structural formula is:

[0092] In another preferred embodiment, the modified mutant CD47 antibody is an activatable antibody.

[0093] In another preferred example, the protease is produced by a tumor close to cells expressing CD47 in a tissue and / or is produced by a tumor that co-localizes with CD47 in a tissue, and wherein when the activatable antibody is exposed to the protease, the protease cleaves P in the activatable antibody.

[0094] In another preferred embodiment, R4 in P is connected to the thiol group of the mutant CD47 antibody (Ab).

[0095] In a second aspect of the present invention, a CD47 antibody or an antigen-binding fragment thereof is provided, wherein the CD47 antibody has a heavy chain variable region and a light chain variable region,

[0096] (1) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0097] HCDR1 SGYDMC (SEQ ID NO. 18),

[0098] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.19),

[0099] HCDR3 GRSSYRLNL (SEQ ID NO. 20), and

[0100] The light chain variable region comprises three light chain variable regions as shown below:

[0101] LCDR1 QASQSISNELS(SEQ ID NO.21),

[0102] LCDR2 RASTLES(SEQ ID NO.22),

[0103] LCDR3 QSTFYGDYGNA (SEQ ID NO. 23); or

[0104] (2) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0105] HCDR1 SGYDMC (SEQ ID NO. 25),

[0106] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.26),

[0107] HCDR3 GRSSYRLNL (SEQ ID NO. 27), and

[0108] The light chain variable region comprises three light chain variable regions as shown below:

[0109] LCDR1 QASQSISNELS(SEQ ID NO.28),

[0110] LCDR2 RASTLES(SEQ ID NO.29),

[0111] LCDR3 QSTFYGDYGNA (SEQ ID NO. 30) or

[0112] (3) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0113] HCDR1 SGYDMC (SEQ ID NO. 31),

[0114] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.32),

[0115] HCDR3 GRSSYRLNL (SEQ ID NO. 33), and

[0116] The light chain variable region comprises three light chain variable regions as shown below:

[0117] LCDR1 QASQSISNELS(SEQ ID NO.34),

[0118] LCDR2 RASTLES(SEQ ID NO.35),

[0119] LCDR3 QSTFYGDYGNA (SEQ ID NO. 36) or

[0120] (4) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0121] HCDR1 SGYDMC (SEQ ID NO. 37),

[0122] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.38),

[0123] HCDR3 GRSSYRLNL (SEQ ID NO. 39), and

[0124] The light chain variable region comprises three light chain variable regions as shown below:

[0125] LCDR1 QASQSISNELS(SEQ ID NO.40),

[0126] LCDR2 RASTLES(SEQ ID NO.41),

[0127] LCDR3 QSTFYGDYGNA (SEQ ID NO. 42) or

[0128] (5) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0129] HCDR1 SGYDMC (SEQ ID NO. 43),

[0130] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.44),

[0131] HCDR3 GRSSYRLNL (SEQ ID NO. 45), and

[0132] The light chain variable region comprises three light chain variable regions as shown below:

[0133] LCDR1 QASQSISNELS(SEQ ID NO.46),

[0134] LCDR2 RASTLES(SEQ ID NO.47),

[0135] LCDR3 QSTFYGDYGNA(SEQ ID NO.48);

[0136] Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain CD47 binding affinity.

[0137] In another preferred example, the wild-type CD47 antibody comprises a heavy chain variable region as shown in SEQ ID NO.1, and a light chain variable region as shown in SEQ ID NO.2.

[0138] In another preferred example, the wild-type CD47 antibody comprises a heavy chain variable region as shown in SEQ ID NO.3, and a light chain variable region as shown in SEQ ID NO.4.

[0139] In another preferred example, the wild-type CD47 antibody comprises a heavy chain variable region as shown in SEQ ID NO.5, and a light chain variable region as shown in SEQ ID NO.6.

[0140] In another preferred example, the wild-type CD47 antibody comprises a heavy chain variable region as shown in SEQ ID NO.7, and a light chain variable region as shown in SEQ ID NO.8.

[0141] In another preferred example, the wild-type CD47 antibody comprises a heavy chain variable region as shown in SEQ ID NO.9, and a light chain variable region as shown in SEQ ID NO.10.

[0142] In another preferred embodiment, the wild-type CD47 antibody comprises a heavy chain variable region whose amino acid sequence has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO.1, 3, 5, 7 or 9.

[0143] In another preferred embodiment, the wild-type CD47 antibody comprises a light chain variable region that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO.2, 4, 6, 8 or 10.

[0144] In another preferred embodiment, the CD47 antibody is a humanized antibody.

[0145] In another preferred embodiment, the heavy chain variable region of the humanized antibody against CD47 has an amino acid sequence selected from the group consisting of SEQ ID NO.11, 12, 13 or 14.

[0146] In another preferred example, the light chain variable region of the humanized antibody against CD47 has an amino acid sequence selected from the group consisting of SEQ ID NO. 15, 16 or 17.

[0147] In another preferred embodiment, the antibody comprises a heavy chain and a light chain.

[0148] In another preferred embodiment, the heavy chain further comprises a heavy chain constant region.

[0149] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0150] In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region.

[0151] In another preferred embodiment, the light chain also includes a light chain constant region.

[0152] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0153] In another preferred embodiment, the light chain constant region is a human antibody light chain kappa constant region.

[0154] The third aspect of the present invention provides a recombinant protein, wherein the recombinant protein comprises:

[0155] (1) the mutant CD47 antibody or modified mutant CD47 antibody according to the first aspect of the present invention or the anti-CD47 antibody according to the second aspect of the present invention;

[0156] (2) any polypeptide molecule or fragment having therapeutic function; and / or

[0157] (3) Optional protein functional domains that help improve the physicochemical and pharmacological properties of the molecule.

[0158] In another preferred embodiment, the recombinant protein further includes: (4) an optional tag sequence for facilitating expression and / or purification.

[0159] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0160] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0161] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are not limited to: polypeptide molecules or fragments targeting targets selected from the following group: Her2, DLL3, CLDN6, EGFR, TGFβ, BCMA, B7H6, GUCY2C, CD478, CD123, CD19, CD20, CD22, B7-H3, GPC3, CD73, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7.

[0162] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0163] In another preferred embodiment, the fusion protein includes a multispecific antibody and a chimeric antibody.

[0164] In another preferred embodiment, the functional domains for improving the physicochemical properties or drugability of proteins include Fc segments, anti-albumin nanoantibodies (HLE), and albumin binding domains (ABD).

[0165] The fourth aspect of the present invention provides an isolated polynucleotide encoding the mutant CD47 antibody according to the first aspect of the present invention or the anti-CD47 antibody according to the second aspect of the present invention.

[0166] The fifth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the fourth aspect of the present invention.

[0167] The sixth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector described in the fifth aspect of the present invention or the polynucleotide described in the fourth aspect of the present invention is integrated into its genome.

[0168] The seventh aspect of the present invention provides a pharmaceutical composition, which contains:

[0169] (I) the mutant CD47 antibody or modified mutant CD47 antibody according to the first aspect of the present invention or the anti-CD47 antibody according to the second aspect of the present invention; and

[0170] (II) a pharmaceutically acceptable carrier.

[0171] In another preferred embodiment, the pharmaceutical composition further comprises:

[0172] (b) Other biologically active drugs, such as drugs for treating tumors.

[0173] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0174] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spraying and aerosol inhalation.

[0175] In another preferred embodiment, the pharmaceutical composition is administered by co-culturing the pharmaceutical composition and immune cells (such as dendritic cells, natural killer cells, lymphocytes, monocytes / macrophages, granulocytes, etc.), and then separating the immune cells for in vivo reinfusion.

[0176] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0177] In another preferred embodiment, the pharmaceutical composition is used for anti-tumor treatment.

[0178] The eighth aspect of the present invention provides an immunoconjugate, comprising:

[0179] (a) the mutant CD47 antibody or modified mutant CD47 antibody according to the first aspect of the present invention or the anti-CD47 antibody according to the second aspect of the present invention; and

[0180] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0181] In another preferred embodiment, the components (a) and (b) are operably linked.

[0182] In another preferred embodiment, the conjugate portion is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0183] The ninth aspect of the present invention provides the use of the mutant CD47 antibody or modified mutant CD47 antibody as described in the first aspect of the present invention, the anti-CD47 antibody as described in the second aspect of the present invention, or the immunoconjugate as described in the eighth aspect of the present invention, for preparing (a) a detection reagent or a kit; and / or (b) preparing a pharmaceutical composition for preventing and / or treating cancer, tumor or autoimmune disease.

[0184] The tenth aspect of the present invention provides a method for preventing or treating tumors, comprising administering to a subject in need thereof the mutant CD47 antibody or modified mutant CD47 antibody as described in the first aspect of the present invention, the pharmaceutical composition as described in the seventh aspect of the present invention, or the immunoconjugate as described in the eighth aspect of the present invention.

[0185] In another preferred embodiment, the tumor includes but is not limited to: breast cancer, liver cancer, gastric cancer, colon cancer, leukemia, lung cancer, kidney tumor, small intestine cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma, bone cancer, adrenal tumor, or bladder tumor.

[0186] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0187] Figure 1 The light chain and heavy chain variable region alignment sequences of each anti-CD47 rabbit antibody are shown.

[0188] Figure 2 The FACS binding experiment of five CD47 antibodies to CHOK1-CD47 cells is shown.

[0189] Figure 3 ELISA binding experiment of CD47 antibody to CD47 antigen is shown.

[0190] Figure 4 Shown is a FACS binding experiment of CD47 antibody to CHOK1-CD47 cells.

[0191] Figure 5 Shown is a FACS binding experiment of CD47 TMEAbody on Raji cells.

[0192] Figure 6 Shown is a binding experiment of CD47 TMEAbody blocking SIRPα on Raji cells.

[0193] Figure 7 It is shown that CD47 TMEAbody blocks the binding of CD47 antibody to erythrocytes.

[0194] Figure 8 RBC agglutination experiments with CD47 TMEAbody are shown.

[0195] Fig. 9 ADCC activity of CD47 TMEAbody on Raji cells is shown.

[0196] Fig.10 Shown are FACS binding experiments of different CD47 humanized antibodies to CHOK1-CD47 cells.

[0197] Fig.11 In vivo efficacy experiments of CD47 TMEAbody in a mouse tumor (Raji) model are shown.

[0198] Fig.12 In vivo efficacy experiments of CD47 TMEAbody in a mouse tumor (SHP-77) model are shown. DETAILED DESCRIPTION

[0199] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that a mutant CD47 antibody is obtained by mutating a specific amino acid in the CDR of the wild-type CD47 antibody variable region to Cys, and the modified mutant CD47 antibody obtained by connecting a masker by chemical coupling can reduce the binding activity with CD47 and achieve specific activation of the tumor microenvironment; the modified mutant CD47 antibody can be activated in the tumor microenvironment and stimulate anti-tumor immune response, and has good safety. On this basis, the present invention was completed.

[0200] the term

[0201] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0202] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0203] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0204] Antibody

[0205] Generally, "antibodies" are also referred to as "immunoglobulins" which may be natural or conventional antibodies in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, λ (l) and κ (k). There are five major heavy chain species (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a heavy chain variable region (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant domain (CL) of the light chain and the constant region (CH) of the heavy chain confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and is composed of the variable portion of a light chain and a heavy chain. The specificity of the antibody depends on the structural complementarity between the antibody binding site and the antigenic determining region. The antibody binding site is composed of residues mainly from the hypervariable region or complementary determining region (CDR). Occasionally, residues from non-hypervariable or framework regions (FR) affect the overall domain structure and thus the binding site. The complementary determining region or CDR refers to the amino acid sequence that jointly defines the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site. The light chain and heavy chain of the immunoglobulin each have three CDRs, which are separately referred to as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H. Conventional antibody antigen binding sites therefore include six CDRs, including a set of CDRs from each heavy chain and light chain v region.

[0206] As used herein, the terms "single domain antibody" and "nanoantibody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0207] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0208] As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., refers to those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. The light chain and heavy chain of an immunoglobulin each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain can therefore be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0209] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence after aligning the candidate sequence with the specific peptide or polypeptide sequence (and introducing gaps, if necessary) to obtain the maximum percentage sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine the percentage of amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software.

[0210] Knowing the amino acid sequence of the CDRs, one skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0211] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0212] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule with a single amino acid composition directed against a specific antigen, and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced by a single clone of a B cell or hybridoma, but can also be recombinant, i.e., produced by protein engineering.

[0213] As used herein, the term "antigen" or "target antigen" refers to a molecule or portion of a molecule that can be bound by an antibody or antibody-like binding protein. The term further refers to a molecule or portion of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein can compete with an intact antibody that recognizes the target antigen.

[0214] As used herein, the term "affinity" is theoretically defined by the equilibrium association between a complete antibody and an antigen. The affinity of the dual antibody of the present invention can be evaluated or determined by a KD value (dissociation constant) (or other determination methods), such as bio-layer interferometry (BLI), using a FortebioRed96 instrument to measure and determine.

[0215] As used herein, the term “EC 50 ", also known as half-maximal effect concentration, refers to the antibody concentration that causes 50% of the maximum effect.

[0216] As used herein, the term "knobs-into-holes (KIH)" refers to the currently mature Fc segment heterodimerization technology. For example, a smaller threonine (T) at position 366 in the CH3 region of an antibody heavy chain is replaced with a larger tryptophan (W) to form a Knobs structure (T366W), and a larger tyrosine (Y) at position 407 in the CH3 region of another antibody heavy chain is mutated into a smaller threonine (T) to form a holes structure (Y407T). The heterologous heavy chain dimerization is promoted by relying on the decrease in the steric hindrance effect after the mutation and the covalent disulfide bond formed in the hinge region, which is the KIH structure. The more commonly used Knobs-into-holes structures in the Fc segment include improving the Y407T single point mutation of the heavy chain hole to a 3-point mutation (T366S, L368A and Y407V) to obtain a stable "1+3" Knobs-into-hole structure, or further adding a S354C site mutation to the heavy chain Knob and a S349C site mutation to the heavy chain hole.

[0217] Tumor microenvironment activating antibodies

[0218] As used herein, "masked antibodies of the present invention", "tumor microenvironment activated antibodies of the present invention", and "TMEAbody (Tumor micro-environment activated antibody) of the present invention" can be used interchangeably, and refer to derivative antibodies comprising the modified mutant CD47 antibody or mutant CD47 antibody (CD47 antibody mutant) described in the first aspect of the present invention, wherein the derivative antibody is a modified biological molecule (including a masking body and a connecting peptide cleavable under pathological conditions), which is activated only in a pathological microenvironment, such as a tumor microenvironment or an inflammatory site. By releasing biological molecules at pathological sites, the targeting efficacy of biological molecules is improved, drug resistance is overcome, and toxicity can be reduced (for example, the toxicity of CD47 antibodies can be reduced).

[0219] It should be understood that the masked antibodies of the present invention include: masked antibodies that are not activated in a pathological microenvironment and masked antibodies that have been activated in a pathological microenvironment (for example, the cleavable connecting peptide is cut by a proteolytic enzyme in the tumor microenvironment to release the masked body and obtain an activated biological molecule).

[0220] Preferably, the modified mutant CD47 antibody of the present invention has reduced binding affinity to CD47 in an unactivated state, but in vitro experiments show that it still maintains a certain CD47 binding ability; and, in an activated state, it restores CD47 binding ability that is equivalent to or better than that of the wild-type CD47 antibody.

[0221] In tumor patients, tumor cells or antigen presenting cells (APCs) carrying tumor antigens partially or completely inhibit the host's immune killing of tumors by binding to T cells. However, the tumor microenvironment activation antibodies of the present invention are activated and released in pathological microenvironments by proteolytic enzymes, especially asparagine endopeptidases or granzymes, or under acidic conditions.

[0222] The antibody used in the present invention may be an antibody known in the art or a functional fragment thereof. For example, the antibody or its functional fragment may be selected from: anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD473 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-TNFα antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-b-CD40 antibody, or anti-ICOS antibody, anti-CD25 antibody, anti-CD470 antibody, anti-CD47 antibody, anti-CD22 antibody, Anti-CCR4 antibody, anti-CD478 antibody, anti-CD52 antibody, anti-complement C5 antibody, anti-RSV F protein, anti-GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib / Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 antibody, anti-Integrinα4 antibody, anti-lgE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR2 antibody, anti-VISTA antibody.

[0223] In the scope of the present invention, the meaning and structure of cytokines are as generally defined in the art. It generally refers to a class of small proteins with a wide range of biological activities, the synthesis and secretion of which are completed by stimulating immune cells, such as monocytes, macrophages, T cells, B cells, NK cells, etc., or non-immune cells, such as endothelial cells, epidermal cells, fibroblasts, etc. Cytokines can regulate cell proliferation, differentiation, action and immune response by binding to corresponding receptors. Suitable cytokines include interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemokines and growth factors, etc.

[0224] Exemplary cytokines include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, TNF-α, TRAP, and TRAIL.

[0225] In a specific embodiment, a CD47 TMEAbody tumor microenvironment activating antibody is provided.

[0226] Furthermore, the modified mutant CD47 antibody has the structure shown below:

[0227] Ab-(P-R1)n

[0228] in,

[0229] Ab is a CD47 antibody mutant;

[0230] R1 is each independently a blocker;

[0231] P is a cleavable connecting peptide;

[0232] n is a positive integer from 1 to 5;

[0233] “-” is a chemical bond, a linker or a connector.

[0234] Preferably, the mask R4 is site-specifically linked to an amino acid site selected from the following group in the CD47 antibody mutant via a cleavable linker peptide P.

[0235] Preferred mutation sites include Y61C in VH and Y49C in VL.

[0236] Within the scope of the present invention, one or more (e.g., within 5, or within 3) amino acid residues in the amino acid sequence of the D1 biomolecule are mutated to cysteine, and covalently linked to the mask (P-R1) through the thiol bond of cysteine. For example, one or two amino acid residues of interest in the biomolecule of interest are mutated to cysteine ​​to be linked to the functional group. For an antibody, the mutation site can be the complementary determining region or the non-complementary determining region of the variable region.

[0237] Preferably, the mutation is a mutation by substitution. More preferably, the mutation occurs in the variable region of the antibody light chain. Generally, a mutant can be prepared first and then tested for its binding activity to the corresponding antigen. If the mutant retains 70% or more, preferably 80% or more, more preferably 90% or more binding activity compared to the wild-type antibody, it is considered that the amino acid residue at the mutation position can be mutated to cysteine ​​to be covalently bonded to the functional group. Alternatively, in certain embodiments, if the conjugate produced by connecting the mutant to R4 retains 80% or more, preferably 90% or more, more preferably 95% or more binding activity, it is considered that the amino acid residue at the mutation position can be mutated to cysteine ​​to be covalently bonded to the functional group.

[0238] As used herein, the term "connector peptide" refers to one or more amino acid residues inserted into an immunoglobulin domain to provide sufficient mobility for the light chain and heavy chain domains to fold into an exchange dual variable region immunoglobulin.

[0239] Suitable examples of linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary with the type of secondary structural elements that need to be achieved in the linker. A preferred linker is as follows: GS, (G4S)n, where n is an integer of 1-4.

[0240] The antibodies of the present invention include not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings of this article, these fragments, derivatives and analogs belong to the well-known scope of those skilled in the art.

[0241] The antibody of the present invention refers to an antibody with anti-CD47 properties. The term also includes variant forms of antibodies having the same functions as the antibody of the present invention and comprising two of the above structures. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and the addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, the addition of one or several amino acids at the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0242] Variant forms of the antibody include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0243] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0244] Table A

[0245]

[0246]

[0247] Encoding nucleic acid and expression vector

[0248] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that only encodes a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0249] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0250] The nucleic acids (and nucleic acid combinations) of the present invention can be used to produce the recombinant antibodies of the present invention in a suitable expression system.

[0251] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0252] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0253] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0254] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0255] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0256] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0257] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0258] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0259] Under early culture conditions, the expression level of bispecific antibodies can reach 3.9 g / L, the purity is above 97%, and lactic acid can be metabolized well during the culture process.

[0260] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0261] The bispecific antibodies of the present invention can be used alone, or combined or coupled with a detectable marker (for diagnostic purposes), a therapeutic agent, or any combination of these substances.

[0262] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0263] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. tumor therapeutic agents (e.g., cisplatin) or any form of anti-tumor drugs, etc.

[0264] Pharmaceutical composition

[0265] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the bispecific antibody or its active fragment or its fusion protein described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, or intracavitary injection.

[0266] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned nanoantibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0267] In the present invention, the bispecific antibody can be used alone, and the dosage regimen can be adjusted to obtain the best desired response, for example, a single administration, or multiple administrations over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0268] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 10 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0269] The main advantages of the present invention include

[0270] (1) The modified mutant CD47 antibody of the present invention has excellent safety, has no effect on hemagglutination of red blood cells, and can effectively reduce the toxic side effects of the antibody.

[0271] (2) The blocked CD47 TMEAbody of the present invention largely blocks the binding of CD47 antibody to CD47 or ADCC activity, whereas the activated CD47 TMEAbody can restore its binding affinity.

[0272] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0273] Example 1: Preparation of rabbit anti-CD47 monoclonal antibody

[0274] New Zealand white rabbits were immunized with human CD47. Single B cells were isolated using a modified protocol from a published literature (Offner et al. PLoS ONE 9(2), 2014). Human CD47+ B cells were sorted into single wells using IgG+ direct flow cytometry. B cell culture supernatants were screened for binding to human CD47 by enzyme-linked immunosorbent assay (ELISA), and B cells were lysed and stored at -80°C.

[0275] The variable regions of the light and heavy chains of CD47-specific B cells were amplified by PCR and cloned into expression vectors described in the published literature (Offner et al. PLoS ONE 9(2), 2014). Each recombinant rabbit monoclonal antibody was expressed in Expi293 cells and purified with protein A. The purified anti-CD47 antibodies were functionally characterized.

[0276] Figure 1 The light chain and heavy chain variable region alignment sequences of each anti-CD47 rabbit antibody are shown.

[0277] The amino acid sequence of the heavy chain variable region VH of 70G1 is SEQ ID NO.1

[0278] QSVKESEGGLFKPTDTLTLTCTVSGFSLS NYAIN WVRQAPGNGLEWIG IINGYGMTYYASWAKS RSTITRNTSENTVTLKLTSLTAADTATYFCAR DRYGSYVGYSSDI WGPGTLVTVSS

[0279] The amino acid sequence of the light chain variable region VL of 70G1 is SEQ ID NO.2

[0280] AYDMTQTPASVEAAVGGTVTIKC QASQNISSRLA WYQQKPGQPPKLLIY GTSTLAS GVPSRFRGSGSGTQFSLTISDLECADAATYYC QHGYYYSSSFTTYNA FGGGTEVVVK

[0281] The amino acid sequence of the heavy chain variable region VH of 71D6 is SEQ ID NO.3

[0282] QSLEESGGDLVKPGASLTLTCTASGFSFS TSYWIY WVRQAPGKGLELIA CIATGSDGSTYYASWASG RFTISKTSSTTVTLQMTSLTAADTATYFCAR EAGNGVYNL WGQGTLVTVSS

[0283] The amino acid sequence of the light chain variable region VL of 71D6 is SEQ ID NO.4

[0284] AYDMTQTPASVEVAVGGTVTIKC QASESIRNALA WYQQKPGQPPKLLIS GASKLAS GVASRFKGSGSATEFTLTISDLECADAATYYC QQYYSSSNVDNG FGGGTEVVVK

[0285] The amino acid sequence of the heavy chain variable region VH of 74F1 is SEQ ID NO.5

[0286] QQQLEESGGDLVKPEGSLTLTCTASGFSFS SRYNMC WVRQAPGKGLEWIA CIYTGSSGSTYYASWAKG RFTISKTSSTVDLKMTSLTAADTATYFCAG GSYSYRLNL WGQGTLVTVSS

[0287] The amino acid sequence of the light chain variable region VL of 74F1 is SEQ ID NO.6

[0288] DVVMTQTPASVSTAVGGTVTIKC QASQSISNELS WYQQKPGQPPKLLIY RASTLES GVSSRFSGSGSGTEFTLTISDLECADAATYYC QATGYGDFGNA FGGGTEVVVK

[0289] 70H1 heavy chain variable region VH amino acid sequence: SEQ ID NO.7

[0290] QQQLEESGGGLVKPEGSLTLTCTASGFSFS SGYDMC WVRQAPGRGLEWIA CIYTRSSGGTYYASWAKG RFTISKTSSTVDLLRMTSLTAADTATYFCAG GRSSYRLNL WGQGALVTVSS

[0291] 70H1 light chain variable region VL amino acid sequence: SEQ ID NO.8

[0292] DVVMTQTPSPVEAAVGGTVTIKC QASQSISNELS WYQQKPGQPPKLLIY RASTLES GVSSRFKGSGSGTEFTLTISDLECADAATYYC QSTFYGDYGNA FGGGTEVVVK

[0293] 69F12 heavy chain variable region VH amino acid sequence: SEQ ID NO.9

[0294] QSVEESEGGLFKPTDTLTLTCTISGFSLS NYDMI WVRQAPGNGLEYIG IISSRDKAGHAAWAKR RSTITRNPKENTVTLKMTSLTVADTATYFCAR RFSSMHGLDI WGPGTLVTVSS

[0295] 69F12 light chain variable region VL amino acid sequence: SEQ ID NO.10

[0296] AYDMTQTPSSVSAAVGGTVTIKC QASQSISNYLS WYQQKPGQPPKLLIY LASTLAS GVPSRFKGSGSGTQFTLTISGVQCADAATYYC QQGYSSDSVDNV FGGGTEVVVK

[0297] Example 2: FACS binding of CD47 antibody to CHOK1-CD47 cells

[0298] The desired overexpression cell line CHOK1-CD47 (RPMI-1640 (ATCC Modification, Gibico, Cat#A10491-01) + 10% FBS (BI, Cat#04-001-1A) + 1×P / S + 6ug / ml Puromecin (Gibico, Cat#a1113803, 10×1ml (10mg / ml)) was expanded to the logarithmic phase, the cells were digested and collected, and the cells were blown into a single cell suspension, centrifuged and the supernatant was discarded. Resuspended with FACS buffer (PBS + 2% FBS), counted, and the cell density was adjusted to 2×10 6 / ml, 100ul / well was added to a 96-well plate (Corning, Cat#3799), centrifuged, the supernatant was discarded, and the antibody sample to be tested (100nM as the starting concentration, 5-fold gradient dilution) 100ul / well was added, mixed with the cells, and incubated at 4°C for 1h. Washed 3 times by centrifugation with FACS buffer, 100ul / well Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscienceTM, Invitrogen, Cat#12-4998-82, 1:200) was added, and incubated at 4°C for 1h. After washing 3 times by centrifugation with FACS buffer, 200ul / well FACS buffer was resuspended and detected using FACS (BECKMAN COULTER, CytoFLEX). Data fitting was performed using software (GraphPad Prism8) to analyze the results.

[0299] like Figure 2 As shown, all five antibodies bound well to CHOK1-CD47 cells, and the EC50 (nM) of 70G1, 71D6, 74F1, 70H1, and 69F12 were 2.88, 3.80, 2.82, 0.91, and 0.80, respectively.

[0300] Example 3: 70H1 TMEAbody site screening

[0301] In this example, the inventors selected 70H1 as the parent antibody, performed TMEA body site screening, and artificially synthesized the corresponding DNA coding sequence (codon optimized) for expression in the host.

[0302] The synthesized DNA was digested and ligated to a modified pTT5 vector (Biovector) to generate pTT5-anti-CD47. Using pTT5-anti-CD47 as a template, cysteine ​​mutations were introduced, primers were designed to replace the codons at the mutation position with cysteine ​​(Cys) codons, PCR was performed, and the mutation fragments were digested to construct a modified pTT5 vector.

[0303] The expression host was HEK293T cells (Life Technologies). Before transfection, HEK293T cells were cultured in complete medium containing 10% FBS (Gibico) at 37°C and 5% CO2. One day before transfection, cells were seeded into 15 cm culture dishes at an appropriate density, and the medium was changed to low IgG FBS (Gibico). 6 h after transfection or on the second day, the medium was changed to Freestyle293 (Gibico).

[0304] On the day of transfection, when the cells reached a certain degree of confluence, the plasmid expressing the target protein was co-transfected into 293T cells using lipofectamine 2000 (Life Technologies) and PEI (Sigma). The antibody expression plasmid includes the heavy chain and light chain of the antibody. The culture supernatant was recovered on the 4th and 6th day after transfection, respectively. The expression and activity of the protein or antibody were detected, and the protein or antibody was purified.

[0305] The hypervariable regions of the variable regions in the heavy and light chains of antibodies (Ab) constitute the antigen (Ag) binding site of the antibody. Because the antigen binding site is complementary to the structure of the antigen epitope, the hypervariable region is also called the complementarity determining region (CDR) of the antibody.

[0306] In the present invention, the activity of each mutant with a point mutation compared with its initial antibody was detected by ELISA. Specifically, a 96-well plate was coated with an antibody antigen overnight, then blocked with 1% BSA blocking agent (Thermo Fisher) at 37°C for 2h, and washed 3 times with PBST. The corresponding antibody or the corresponding mutant was added, combined for 1h at 37°C, and then washed 3 times with PBST. HRP enzyme-linked anti-human IgG was added, combined for 1h at 37°C, and washed 3 times with PBST. TMB substrate (Solarbio., Inc.) was used to detect the absorbance at 450nm.

[0307] The effect on the binding strength of the mutant was calculated by EC50 post-mutation / EC50 wild type.

[0308] According to the above method, the binding activities of the anti-CD47 antibody 70H1 mutants with mutations at different positions shown in Table 1 were detected.

[0309] 70H1 heavy chain variable region VH amino acid sequence: SEQ ID NO.7

[0310] QQQLEESGGGLVKPEGSLTLTCTASGFSFS SGYDMC WVRQAPGRGLEWIA CIYTSSGGTYYASWAKG RFTISKTSSTVDLLRMTSLTAADTATYFCAG GRSSYRLNL WGQGALVTVSS

[0311] 70H1 light chain variable region VL amino acid sequence: SEQ ID NO.8

[0312] DVVMTQTPSPVEAAVGGTVTIKC QASQSISNELS WYQQKPGQPPKLLIY RASTLES GVSSRFKGSGSGTEFTLTISDLECADAATYYC QSTFYGDYGNA FGGGTEVVVK

[0313] Table 1: Effect of mutation position in antibody heavy chain variable region / light chain variable region on binding activity

[0314]

[0315]

[0316] Among them, VL-49 is located in the FR region.

[0317] Example 4. CD47 TMEAbody reduces binding affinity to CD47 and can restore

[0318] Based on the structural research and screening of the inventors, the results show that the VH-61 (YC) site has an excellent blocking effect (the site is the 61st position in VH3, see the 61st Y site underlined in the VH3 sequence in Table 6). In this example, the coding sequence of the corresponding CD47 antibody (70H1.IgG1) containing VH-61 (Y61C mutation) was synthesized and expressed to obtain a recombinant antibody. In addition, the obtained CD47 antibody was coupled with PEG (molecular weight of about 20K or other molecular weight PEG) to obtain CD47 TMEAbody.

[0319] 4.1ELISA detection of CD47 antibody

[0320] Dilute the recombinant human CD47 protein (DIMA BIOTECH, Cat#PME-100008) with PBS to 1ug / ml, coat 100ul / well on the ELISA plate, and incubate overnight at 4℃. Wash the plate three times with 300ul / well PBST the next day. Add 300ul / well PBST (W / W) blocking solution containing 1% BSA, block at 37℃ for 2h. After discarding the blocking solution, wash the plate three times with PBST. Add 100ul / well of antibody (5-fold gradient dilution, eight points), incubate at 37℃ for 1h. Discard the antibody and wash the plate three times with PBST. Add Goat anti-HumanIgG Fc Highly Cross-Adsorbed Secondary Antibody, HRP (Invitrogen, Cat#A18829, 1:5000, 100ul / well), incubate at 37℃ for 0.5h, and wash three times with PBST. Add 100ul / well TMB colorimetric substrate (solarbio, Cat#PR1200), incubate at 37°C for 5min, and then add 50ul / well ELISA stop solution (solarbio, Cat#C1058). Use a microplate reader (ELx800 Absorbance Microplate Reader, BioTek) to read OD450. Use software (GraphPadPrism8) to analyze, perform data fitting, and analyze the results.

[0321] like Figure 3 As shown in Table 2.1, the binding affinity of CD47 TMEAbody (70H1.IgG1+20K) to human CD47 protein is about 7 times weaker than that of 70H1, which can be restored by legumain (leguminous protein, also known as asparaginylendopeptidase (AEP)).

[0322] Table 2.1

[0323] Antibody Name EC50(nM) 70H1.IgG1 0.12 70H1.IgG1.Y61C 0.07 CD47TMEAbody 0.72 CD47TMEAbody activated 0.15 Human IgG1 NA

[0324] 4.2 Affinity detection of CD47 antibody

[0325] The binding strength between antigen and antibody was detected by BIAcore T200 instrument, and the binding strength between CD47 antigen and antibody was detected by amino coupling of CM5 chip (purchased from Cytiva, 29149603). First, CD47 antigen was fixed to CM5 chip, and according to the instructions of Amine Coupling Kit (purchased from Cytiva, BR100050), PBST was used as mobile phase, NHS and EDC were mixed, the chip was activated for about 420s, CD47 antigen was diluted to 10μg / mL with 10mM sodium acetate pH 5.5, and the sample was injected to reach 141.2RU level, and finally 1M ethanolamine (pH8.5) was used to block the remaining activation sites. Then, the affinity of antibody and CD47 antigen was determined by multi-cycle kinetic method. In each cycle, a single concentration of antibody protein was injected, and the binding and dissociation process of antibody and antigen protein was recorded. Finally, the chip was regenerated with Glycine-HCl pH 1.5, where the mobile phase was PBST, the flow rate was 30μL / min, the regeneration time was 15s, and the detection temperature was 25°C. Finally, according to the 1:1binding model, the data was analyzed and the antibody-antigen binding kinetic parameters were fitted, including the binding rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax.

[0326] The binding rate (Ka), dissociation rate (Kd) and binding affinity (KD) of CD47 antibody to human CD47 antigen are shown in Table 2.2. Compared with 70H1, the affinity of CD47 TMEAbody decreased by 470 times.

[0327] Table 2.2 Affinity of CD47 antibody and human CD47 antigen detected by SPR (biacore)

[0328] Antibody Name Ka(1 / Ms) Kd(1 / s) KD(M) 70H1 4.54E+06 1.40E-04 3.08E-11 CD47TMEAbody 2.44E+04 3.43E-04 1.41E-08

[0329] 4.3CD47 TMEAbody weakens the binding of CD47 antibody to CHOK1-CD47 cells and can restore

[0330] The desired overexpression cell line CHOK1-CD47 (RPMI-1640 (ATCC Modification, Gibico, Cat#A10491-01) + 10% FBS (BI, Cat#04-001-1A) + 1×P / S + 6ug / ml Puromecin (Gibico, Cat#A1113803, 10×1ml (10mg / ml))) was expanded to the logarithmic phase, the cells were digested and collected and pipetted into a single cell suspension, the cells were counted and centrifuged, and the supernatant was discarded. Resuspended in FACS buffer (PBS + 2% FBS) to 2×106 / ml, 100ul / well was added to a 96-well plate (Corning, Cat#3799), centrifuged, the supernatant was discarded, 100ul / well of the antibody sample to be tested (100nM as the starting concentration, 5-fold gradient dilution, eight points) was added, mixed with the cells, and incubated at 4°C for 1h. Washed 3 times by centrifugation with FACS buffer, 100ul / well Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscienceTM, Invitrogen, Cat#12-4998-82, 1:200) was added, and incubated at 4°C for 1h. After washing 3 times by centrifugation with FACS buffer, 200ul / well FACS buffer was resuspended and detected using FACS (BECKMAN COULTER, CytoFLEX). Data fitting was performed using software (GraphPad Prism8) to analyze the results.

[0331] like Figure 4 As shown in Table 3, the binding of 70H1 TMEAbody to cells was reduced by about 20 times compared with 70H1. However, when activated by asparagine endopeptidase, the activated 70H1 TMEAbody restored the binding equivalent to that of 70H1IgG1-mutation.

[0332] Therefore, CD47 TMEAbody (70H1.IgG1+20K) can largely block the binding ability of 70H1 antibody to CHOK1-CD47 cells. At the same time, activated CD47 TMEAbody can restore its binding ability to CHOK1-CD47 cells.

[0333] Table 3

[0334]

[0335]

[0336] Example 5 CD47 TMEAbody attenuates the blocking activity of CD47 antibody and can restore

[0337] 5.1 Dose-dependent binding of CD47 antibody to Raji cells

[0338] Expand the desired cell line Raji (RPMI-1640 (ATCC Modification, Gibico, Cat#A10491-01) + 10% FBS (BI, Cat#04-001-1A) + 1×P / S) to the logarithmic phase, collect the cells and pipette into a single cell suspension, centrifuge and discard the supernatant. Resuspend with FACS buffer (PBS + 2% FBS), count, and adjust the cell density to 2×10 6 / ml, 100ul / well was added to a 96-well plate (Corning, Cat#3799), centrifuged, the supernatant was discarded, and the antibody sample to be tested (100nM as the starting concentration, 5-fold gradient dilution) 100ul / well was added, mixed with the cells, and incubated at 4°C for 1h. Washed 3 times by centrifugation with FACS buffer, 100u / well lGoat anti-Human IgG Fc Secondary Antibody, PE (eBioscienceTM, Invitrogen, Cat#12-4998-82, 1:200) was added, and incubated at 4°C for 1h. After washing 3 times by centrifugation with FACS buffer, 200ul / well FACS buffer was resuspended and detected using FACS (BECKMAN COULTER, CytoFLEX). Data fitting was performed using software (GraphPad Prism8) to analyze the results.

[0339] like Figure 5 As shown in Table 4, the binding of CD47 TMEAbody to Raji cells was reduced by about 60 times compared with 70H1. However, when activated by asparagine endopeptidase, the activated 70H1 TMEAbody restored the binding equivalent to that of 70H1IgG1-mutation.

[0340] Table 4

[0341] Antibody Name EC50(nM) 70H1.IgG1.Y61C 0.47 CD47TMEAbody 31.94 CD47TMEAbody activated 0.24 Human IgG1 NA

[0342] 5.2 Detection of blocking function activity of CD47 antibody

[0343] Expand the desired cell line Raji (RPMI-1640 (ATCC Modification, Gibico, Cat#A10491-01) + 10% FBS (BI, Cat#04-001-1A) + 1×P / S) to the logarithmic phase, collect the cells and pipette into a single cell suspension, centrifuge and discard the supernatant. Resuspend with FACS buffer (PBS + 2% FBS), count, and adjust the cell density to 2×10 6 / ml, 100ul / well was added to a 96-well plate (Corning, Cat#3799), centrifuged, the supernatant was discarded, and the antibody sample to be tested (CD47 Antibodies 200nM as the starting concentration, 5-fold gradient dilution, eight points and Human SIPR alpha, mFc (DIMA Biotech, PME100531) 16ug / ml were mixed in equal volumes) 100ul / well, mixed with cells, and incubated at 4°C for 1h. Washed by centrifugation with FACS buffer 3 times, 100ul / well Goat F(ab')2Anti-Mouse IgG-Fc(PE), pre-adsorbed (abcam, ab98742, 1:200) was added, and incubated at 4°C for 1h. After centrifugation and washing with FACS buffer 3 times, 200ul / well FACS buffer was resuspended and detected using FACS (BECKMAN COULTER, CytoFLEX). Data fitting was performed using software (GraphPad Prism8) to analyze the results.

[0344] like Figure 6 As shown, the activity of CD47 TMEAbody (70H1.IgG1-mutantion+20K) in blocking the binding of CD47 to SIRPα on Raji cells was greatly reduced, and the blocking activity of CD47 TMEAbody was restored after being activated.

[0345] Example 6. CD47 TMEAbody blocks the binding of CD47 antibody to red blood cells

[0346] Human red blood cells (RBC) (XFB-RBC, 10 ml) were centrifuged and the supernatant was discarded. Resuspended with FACS buffer (PBS + 2% FBS), counted, and the cell density was adjusted to 2×10 6 / ml, add 100ul / well to a 96-well plate, centrifuge, discard the supernatant, add the antibody to be tested (100nM as the starting concentration, 5-fold gradient dilution, eight points), 100ul / well, and mix with the cells. Incubate at 4℃ for 1h. Wash three times by centrifugation with FACS buffer, add 100u / well Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscienceTM, Invitrogen, Cat#12-4998-82, 1:200), and incubate at 4℃ for 1h. After washing three times by centrifugation with FACS buffer, resuspend in 200ul / well FACS buffer and detect using FACS (BECKMAN COULTER, CytoFLEX). Data fitting and analysis of results were performed using software (GraphPad Prism8).

[0347] like Figure 7 As shown, CD47 antibody binds strongly to erythrocytes, while CD47 TMEAbody hardly binds to erythrocytes.

[0348] Example 7 Human red blood cell (RBC) agglutination test

[0349] Human red blood cells (RBC) (XFB-RBC, 10 ml) were centrifuged and the supernatant was discarded. The cells were washed twice with washing buffer (1×PBS+0.05MEDTA), resuspended in PBS and counted. The cell density was adjusted to 2×10 6 / ml. Add 100ul / well to a 96-well plate (Corning, Cat#3799), centrifuge and discard the supernatant. Add 50ul / well of the antibody to be tested (100nM as the starting concentration, 5-fold gradient dilution, 7 points + 0 point), and incubate at 37°C for 1h.

[0350] like Figure 8 As shown, except for the positive antibody 5F9 which causes erythrocyte agglutination at high concentrations, the other CD47 antibodies do not cause erythrocyte agglutination.

[0351] Example 8. CD47 TMEAbody loses antibody-dependent cell-mediated cytotoxicity (ADCC) and can be restored

[0352] Raji (RPMI-1640 (ATCC Modification, Gibico, Cat#A10491-01) + 10% FBS (BI, Cat#04-001-1A) + 1×P / S) was expanded to the logarithmic phase, the cells were collected and pipetted into a single cell suspension, the supernatant was discarded after centrifugation, the cells were resuspended in culture medium and counted, and the cell density was adjusted to 2×10 5 / ml. 50ul / well was added to a 96-well cell culture plate (Corning, Cat#3599) and incubated overnight at 37°C and 5% CO2. PBMC (XF, XC11100W) was resuspended in 10% FBS-RPMI1640 medium, centrifuged and the supernatant was discarded, and the cell density was adjusted to 8×10 6 / ml. Add 50μl / well of the antibody to be tested and 50μl of PBMC cells (E:T ratio is 20:1) to the culture plate. Place the culture plate in a CO2 incubator and incubate for 24h. ADCC experiment LDH kit detection (Cytotoxicity Detection KitPLUS (LDH), Roche, Cat#4744934001).

[0353] like Fig. 9,As shown in Table 5, compared with the 70H1 antibody, the CD47 TMEAbody lost its ADCC activity and recovered its ADCC activity after being activated.

[0354] Table 5

[0355] Antibody Name EC50(nM) 70H1.IgG1.Y61C 0.17 CD47TMEAbody 16.32 CD47TMEAbody activated 0.14 Human IgG1 NA

[0356] Example 9. Humanization of 70H1

[0357] The humanized light chain template of the rabbit antibody 70H1 was IGKV1_5, and the humanized heavy chain template was IGHV3_NL1. The CDRs of the rabbit antibody 70H1 were transplanted into the corresponding human templates, and then the key amino acids in the FR region sequence of the antibody were back mutated to ensure the original affinity. The specific mutation design is shown in Table 6. Finally, the different heavy chains and light chains obtained by the back mutation of the humanized antibody were combined to obtain 12 humanized antibodies.

[0358] Table 6.

[0359]

[0360] SEQ. represents SEQ ID NO.

[0361] Fig.10 The FACS binding of 12 different humanized antibodies to CHOK1-CD47 cells was shown, and the FACS EC50 of different antibodies is shown in Table 7. The final preferred antibody was 70H1-VH3VL1.

[0362] Table 7

[0363]

[0364]

[0365] Example 10. In vivo efficacy of CD47 TMEAbody in mouse tumor (RAJI) model

[0366] Human Burkitt's lymphoma cells RAJI cells were subcutaneously transplanted into NOD-SCID mice to construct an in vivo model. RAJI cells were cultured according to the culture conditions, and cells were collected and counted in the exponential growth phase. Each mouse was inoculated with 1×10 7 Burkitt's lymphoma cells RAJI (suspended in 0.1 ml basal medium) were used for inoculation. After the tumor grew to 80-114 mm 3 At 7 days after tumor loading, mice were randomly divided into two groups according to their weight and tumor volume: PBS group,

[0367] 70H1.IgG1.Y61C (1 mg / kg) group,

[0368] 70H1.IgG1.Y61C+20K (0.3 mg / kg) group,

[0369] 70H1.IgG1.Y61C+20K (1 mg / kg) group,

[0370] 70H1.IgG1.Y61C+20K (3 mg / kg) group.

[0371] Among them, 70H1.IgG1.Y61C+20K is a CD47 TMEAbody coupled with PEG with a molecular weight of about 20K.

[0372] Treatment started on the day of grouping. The mice were weighed and the tumor volume was measured twice a week. The volume was calculated using the formula tumor volume = 1 / 2 × length × width × width (mm 3 ). Tumor inhibition rate TGI (%) = [1-(Tt-T0) / (Ct-C0)] × 100%; tumor inhibition rate T / C (%) = (Tt / T0) / (Ct / C0) × 100%. T0 is the tumor volume of the test antibody group at the time of grouping; Tt is the tumor volume of the test antibody group during each measurement; C0 is the average tumor volume of the PBS group at the time of grouping; Ct is the average tumor volume of the PBS group at each measurement.

[0373] like Fig.11 As shown, 70H1.IgG1.Y61C+20K showed significant tumor growth inhibition in the subcutaneous RAJI transplanted tumor model of mice. After 31 days of treatment, the inhibition rates of 70H1.IgG1.Y61C (1 mg / kg), 70H1.IgG1.Y61C+20K (0.3 mg / kg), 70H1.IgG1.Y61C+20K (1 mg / kg), 70H1.IgG1.Y61C+20K (3 mg / kg), and 5F9 (1 ​​mg / kg) were 60.91%, 6.45%, 69.46%, 98.97%, and 74.55%, respectively, and the T / C was 41.81%, 93.73%, 33.06%, 4.51%, and 28.36%, respectively. There was no significant change in body weight during treatment.

[0374] like Fig.11 As shown, the results show that 70H1.IgG1.Y61C+20K can be activated in the tumor microenvironment and stimulate anti-tumor immune response, and it has similar in vivo efficacy to 70H1.IgG1.Y61C while increasing the administration dose. The specific data of tumor volume on different days are shown in Table 8.1 or Table 8.2 below.

[0375] Table 8.1. Detailed data of tumor volume on different days

[0376]

[0377] Table 8.2. Detailed data of tumor volume on different days

[0378]

[0379] Example 11. In vivo efficacy of CD47 TMEAbody in a mouse tumor (SHP-77) model

[0380] Human small cell lung cancer cells SHP-77 cells were subcutaneously transplanted into CB17-SCID mice to construct an in vivo model. SHP-77 cells were cultured according to the culture conditions, and cells were collected and counted in the exponential growth phase. Each mouse was inoculated with 2.5×10 6 Human small cell lung cancer cells SHP-77 cells (suspended in 0.1 ml of basal culture medium). After inoculation, wait until the tumor grows to 60-103 mm 3 At 5 days after tumor loading, mice were randomly divided into groups according to their weight and tumor volume. The experiment was divided into:

[0381] PBS group,

[0382] 70H1.IgG1.Y61C+20K (0.3 mg / kg) group,

[0383] 70H1.IgG1.Y61C+20K (1 mg / kg) group,

[0384] 70H1.IgG1.Y61C+20K (3 mg / kg) group,

[0385] Among them, 70H1.IgG1.Y61C+20K is a CD47 TMEAbody coupled with PEG with a molecular weight of about 20K.

[0386] Treatment started on the day of grouping. The mice were weighed and the tumor volume was measured twice a week. The volume was calculated using the formula tumor volume = 1 / 2 × length × width × width (mm 3 ). Tumor inhibition rate TGI (%) = [1-(Tt-T0) / (Ct-C0)] × 100%; tumor inhibition rate T / C (%) = (Tt / T0) / (Ct / C0) × 100%. T0 is the tumor volume of the test antibody group at the time of grouping; Tt is the tumor volume of the test antibody group during each measurement; C0 is the average tumor volume of the PBS group at the time of grouping; Ct is the average tumor volume of the PBS group at each measurement.

[0387] like Fig.12As shown, 70H1.IgG1.Y61C+20K showed significant tumor growth inhibition in the subcutaneous SHP77 transplanted tumor model of mice. After 38 days of treatment, the inhibition rates TGI of the 70H1.IgG1.Y61C+20K (0.3 mg / kg) group, the 70H1.IgG1.Y61C+20K (1 mg / kg) group and the 70H1.IgG1.Y61C+20K (3 mg / kg) group were 75.38%, 105.46% and 105.53%, respectively, and the T / C was 29.52%, 1.13% and 1.02%, respectively. There was no significant change in body weight during the treatment.

[0388] like Fig.12 As shown, the results show that 70H1.IgG1.Y61C+20K can be activated in the tumor microenvironment and stimulate anti-tumor immune response. Among them, the specific data of tumor volume on different days are shown in Tables 9.1 and 9.2 below.

[0389] Table 9.1. Detailed data of tumor volume on different days

[0390]

[0391] Table 9.2. Detailed data of tumor volume on different days

[0392]

[0393] The sequence involved in the present invention is as follows:

[0394] SEQ ID NO.1 70G1 VH

[0395] QSVKESEGGLFKPTDTLTLTCTVSGFSLSNYAINWVRQAPGNGLEWIGIINGYGMTYYASWAKSRSTITRNTSENTVTLKLTSLTAADTATYFCARDRYGSYVGYSSDIWGPGTLVTVSS

[0396] SEQ ID NO.2 70G1 VL

[0397] AYDMTQTPASVEAAVGGTVTIKCQASQNISSRLAWYQQKPGQPPKLLIYGTSTLASGVPSRFRGSGSGTQFSLTISDLECADAATYYCQHGYYYSSSFTTYNAFGGGTEVVVK

[0398] SEQ ID NO.3 71D6 VH

[0399] QSLEESGGDLVKPGASLTLTCTASGFSFSTSYWIYWVRQAPGKGLELIACIATGSDGSTYYASWASGRFTISKTSSTTVTLQMTSLTAADTATYFCAREAGNGVYNLWGQGTLVTVSS

[0400] SEQ ID NO.4 71D6 VL

[0401] AYDMTQTPASVEVAVGGTVTIKCQASESIRNALAWYQQKPGQPPKLLISGASKLASGVASRFKGSGSATEFTLTISDLECADAATYYCQQYYSSSNVDNGFGGGTEVVVK

[0402] SEQ ID NO.5 74F1 VH

[0403] QQQLEESGGDLVKPEGSLTLTCTASGFSFSSRYNMCWVRQAPGKGLEWIACIYTGSSGSTYYASWAKGRFTISKTSSTVDLKMTSLTAADTATYFCAGGSYSYRLNLWGQGTLVTVSS

[0404] SEQ ID NO.6 74F1 VL

[0405] DVVMTQTPASVSTAVGGTVTIKCQASQSISNELSWYQQKPGQPPKLLIYRASTLESGVSSRFSGSGSGTEFTLTISDLECADAATYYCQATGYGDFGNAFGGGTEVVVK

[0406] SEQ ID NO.7 70H1 VH

[0407] QQQLEESGGGLVKPEGSLTLTCTASGFSFSSGYDMCWVRQAPGRGLEWIACIYTRSSGGTYYASWAKGRFTISKTSSTVDLRMTSLTAADTATYFCAGGRSSYRLNLWGQGALVTVSS

[0408] SEQ ID NO.8 70H1 VL

[0409] DVVMTQTPSPVEAAVGGTVTIKCQASQSISNELSWYQQKPGQPPKLLIYRASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTFYGDYGNAFGGGTEVVVK

[0410] SEQ ID NO.9 69F12 VH

[0411] QSVEESEGGLFKPTDTLTLTCTISGFSLSNYDMIWVRQAPGNGLEYIGIISSRDKAGHAAWAKRRSTITRNPKENTVTLKMTSLTVADTATYFCARRFSSMHGLDIWGPGTLVTVSS

[0412] SEQ ID NO.10 69F12 VL

[0413] AYDMTQTPSSVSAAVGGTVTIKCQASQSISNYLSWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTQFTLTISGVQCADAATYYCQQGYSSDSVDNVFGGGTEVVVK

[0414] SEQ ID NO.11 70H1 VH1

[0415] QVQLVESGGGVVQPGGSLRLSCAASGFTFSSGYDMCWVRQAPGKGLEWVSCIYTRSSGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRSSYRLNLWGQGTLVTVSS

[0416] SEQ ID NO.12 70H1 VH2

[0417] QQQLVESGGGVVQPGGSLRLSCAASGFTFSSGYDMCWVRQAPGKGLEWIACIYTRSSGGTYYASWAKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKGRSSYRLNLWGQGTLVTVSS

[0418] SEQ ID NO.13 70H1 VH3

[0419] <h2 style=";text-align:left;direction:ltr">QQQLVESGGGVVQPGGSLRLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIYTRSSGGTYYASWAKGRFTISKDNSKNTLYLQMNSLRAEDTAVYFCAKGRSSYRLNLWGQGTLVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0420] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.14 70H1 VH4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0421] <h2 style=";text-align:left;direction:ltr"> QQQLVESGGGVVQPGGSLRLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIYTRSSGGTYYASWAKGRFTISKDNSKNTVYLQMNSLRAEDTAVYFCAKGRSSYRLNLWGQGTLVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0422] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.15 70H1 VL1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0423] <h2 style=";text-align:left;direction:ltr"> IK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0424] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.16 70H1 VL2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0425] <h2 style=";text-align:left;direction:ltr"> IK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0426] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.17 70H1 VL3 D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0427] <h2 style=";text-align:left;direction:ltr"> SGYDMC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0428] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.19 70H1 HCDR2 CIYTRSSGGTYYASWAKG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0429] SEQ ID NO.20 70H1 HCDR3 GRSSYRLNL

[0430] SEQ ID NO.21 70H1 LCDR1 QASQSISNELS

[0431] SEQ ID NO.22 70H1 LCDR2 PLANTS

[0432] SEQ ID NO.23 70H1 LCDR3 QSTFYGDYGNA

[0433] SEQ ID NO.24 70H1 HCDR2'CIYTSSGGTYCASWAKG

[0434] SEQ ID NO.25 70G1 HCDR1 NYAIN

[0435] SEQ ID NO.26 70G1 HCDR2 IINGYGMTYYASWAKS

[0436] SEQ ID NO.27 70G1 HCDR3 DRYGSYVGYSSDI

[0437] SEQ ID NO.28 70G1 LCDR1 QASQNISSRLA

[0438] SEQ ID NO.29 70G1 LCDR2 GTSTLAS

[0439] SEQ ID NO.30 70G1 LCDR3 QHGYYYSSSFTTYNA

[0440] SEQ ID NO.31 71D6 HCDR1 TSYWIY

[0441] SEQ ID NO.32 71D6 HCDR2 CIATGSDGSTYYASWASG

[0442] SEQ ID NO.33 71D6 HCDR3 EAGNGVYNL

[0443] SEQ ID NO.34 71D6 LCDR1 QASESIRNALA

[0444] SEQ ID NO.35 71D6 LCDR2 GASKLAS

[0445] SEQ ID NO.36 71D6 LCDR3 QQYYSSSNVDNG

[0446] SEQ ID NO.37 74F1 HCDR1 SRYNMC

[0447] SEQ ID NO.38 74F1 HCDR2 CIYTGSSGSTYYASWAKG

[0448] SEQ ID NO.39 74F1 HCDR3 GSYSYRLNL

[0449] SEQ ID NO.40 74F1 LCDR1 QASQSISNELS

[0450] SEQ ID NO.41 74F1 LCDR2 RASTLES

[0451] SEQ ID NO.42 74F1 LCDR3 QATGYGDFGNA

[0452] SEQ ID NO.43 69F12 HCDR1 NYDMI

[0453] SEQ ID NO.44 69F12 HCDR2 IISSRDKAGHAAWAKR

[0454] SEQ ID NO.45 69F12 HCDR3 RFSSMHGLDI

[0455] SEQ ID NO.46 69F12 LCDR1 QASQSISNYLS

[0456] SEQ ID NO.47 69F12 LCDR2 LASTLAS

[0457] SEQ ID NO.48 69F12 LCDR3 QQGYSSDSVDNV

[0458] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A mutant CD47 antibody, characterized in that: The mutant CD47 antibody introduces one or more mutations in the complementarity determining region (CDR region) of the heavy chain variable region and / or the light chain variable region relative to the wild-type CD47 antibody sequence, and the mutation is selected from the following group: The Y33, R55, Y61, W65, Y105 or L107 position of the heavy chain variable region; and / or the Q27, Y49, T53, F92, Y93, Y96 or N98 position of the light chain variable region comprises a mutation in which one or more amino acids are replaced with cysteine ​​(Cys); Wherein, the affinity of the wild-type CD47 antibody binding to CD47 is E0, the affinity of the mutant CD47 antibody binding to CD47 is E1, and E1 / E0≤5.5 (preferably E1 / E0≤3, preferably ≤1.5, and more preferably E1 / E0≤1); Wherein, the wild-type CD47 antibody has a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises three heavy chain variable regions as shown below: HCDR1 SGYDMC (SEQ ID NO. 18), HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.19), HCDR3 GRSSYRLNL (SEQ ID NO. 20), and The light chain variable region comprises three light chain variable regions as shown below: LCDR1 QASQSISNELS(SEQ ID NO.21), LCDR2 RASTLES(SEQ ID NO.22), LCDR3 QSTFYGDYGNA(SEQ ID NO.23); Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain CD47 binding affinity.

2. The mutant CD47 antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the mutant CD47 antibody has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the heavy chain variable region of SEQ ID NO.7, 11, 12, 13 or 14; and The amino acid sequence of the light chain variable region of the mutant CD47 antibody has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the light chain variable region of SEQ ID NO.8, 15, 16 or 17.

3. A CD47 antibody or an antigen-binding fragment thereof, characterized in that: The CD47 antibody has a heavy chain variable region and a light chain variable region, (1) The heavy chain variable region comprises three heavy chain variable regions as shown below, HCDR1 SGYDMC (SEQ ID NO. 18), HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.19), HCDR3 GRSSYRLNL (SEQ ID NO. 20), and The light chain variable region comprises three light chain variable regions as shown below: LCDR1 QASQSISNELS(SEQ ID NO.21), LCDR2 RASTLES(SEQ ID NO.22), LCDR3 QSTFYGDYGNA (SEQ ID NO. 23); or (2) The heavy chain variable region comprises three heavy chain variable regions as shown below, HCDR1 SGYDMC (SEQ ID NO. 25), HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.26), HCDR3 GRSSYRLNL (SEQ ID NO. 27), and The light chain variable region comprises three light chain variable regions as shown below: LCDR1 QASQSISNELS(SEQ ID NO.28), LCDR2 RASTLES(SEQ ID NO.29), LCDR3 QSTFYGDYGNA (SEQ ID NO. 30) or (3) The heavy chain variable region comprises three heavy chain variable regions as shown below, HCDR1 SGYDMC (SEQ ID NO. 31), HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.32), HCDR3 GRSSYRLNL (SEQ ID NO. 33), and The light chain variable region comprises three light chain variable regions as shown below: LCDR1 QASQSISNELS(SEQ ID NO.34), LCDR2 RASTLES(SEQ ID NO.35), LCDR3 QSTFYGDYGNA (SEQ ID NO. 36) or (4) The heavy chain variable region comprises three heavy chain variable regions as shown below, HCDR1 SGYDMC (SEQ ID NO. 37), HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.38), HCDR3 GRSSYRLNL (SEQ ID NO. 39), and The light chain variable region comprises three light chain variable regions as shown below: LCDR1 QASQSISNELS(SEQ ID NO.40), LCDR2 RASTLES(SEQ ID NO.41), LCDR3 QSTFYGDYGNA (SEQ ID NO. 42) or (5) The heavy chain variable region comprises three heavy chain variable regions as shown below, HCDR1 SGYDMC (SEQ ID NO. 43), HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.44), HCDR3 GRSSYRLNL (SEQ ID NO. 45), and The light chain variable region comprises three light chain variable regions as shown below: LCDR1 QASQSISNELS(SEQ ID NO.46), LCDR2 RASTLES(SEQ ID NO.47), LCDR3 QSTFYGDYGNA(SEQ ID NO.48); Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain CD47 binding affinity.

4. A recombinant protein, characterized in that The recombinant protein comprises: (1) The mutant CD47 antibody or modified mutant CD47 antibody according to claim 1 or 2, or the anti-CD47 antibody according to claim 3; (2) any polypeptide molecule or fragment having therapeutic function; and / or (3) Optional protein functional domains that help improve the physicochemical and pharmacological properties of the molecule.

5. An isolated polynucleotide, characterized in that The polynucleotide encodes the mutant CD47 antibody according to claim 1 or 2 or the anti-CD47 antibody according to claim 3.

6. A carrier, characterized in that The vector contains the polynucleotide according to claim 5.

7. A genetically engineered host cell, characterized in that The host cell contains the vector of claim 6 or the polynucleotide of claim 5 is integrated into its genome.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (I) the mutant CD47 antibody according to claim 1 or 2 or the anti-CD47 antibody according to claim 3; and (II) a pharmaceutically acceptable carrier.

9. An immunoconjugate, characterized in that: The immunoconjugate comprises: (a) the mutant CD47 antibody according to claim 1 or 2 or the anti-CD47 antibody according to claim 3; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

10. Use of the mutant CD47 antibody according to claim 1 or 2, the anti-CD47 antibody according to claim 3, the pharmaceutical composition according to claim 8 or the immunoconjugate according to claim 9, characterized in that: Used for preparing (a) a detection reagent or a kit; and / or (b) a pharmaceutical composition for preventing and / or treating cancer, tumor or autoimmune disease.

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