Tumor microenvironment specifically activated CD47 antibody

Abstract

Provided is a tumor microenvironment specifically activated CD47 antibody. Specifically, provided is a bispecific antibody which is, in a chemical coupling manner, linked to a substrate and a blocker which are highly expressed in a tumor environment and can recognize cleaving. The antibody activity of the bispecific antibody in a normal tissue is suppressed, and the antibody activity of the bispecific antibody is activated in a tumor environment. In addition, the bispecific antibody also has a significantly reduced toxic and side effect.

Description

CD47 antibodies specifically activated by the tumor microenvironment Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a CD47 antibody specifically activated by the 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 the ligand and an intracellular inhibitory motif containing ITIM (immunoreceptor tyrosine-based inhibitory motif), which can bind to SHP-1 (containing sh2 motif) Protein Tyrosine Phosphatase 1) or SHP2 (also known as PTPN11) to transmit information.

[0004] Ovarian cancer tumor heterogeneity and the surrounding tumor microenvironment (TME) have been reported to influence the efficacy of immunotherapy and patient prognosis. The TME of ovarian cancer is inherently heterogeneous. CD47 plays an important role in cell functional behavior and immune homeostasis related to 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 activation of SHP1 and SHP2 by tyrosine phosphorylation of the SIRPαITIM, leading to dephosphorylation of myosin IIA 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 red blood cells were removed from CD47-deficient mice and given to wild-type mice, the CD47-deficient red blood cells were quickly cleared, and inhibiting macrophages reversed this effect, indicating that macrophages play a crucial role in phagocytosis.

[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). CD47 is not directly involved in regulating tumor cell proliferation and survival. Antibody inhibition of CD47 can increase tumor cell phagocytosis even in the absence of T cells, B cells, and NK cells, suggesting that tumor cells can evade macrophage phagocytosis and thus avoid clearance by macrophages.

[0008] The anti-CD47 antibody studied in the present invention has low toxicity and high safety to human body.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a CD47 antibody that specifically activates the tumor microenvironment.

[0011] In a first aspect of the present invention, a mutant CD47 antibody is provided, wherein the mutant CD47 antibody has one or more mutations introduced into the complementarity determining regions (CDR regions) of the heavy chain variable region and / or the light chain variable region relative to the wild-type CD47 antibody sequence, wherein the mutations are selected from the following group:

[0012] a mutation comprising one or more amino acid substitutions to cysteine ​​(Cys) at positions Y33, R55, Y61, W65, Y105, or L107 of the heavy chain variable region; and / or at positions Q27, Y49, T53, F92, Y93, Y96, or N98 of the light chain variable region;

[0013] The wild-type CD47 antibody binds to CD47 with an affinity of E0, the mutant CD47 antibody binds to CD47 with an affinity of E1, and E1 / E0 ≤ 5.5 (preferably E1 / E0 ≤ 3, preferably ≤ 1.5, and more preferably E1 / E0 ≤ 1);

[0014] 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:

[0015] HCDR1 SGYDMC (SEQ ID NO. 18),

[0016] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.19),

[0017] HCDR3 GRSSYRLNL (SEQ ID NO. 20), and

[0018] The light chain variable region comprises three light chain variable regions as shown below:

[0019] LCDR1 QASQSISNELS(SEQ ID NO.21),

[0020] LCDR2 RASTLES(SEQ ID NO.22),

[0021] LCDR3 QSTFYGDYGNA(SEQ ID NO.23);

[0022] Wherein, any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD47 binding affinity.

[0023] 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 derived CDR sequence can retain the affinity for binding to CD47.

[0024] 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).

[0025] 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 capable of retaining CD47 binding affinity is an amino acid sequence with a homology or sequence identity of at least 85%.

[0026] In another preferred embodiment, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0027] 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.

[0028] In another preferred embodiment, the heavy chain variable region of the mutant CD47 antibody comprises three heavy chain variable regions as shown below:

[0029] HCDR1 SGYDMC (SEQ ID NO. 18),

[0030] HCDR2 CIYTRSSGGTYCASWAKG(SEQ ID NO.24),

[0031] HCDR3 GRSSYRLNL (SEQ ID NO. 20), and

[0032] The light chain variable region comprises three light chain variable regions as shown below:

[0033] LCDR1 QASQSISNELS(SEQ ID NO.21),

[0034] LCDR2 RASTLES(SEQ ID NO.22),

[0035] LCDR3 QSTFYGDYGNA(SEQ ID NO.23);

[0036] Wherein, any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD47 binding affinity.

[0037] In another preferred embodiment, the mutant CD47 antibody includes a PEGylated CD47 antibody.

[0038] In another preferred embodiment, the PEGylation is carried out using PEG with a molecular weight of 5-100 kD (preferably 10-50 kD).

[0039] In another preferred embodiment, the PEGylation includes site-directed PEGylation or non-site-directed PEGylation.

[0040] In another preferred embodiment, the affinity for binding to CD47 is the EC50 value for binding to CD47.

[0041] In another preferred embodiment, 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.

[0042] In another preferred embodiment, the wild-type CD47 antibody is a CD47 mouse antibody or a CD47 humanized antibody.

[0043] In another preferred embodiment, 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.

[0044] 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.

[0045] 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.

[0046] 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

[0047] 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

[0048] A light chain variable region amino acid sequence 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. 15, 16 or 17.

[0049] In another preferred embodiment, the mutant CD47 antibody comprises one or more mutations relative to 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.

[0050] In another preferred example, the mutant CD47 antibody comprises a mutation in which one or more amino acids are substituted 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 substituted with cysteine ​​(Cys) in the complementarity determining region (CDR) of the light chain variable region relative to the wild-type CD47 antibody.

[0051] In another preferred embodiment, the mutant CD47 antibody comprises a mutation selected from the group consisting of Y33C, R55C, Y61C, W65C, Y105C or L107C, or a combination thereof, in the complementarity determining region of the heavy chain variable region corresponding to the wild-type CD47 antibody, preferably comprising a Y61C mutation; and / or

[0052] 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.

[0053] In another preferred embodiment, all position numbers of amino acids mentioned for amino acid residues of the antibody are based on the Kabat numbering.

[0054] 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

[0055] 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.

[0056] 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.

[0057] 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.

[0058] In another preferred embodiment, the mutant CD47 antibody is a bispecific, trispecific or multispecific antibody.

[0059] In another preferred embodiment, the mutant CD47 antibody includes a modified mutant CD47 antibody.

[0060] In another preferred embodiment, the modified mutant CD47 antibody is a tumor microenvironment-specific activated antibody (TMEAbody), that is, a masked antibody.

[0061] In another preferred embodiment, the modified mutant CD47 antibody includes, but is not limited to, linking a blocker to the cysteine ​​at the mutation site of the mutant CD47 antibody via a cleavable linker 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.

[0062] In another preferred embodiment, the mutant CD47 antibody includes a modified mutant CD47 antibody having the following structure:

[0063] Ab-(P-R1)n

[0064] in,

[0065] Ab is a mutant CD47 antibody;

[0066] R1 is each independently a blocker;

[0067] P is a cleavable connecting peptide;

[0068] n is a positive integer from 1 to 5;

[0069] “-” represents a chemical bond, a linker, or a connector.

[0070] In another preferred embodiment, the masking body refers to a group that prevents Ab from binding to its ligand or receptor.

[0071] In another preferred embodiment, the masking body is selected from polyethylene glycol-C 1-5 -alkylcarbonyl group:

[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 agent 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 structure shown below:

[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 a chemical bond that can be activated by acid in a pathological microenvironment;

[0078] R3 is absent, or is a linker arm that automatically falls off after R2 is cleaved or R2 is activated by acid in a pathological microenvironment; when R2 is absent, 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 group consisting of 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, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidin, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, peptidase, interstitial protease, plasma protease, pitcher plant 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] wherein X and Y are independently NR' or O, Z is H or C1-10 alkyl, and R' is H or C1-4 alkyl; and R3 is connected to R1 and R4 through X and Y in the form of amide, ester, carbamate, urea or hydrazone bonds.

[0086] In another preferred embodiment, R4 is

[0087] In another preferred embodiment, the structure of P-R1 is as follows:

[0088] Wherein R1 is PEG20K, and the structural formula is:

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

[0090] Wherein R1 is PEG40K, and the structural formula is:

[0091] In another preferred embodiment, the modified mutant CD47 antibody is an activatable antibody.

[0092] In another preferred embodiment, the protease is produced by a tumor proximal 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.

[0093] In another preferred embodiment, R4 in the P is connected to the sulfhydryl group of the mutant CD47 antibody (Ab).

[0094] 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.

[0095] (1) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0096] HCDR1 SGYDMC (SEQ ID NO. 18),

[0097] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.19),

[0098] HCDR3 GRSSYRLNL (SEQ ID NO. 20), and

[0099] The light chain variable region comprises three light chain variable regions as shown below:

[0100] LCDR1 QASQSISNELS(SEQ ID NO.21),

[0101] LCDR2 RASTLES(SEQ ID NO.22),

[0102] LCDR3 QSTFYGDYGNA (SEQ ID NO. 23); or

[0103] (2) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0104] HCDR1 SGYDMC (SEQ ID NO. 25),

[0105] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.26),

[0106] HCDR3 GRSSYRLNL (SEQ ID NO. 27), and

[0107] The light chain variable region comprises three light chain variable regions as shown below:

[0108] LCDR1 QASQSISNELS(SEQ ID NO.28),

[0109] LCDR2 RASTLES(SEQ ID NO.29),

[0110] LCDR3 QSTFYGDYGNA (SEQ ID NO. 30) or

[0111] (3) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0112] HCDR1 SGYDMC (SEQ ID NO. 31),

[0113] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.32),

[0114] HCDR3 GRSSYRLNL (SEQ ID NO. 33), and

[0115] The light chain variable region comprises three light chain variable regions as shown below:

[0116] LCDR1 QASQSISNELS(SEQ ID NO.34),

[0117] LCDR2 RASTLES(SEQ ID NO.35),

[0118] LCDR3 QSTFYGDYGNA (SEQ ID NO. 36) or

[0119] (4) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0120] HCDR1 SGYDMC (SEQ ID NO. 37),

[0121] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.38),

[0122] HCDR3 GRSSYRLNL (SEQ ID NO. 39), and

[0123] The light chain variable region comprises three light chain variable regions as shown below:

[0124] LCDR1 QASQSISNELS(SEQ ID NO.40),

[0125] LCDR2 RASTLES(SEQ ID NO.41),

[0126] LCDR3 QSTFYGDYGNA (SEQ ID NO. 42) or

[0127] (5) The heavy chain variable region comprises three heavy chain variable regions as shown below,

[0128] HCDR1 SGYDMC (SEQ ID NO. 43),

[0129] HCDR2 CIYTRSSGGTYYASWAKG (SEQ ID NO.44),

[0130] HCDR3 GRSSYRLNL (SEQ ID NO. 45), and

[0131] The light chain variable region comprises three light chain variable regions as shown below:

[0132] LCDR1 QASQSISNELS(SEQ ID NO.46),

[0133] LCDR2 RASTLES(SEQ ID NO.47),

[0134] LCDR3 QSTFYGDYGNA(SEQ ID NO.48);

[0135] Wherein, any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD47 binding affinity.

[0136] In another preferred embodiment, 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.

[0137] In another preferred embodiment, 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.

[0138] In another preferred embodiment, 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.

[0139] In another preferred embodiment, 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.

[0140] In another preferred embodiment, 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.

[0141] 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.

[0142] 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.

[0143] In another preferred embodiment, the CD47 antibody is a humanized antibody.

[0144] 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.

[0145] In another preferred embodiment, 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.

[0146] In another preferred embodiment, the antibody comprises a heavy chain and a light chain.

[0147] In another preferred embodiment, the heavy chain further comprises a heavy chain constant region.

[0148] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0149] In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region.

[0150] In another preferred embodiment, the light chain further comprises a light chain constant region.

[0151] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0152] In another preferred embodiment, the light chain constant region is a human antibody light chain kappa constant region.

[0153] A third aspect of the present invention provides a recombinant protein, comprising:

[0154] (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;

[0155] (2) Optional polypeptide molecules or fragments with therapeutic functions; and / or

[0156] (3) Optional protein functional domains that help improve the physicochemical and pharmaceutical properties of the molecule.

[0157] In another preferred embodiment, the recombinant protein further comprises: (4) an optional tag sequence for assisting expression and / or purification.

[0158] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0159] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0160] 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.

[0161] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0162] In another preferred embodiment, the fusion protein includes a multispecific antibody or a chimeric antibody.

[0163] 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).

[0164] 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.

[0165] The fifth aspect of the present invention provides a vector comprising the polynucleotide according to the fourth aspect of the present invention.

[0166] 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.

[0167] The seventh aspect of the present invention provides a pharmaceutical composition, which contains:

[0168] (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

[0169] (II) a pharmaceutically acceptable carrier.

[0170] In another preferred embodiment, the pharmaceutical composition further comprises:

[0171] (b) Other biologically active drugs, such as drugs for treating tumors.

[0172] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0173] 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.

[0174] In another preferred embodiment, the pharmaceutical composition is administered by co-culturing the pharmaceutical composition with immune cells (such as dendritic cells, natural killer cells, lymphocytes, monocytes / macrophages, granulocytes, etc.), and then isolating the immune cells for in vivo reinfusion.

[0175] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0176] In another preferred embodiment, the pharmaceutical composition is used for anti-tumor treatment.

[0177] In an eighth aspect, the present invention provides an immunoconjugate comprising:

[0178] (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

[0179] (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.

[0180] In another preferred embodiment, the components (a) and (b) are operably linked.

[0181] 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.

[0182] In the ninth aspect, 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 kit; and / or (b) a pharmaceutical composition for preventing and / or treating cancer, tumor, or autoimmune disease.

[0183] 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.

[0184] In another preferred embodiment, the tumor includes but is not limited to: breast cancer, liver cancer, gastric cancer, colorectal 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.

[0185] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0186] Figure 1 shows the sequence alignment of the light chain and heavy chain variable regions of each anti-CD47 rabbit antibody.

[0187] FIG2 shows a FACS binding experiment of five CD47 antibodies to CHOK1-CD47 cells.

[0188] FIG3 shows an ELISA binding experiment of CD47 antibody and CD47 antigen.

[0189] FIG4 shows a FACS binding experiment of CD47 antibody to CHOK1-CD47 cells.

[0190] FIG5 shows a FACS binding experiment of CD47 TMEAbody on Raji cells.

[0191] FIG6 shows a binding experiment of CD47 TMEAbody blocking SIRPα on Raji cells.

[0192] FIG7 shows that CD47 TMEAbody blocks the binding of CD47 antibody to erythrocytes.

[0193] FIG8 shows an RBC agglutination experiment using CD47 TMEAbody.

[0194] FIG9 shows the ADCC activity of CD47 TMEAbody on Raji cells.

[0195] FIG10 shows FACS binding experiments of different humanized CD47 antibodies to CHOK1-CD47 cells.

[0196] FIG11 shows the in vivo efficacy experiment of CD47 TMEAbody in a mouse tumor (Raji) model.

[0197] FIG12 shows the in vivo efficacy experiment of CD47 TMEAbody in a mouse tumor (SHP-77) model.

[0198] FIG13 shows the in vivo efficacy experiment of another CD47 TMEAbody in a mouse tumor (SHP-77) model.

[0199] FIG14 shows a graph of the analysis results of changes in the number of red blood cells (RBC) and platelets (PLT) in hSIRPA / hCD47 mice according to the number of days of antibody administration. DETAILED DESCRIPTION

[0200] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that a mutant CD47 antibody, obtained by mutating specific amino acids in the CDR variable region of a wild-type CD47 antibody to Cys, can be chemically coupled to a masking agent to obtain a modified mutant CD47 antibody with reduced binding activity to CD47 and achieve tumor microenvironment-specific activation. This modified mutant CD47 antibody can be activated in the tumor microenvironment and stimulate an anti-tumor immune response while exhibiting a good safety profile. This led to the completion of the present invention.

[0201] the term

[0202] 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.

[0203] 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.

[0204] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0205] Antibody

[0206] Generally, "antibodies" are also called "immunoglobulins" and can be natural or conventional antibodies, in which two heavy chains are connected to each other by disulfide bonds and each heavy chain is connected 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), which 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, the variable domain (VL) and the constant domain (CL). The heavy chain includes four domains, the 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 for 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 one light chain and one heavy chain. The specificity of an antibody depends on the structural complementarity between the antibody binding site and the antigenic determinant region. The antibody binding site is composed primarily of residues from the hypervariable region or complementarity determining region (CDR). Occasionally, residues from the non-hypervariable or framework region (FR) influence the overall domain structure and, in turn, the binding site. The complementarity determining region or CDR refers to the amino acid sequence that collectively defines the binding affinity and specificity of the native Fv region of the natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, separately referred to as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H. A conventional antibody antigen binding site therefore includes six CDRs, comprising a set of CDRs from each heavy and light chain v region.

[0207] As used herein, the terms "single-domain antibody" and "nanobody" have synonymous meanings and refer to the construction of a single-domain antibody consisting solely of a single heavy chain variable region by cloning the variable region of an antibody heavy chain. This is the smallest fully functional antigen-binding fragment. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy chain variable region.

[0208] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do 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.

[0209] As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between the CDRs, that is, it refers to those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. The light and heavy chains of immunoglobulins 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 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 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%.

[0210] "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 achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment for determination of percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software.

[0211] 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.

[0212] 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.

[0213] 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 production of the antibody 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.

[0214] 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.

[0215] As used herein, the term "affinity" is theoretically defined by the equilibrium association between an intact antibody and an antigen. The affinity of the bispecific antibodies of the present invention can be assessed or determined by KD values ​​(dissociation constants) (or other assays), such as bio-layer interferometry (BLI), using a FortebioRed96 instrument.

[0216] 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.

[0217] As used herein, the term "knobs-into-holes (KIH)" refers to the currently mature Fc segment heterodimerization technology. For example, the small threonine (T) at position 366 of the CH3 region of an antibody heavy chain is replaced with a larger tryptophan (W) to form a knob structure (T366W), and the large tyrosine (Y) at position 407 of the CH3 region of another antibody heavy chain is mutated to a smaller threonine (T) to form a holes structure (Y407T). Relying on the reduced steric hindrance effect after the mutation and the covalent disulfide bond formed in the hinge region, the heterologous heavy chain dimerization is promoted, which is the KIH structure. Currently, the more commonly used knob-into-hole structures in the Fc segment include improving the Y407T single point mutation of the heavy chain hole to a three-point mutation (T366S, L368A and Y407V) to obtain a stable "1+3" knob-into-hole structure, or further adding an S354C site mutation to the heavy chain knob and an S349C site mutation to the heavy chain hole.

[0218] Tumor microenvironment-activating antibodies

[0219] As used herein, the terms "masked antibody of the present invention," "tumor microenvironment activated antibody of the present invention," and "TMEAbody (Tumor micro-environment activated antibody) of the present invention" are 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 biomolecule (including a masking body and a linker peptide cleavable under pathological conditions) that is activated only in a pathological microenvironment, such as a tumor microenvironment or inflammatory site. By releasing the biomolecule at the pathological site, the targeting efficacy of the biomolecule is improved, drug resistance is overcome, and toxicity can be reduced (e.g., the toxicity of the CD47 antibody is reduced).

[0220] It should be understood that the masked antibodies of the present invention include: masked antibodies that have not been 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).

[0221] Preferably, the modified mutant CD47 antibody of the present invention has reduced binding affinity to CD47 in the inactivated state, but in vitro experiments show that it still maintains a certain CD47 binding ability; and, in the activated state, it restores CD47 binding ability that is equivalent to or better than that of the wild-type CD47 antibody.

[0222] 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-activating antibodies of the present invention are activated and released in a pathological microenvironment by proteolytic enzymes, particularly asparagine endopeptidases or granzymes, or under acidic conditions.

[0223] The antibody used in the present invention can be an antibody known in the art or a functional fragment thereof. For example, the antibody or its functional fragment can 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. body, 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-Integrin α4 antibody, anti-IgE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR2 antibody, anti-VISTA antibody.

[0224] Within the scope of this disclosure, the meaning and structure of cytokines are as generally defined in the art. Cytokines generally refer to a class of small proteins with a wide range of biological activities, whose synthesis and secretion are achieved 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, function, and immune response by binding to corresponding receptors. Suitable cytokines include interleukins, interferons, tumor necrosis factors, colony-stimulating factors, chemokines, and growth factors.

[0225] 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.

[0226] In a specific embodiment, a CD47 TMEAbody tumor microenvironment activating antibody is provided.

[0227] Furthermore, the modified mutant CD47 antibody has the structure shown below:

[0228] Ab-(P-R1)n

[0229] in,

[0230] Ab is a CD47 antibody mutant;

[0231] R1 is each independently a blocker;

[0232] P is a cleavable connecting peptide;

[0233] n is a positive integer from 1 to 5;

[0234] “-” represents a chemical bond, a linker, or a connector.

[0235] Preferably, the mask R4 is site-specifically linked to an amino acid site selected from the following group of sites in the CD47 antibody mutant via a cleavable linker peptide P.

[0236] Preferred mutation sites include Y61C in VH and Y49C in VL.

[0237] Within the scope of the present disclosure, one or more (e.g., up to five, or up to three) amino acid residues in the amino acid sequence of the D1 biomolecule are mutated to cysteine ​​residues, and covalently linked to the mask (P-R1) via a thiol bond of the cysteine ​​residues. For example, one or two amino acid residues of interest in the biomolecule of interest are mutated to cysteine ​​residues to link to the functional group. For an antibody, the mutation site can be in the complementarity determining region or the non-complementarity determining region of the variable region.

[0238] 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 linked 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 linked to the functional group.

[0239] As used herein, the term "linker peptide" refers to one or more amino acid residues inserted into an immunoglobulin domain to provide sufficient mobility for the light and heavy chain domains to fold into an exchange dual variable region immunoglobulin.

[0240] Suitable linker examples 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 desired to be achieved in the linker. A preferred linker is as follows: GS, (G4S)n, where n is an integer from 1 to 4.

[0241] The antibodies of the present invention include not only intact antibodies, but also immunologically active antibody fragments or fusion proteins formed by antibodies with 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 retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention can be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent 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 extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of additional amino acid sequences to the polypeptide sequence (such as a leader sequence or secretory sequence, a sequence used to purify the polypeptide, a proprotein sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0242] The antibodies of the present invention refer to antibodies with anti-CD47 properties. The term also includes variant forms of antibodies comprising two of the above structures that have the same functions as the antibodies of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or similar properties generally does not change the function of the protein. For another example, addition of one or more amino acids to 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.

[0243] 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.

[0244] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced 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 making amino acid substitutions according to Table A.

[0245] Table A

[0246] Encoding nucleic acid and expression vector

[0247] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. Polynucleotides encoding the mature polypeptides of the present invention include: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0248] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0249] 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.

[0250] The present invention also relates to polynucleotides that hybridize to 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 hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0251] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0252] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0253] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This 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 protein sequences of the present invention by chemical synthesis.

[0254] 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.

[0255] Host cells can be prokaryotic cells, such as bacterial cells; 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 such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0256] 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 capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0257] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used 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 using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0258] Under early culture conditions, the expression level of bispecific antibodies can reach 3.9g / L, the purity is above 97%, and lactic acid can be metabolized well during the culture process.

[0259] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing 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, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0260] The bispecific antibodies of the present invention can be used alone or in combination with a detectable marker (for diagnostic purposes), a therapeutic agent, or any combination thereof.

[0261] 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.

[0262] 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.

[0263] Pharmaceutical composition

[0264] 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 depending on 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, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection), intracranial injection, or intracavitary injection.

[0265] 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.

[0266] In the present invention, the bispecific antibody can be used alone, and the dosage regimen can be adjusted to achieve the optimal desired response, for example, a single administration, or multiple administrations over a period of time, or the dosage can be proportionally reduced or increased depending on the urgency of the therapeutic situation.

[0267] 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 generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0268] The main advantages of the present invention include

[0269] (1) The modified mutant CD47 antibody of the present invention has excellent safety, has no effect on erythrocyte agglutination, and can effectively reduce the toxic side effects of the antibody.

[0270] (2) The blocked CD47 TMEAbody of the present invention largely blocks the binding of CD47 antibody to CD47 or ADCC activity, whereas activated CD47 TMEAbody can restore its binding affinity.

[0271] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0272] Example 1: Preparation of rabbit anti-CD47 monoclonal antibody

[0273] 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 individual wells using IgG+ direct flow cytometry. B cell culture supernatants were screened for binding to human CD47 by enzyme-linked immunosorbent assay (ELISA). B cells were lysed and stored at -80°C.

[0274] 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 a published article (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.

[0275] Figure 1 shows the sequence alignment of the light chain and heavy chain variable regions of each anti-CD47 rabbit antibody.

[0276] The amino acid sequence of the heavy chain variable region VH of 70G1 is SEQ ID NO.1

[0277] The amino acid sequence of the light chain variable region VL of 70G1 is SEQ ID NO.2

[0278] The amino acid sequence of the heavy chain variable region VH of 71D6 is SEQ ID NO.3

[0279] The amino acid sequence of the light chain variable region VL of 71D6 is SEQ ID NO.4

[0280] The amino acid sequence of the heavy chain variable region VH of 74F1 is SEQ ID NO.5

[0281] The amino acid sequence of the light chain variable region VL of 74F1 is SEQ ID NO.6

[0282] 70H1 heavy chain variable region VH amino acid sequence: SEQ ID NO.7

[0283] 70H1 light chain variable region VL amino acid sequence: SEQ ID NO.8

[0284] 69F12 heavy chain variable region VH amino acid sequence: SEQ ID NO.9

[0285] 69F12 light chain variable region VL amino acid sequence: SEQ ID NO.10

[0286] Example 2: FACS binding of CD47 antibodies to CHOK1-CD47 cells

[0287] The desired overexpressing cell line CHOK1-CD47 was expanded to the logarithmic phase (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)). The cells were digested and collected and pipetted into a single cell suspension. The supernatant was discarded after centrifugation. The cells were resuspended in 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 100ul / well of the antibody sample to be tested (100nM as the starting concentration, 5-fold serial dilution) was added, mixed with the cells, and incubated at 4℃ for 1h. Washed by centrifugation with FACS buffer 3 times, 100ul / well of Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscience TM, Invitrogen, Cat#12-4998-82, 1:200) and incubated at 4°C for 1 hour. After washing three times by centrifugation in FACS buffer, cells were resuspended in 200 μl / well of FACS buffer and analyzed using FACS (BECKMAN COULTER, CytoFLEX). Data were fitted and analyzed using GraphPad Prism 8 software.

[0288] As shown in FIG2 , all five antibodies bound well to CHOK1-CD47 cells, with EC50 (nM) of 70G1, 71D6, 74F1, 70H1, and 69F12 being 2.88, 3.80, 2.82, 0.91, and 0.80, respectively.

[0289] Example 3: 70H1 TMEAbody site screening

[0290] In this example, the inventors selected 70H1 as the parent antibody, screened for TMEA body sites, and artificially synthesized the corresponding DNA coding sequence (codon optimized) for expression in a host.

[0291] The synthesized DNA was digested and ligated into a modified pTT5 vector (Biovector) to generate pTT5-anti-CD47. Using pTT5-anti-CD47 as a template, cysteine ​​mutations were introduced by designing primers to replace the codon at the mutation site with a cysteine ​​(Cys) codon. PCR was then performed to digest the mutant fragment and construct a modified pTT5 vector.

[0292] HEK293T cells (Life Technologies) were used as the expression host. Prior to 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 at an appropriate density in 15 cm culture dishes, and the medium was changed to IgG-reduced FBS (Gibico). Six hours after transfection or two days later, the medium was changed to Freestyle 293 (Gibico).

[0293] On the day of transfection, when cells reached confluence, 293T cells were co-transfected with a plasmid expressing the target protein using lipofectamine 2000 (Life Technologies) and PEI (Sigma). The antibody expression plasmid contained both the heavy and light chains of the antibody. Culture supernatants were collected on days 4 and 6 after transfection, respectively. Protein or antibody expression and activity were assayed and purified.

[0294] The hypervariable regions of the variable regions within the heavy and light chains of antibodies (Abs) constitute the antibody's antigen (Ag) binding site. Because the antigen binding site is structurally complementary to the antigen epitope, the hypervariable regions are also called the complementarity determining regions (CDRs) of the antibody.

[0295] 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 the antibody antigen overnight, then blocked with 1% BSA blocking agent (Thermo Fisher) at 37°C for 2 hours, and washed 3 times with PBST. The corresponding antibody or the corresponding mutant was added, bound at 37°C for 1 hour, and then washed 3 times with PBST. HRP enzyme-linked anti-human IgG was added, bound at 37°C for 1 hour, and washed 3 times with PBST. TMB substrate (Solarbio., Inc.) was used to detect the absorbance at 450nm.

[0296] The effect on the binding strength of the mutant was calculated by EC50 post-mutation / EC50 wild type.

[0297] 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 tested.

[0298] 70H1 heavy chain variable region VH amino acid sequence: SEQ ID NO.7

[0299] 70H1 light chain variable region VL amino acid sequence: SEQ ID NO.8

[0300] Table 1: Effect of mutation position in antibody heavy chain variable region / light chain variable region on binding activity

[0301] Among them, VL-49 is located in the FR region.

[0302] Example 4. CD47 TMEAbody reduces binding affinity to CD47 and can restore

[0303] Based on the inventors' structural research and screening, the results indicate that the VH-61 (YC) site has excellent blocking effects (this site is position 61 in VH3, as shown in Table 6, where the Y site at position 61 is underlined in the VH3 sequence). In this example, the coding sequence of a corresponding CD47 antibody (70H1.IgG1) containing the VH-61 (Y61C mutation) was synthesized and expressed to obtain a recombinant antibody (70H1.IgG1.Y61C). Furthermore, the obtained CD47 antibody was conjugated with PEG (molecule weight approximately 20K or other PEG molecules) to obtain a CD47 TMEAbody.

[0304] 4.1 ELISA detection of CD47 antibodies

[0305] Recombinant human CD47 protein (DIMA BIOTECH, Cat# PME-100008) was diluted to 1 μg / ml in PBS and coated onto an ELISA plate at 100 μl / well. Incubate overnight at 4°C. The next day, wash the plate three times with 300 μl / well of PBST. Add 300 μl / well of blocking buffer containing 1% BSA in PBST (w / w) and block at 37°C for 2 hours. Discard the blocking buffer and wash the plate three times with PBST. Add 100 μl / well of antibody (5-fold serial dilutions, eight steps) and incubate at 37°C for 1 hour. Discard the antibody and wash the plate three times with PBST. Add 100 μl / well of Goat anti-Human IgG Fc Highly Cross-Adsorbed Secondary Antibody, HRP (Invitrogen, Cat# A18829, 1:5000, 100 μl / well), incubate at 37°C for 0.5 hours, and wash three times with PBST. Add 100 μl / well of TMB chromogenic substrate (solarbio, Cat#PR1200) and incubate at 37°C for 5 min. Then add 50 μl / well of ELISA stop buffer (solarbio, Cat#C1058). OD450 values ​​were read using a microplate reader (ELx800 Absorbance Microplate Reader, BioTek). GraphPad Prism 8 software was used to analyze the data and fit the results.

[0306] As shown in Figure 3 and Table 2.1, the binding affinity of CD47 TMEAbody (70H1.IgG1+20K) to human CD47 protein is approximately 7-fold weaker than that of 70H1, which can be restored by legumain (legumin, also known as asparaginyl endopeptidase (AEP)).

[0307] Table 2.1

[0308] 4.2 Affinity testing of CD47 antibodies

[0309] The binding strength of the antigen and antibody was determined using a BIAcore T200 instrument. The binding strength of the CD47 antigen and antibody was determined using amino coupling on a CM5 chip (purchased from Cytiva, 29149603). First, the CD47 antigen was immobilized on a CM5 chip. Following the instructions for the Amine Coupling Kit (purchased from Cytiva, BR100050), PBST was used as the mobile phase. After mixing NHS and EDC, the chip was activated for approximately 420 seconds. The CD47 antigen was diluted to 10 μg / mL with 10 mM sodium acetate, pH 5.5, and injected to achieve a level of 141.2 RU. Finally, the remaining active sites were blocked with 1 M ethanolamine (pH 8.5). The affinity of the antibody for the CD47 antigen was then determined using a multi-cycle kinetic method. During each cycle, a single concentration of antibody protein was injected, and the binding and dissociation processes between the antibody and antigen protein were recorded. Finally, the chip was regenerated with Glycine-HCl pH 1.5. The mobile phase was PBST at a flow rate of 30 μL / min, the regeneration time was 15 seconds, and the detection temperature was 25°C. Finally, the data was analyzed according to a 1:1 binding model, and the antibody-antigen binding kinetic parameters, including the association rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax, were fitted.

[0310] The association rate (Ka), dissociation rate (Kd), and binding affinity (KD) of the CD47 antibody to the human CD47 antigen are shown in Table 2.2. Compared with 70H1, the affinity of the CD47 TMEAbody was reduced by 470-fold.

[0311] Table 2.2 Affinity of CD47 antibodies and human CD47 antigen detected by SPR (biacore)

[0312] 4.3 CD47 TMEAbody weakens the binding of CD47 antibody to CHOK1-CD47 cells and can restore

[0313] The desired overexpression cell line CHOK1-CD47 was expanded to the logarithmic phase (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))). The cells were digested and collected and pipetted into a single cell suspension. After counting the cells, centrifugation was performed and the supernatant was discarded. The cells were resuspended in FACS buffer (PBS + 2% FBS) to 2×10 6 / 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 serial dilution, eight points) was added, mixed with the cells, and incubated at 4℃ for 1h. Washed three times by centrifugation with FACS buffer, 100ul / well of Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscience TM , Invitrogen, Cat#12-4998-82, 1:200) and incubated at 4°C for 1 hour. After washing three times by centrifugation in FACS buffer, cells were resuspended in 200 μl / well of FACS buffer and analyzed using FACS (BECKMAN COULTER, CytoFLEX). Data were fitted and analyzed using GraphPad Prism 8 software.

[0314] As shown in Figure 4 and Table 3, the binding of 70H1 TMEAbody to cells was reduced by approximately 20-fold compared to 70H1. However, when activated with asparagine endopeptidase, the activated 70H1 TMEAbody restored binding comparable to that of the 70H1 IgG1-mutant.

[0315] Therefore, CD47 TMEAbody (70H1.IgG1+20K) can largely block the binding of 70H1 antibody to CHOK1-CD47 cells. Meanwhile, activated CD47 TMEAbody can restore its binding ability to CHOK1-CD47 cells.

[0316] Table 3

[0317] Example 5 CD47 TMEAbody attenuates the blocking activity of CD47 antibody and can restore it

[0318] 5.1 Dose-dependent binding of CD47 antibodies to Raji cells

[0319] 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 in 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 100ul / well of the antibody sample to be tested (100nM as the starting concentration, 5-fold serial dilution) was added, mixed with the cells, and incubated at 4℃ for 1h. Washed with FACS buffer by centrifugation 3 times, 100u / well of Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscience TM , Invitrogen, Cat#12-4998-82, 1:200) and incubated at 4°C for 1 hour. After washing three times by centrifugation in FACS buffer, cells were resuspended in 200 μl / well of FACS buffer and analyzed using FACS (BECKMAN COULTER, CytoFLEX). Data were fitted and analyzed using GraphPad Prism 8 software.

[0320] As shown in Figure 5 and Table 4, the binding of CD47 TMEAbody to Raji cells was approximately 60-fold lower than that of 70H1. However, when activated with asparagine endopeptidase, the activated 70H1 TMEAbody restored binding comparable to that of the 70H1 IgG1-mutant.

[0321] Table 4

[0322] 5.2 CD47 Antibody Blocking Activity Assay

[0323] 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 in FACS buffer (PBS + 2% FBS), count, and adjust the cell density to 2×10 6100 μl / well of the test antibody sample (CD47 Antibodies starting at 200 nM, diluted 5-fold at eight points, and mixed with Human SIPR alpha, mFc (DIMA Biotech, PME100531) 16 μg / ml) at equal volumes) was added to a 96-well plate (Corning, Cat#3799) and centrifuged. The cells were mixed well and incubated at 4°C for 1 hour. The cells were washed three times with FACS buffer by centrifugation. Pre-adsorbed Goat F(ab')2 Anti-Mouse IgG-Fc (PE) (abcam, ab98742, 1:200) was added and incubated at 4°C for 1 hour. After washing three times with FACS buffer by centrifugation, the cells were resuspended in 200 μl / well of FACS buffer and analyzed using FACS (BECKMAN COULTER, CytoFLEX). Data fitting and analysis were performed using software (GraphPad Prism 8).

[0324] As shown in FIG6 , 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 was restored after activation of CD47 TMEAbody.

[0325] Example 6. CD47 TMEAbody blocks the binding of CD47 antibody to erythrocytes

[0326] 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 serial dilution, eight points), 100ul / well, and mix with the cells. Incubate at 4℃ for 1h. Wash three times with FACS buffer by centrifugation, add 100u / well l Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscience TM , Invitrogen, Cat#12-4998-82, 1:200) and incubated at 4°C for 1 hour. After washing three times by centrifugation in FACS buffer, cells were resuspended in 200 μl / well of FACS buffer and analyzed using FACS (BECKMAN COULTER, CytoFLEX). Data were fitted and analyzed using GraphPad Prism 8 software.

[0327] As shown in FIG7 , the CD47 antibody binds relatively strongly to erythrocytes, while the CD47 TMEAbody hardly binds to erythrocytes.

[0328] Example 7 Human red blood cell (RBC) agglutination experiment

[0329] 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+0.05M EDTA), resuspended in PBS, and counted. The cell density was adjusted to 2×10 6 / ml. Add 100 μl / well to a 96-well plate (Corning, Cat#3799), centrifuge, and discard the supernatant. Add 50 μl / well of the test antibody (100 nM as the starting concentration, 5-fold serial dilution, 7 points + 0 point), and incubate at 37°C for 1 hour.

[0330] As shown in FIG8 , except for the positive antibody 5F9 which caused erythrocyte agglutination at high concentrations, the other CD47 antibodies did not cause erythrocyte agglutination.

[0331] Example 8. CD47 TMEAbody loses antibody-dependent cell-mediated cytotoxicity (ADCC) and can be restored

[0332] Raji cells were expanded to the logarithmic phase using RPMI-1640 (ATCC Modification, Gibico, Cat#A10491-01) + 10% FBS (BI, Cat#04-001-1A) + 1×P / S. The cells were collected and pipetted into a single-cell suspension. After centrifugation, the supernatant was discarded and the cells were resuspended in culture medium and counted. 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 in 5% CO2. PBMC (XF, XC11100W) were resuspended in 10% FBS-RPMI1640 medium, centrifuged and the supernatant was discarded. The cell density was adjusted to 8×10 6 / ml. Add 50 μl / well of the test antibody and 50 μl of PBMC cells (E:T ratio 20:1) to the culture plate. Place the culture plate in a CO2 incubator and incubate for 24 hours. ADCC assay using the Cytotoxicity Detection Kit PLUS (LDH), Roche, Cat# 4744934001.

[0333] As shown in FIG9 and Table 5, compared with the 70H1 antibody, the CD47 TMEAbody lost ADCC activity and recovered ADCC activity after activation.

[0334] Table 5

[0335] Example 9. Humanization of 70H1

[0336] 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. Then, key amino acids in the FR region sequence of the antibody were backmutated to ensure the original affinity. The specific mutation design is shown in Table 6. Finally, the different heavy and light chains obtained by backmutation of the humanized antibody were combined to obtain 12 humanized antibodies.

[0337] Table 6.

[0338] SEQ. represents SEQ ID NO.

[0339] Figure 10 shows the FACS binding of 12 different humanized antibodies to CHOK1-CD47 cells, and the FACS EC50 of different antibodies are shown in Table 7. The final preferred antibody was 70H1-VH3VL1.

[0340] Table 7

[0341] Example 10. In vivo efficacy of CD47 TMEAbody in a mouse tumor (RAJI) model

[0342] Human Burkitt's lymphoma RAJI cells were subcutaneously transplanted into NOD-SCID mice to establish an in vivo model. RAJI cells were cultured according to the culture conditions, and cells were collected and counted during the exponential growth phase. Each mouse was inoculated with 1×10 7 Individual Burkitt's lymphoma RAJI cells (suspended in 0.1 ml basal medium) were inoculated and the tumors were grown to 80-114 mm. 3 At 7 days after tumor loading, mice were randomly divided into groups according to their body weight and tumor volume: PBS group,

[0343] 70H1.IgG1.Y61C (1 mg / kg) group,

[0344] 70H1.IgG1.Y61C+20K (0.3 mg / kg) group,

[0345] 70H1.IgG1.Y61C+20K (1 mg / kg) group,

[0346] 70H1.IgG1.Y61C+20K (3 mg / kg) group.

[0347] Among them, 70H1.IgG1.Y61C+20K is a CD47 TMEAbody coupled with PEG with a molecular weight of approximately 20K.

[0348] 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 during each measurement.

[0349] As shown in Figure 11, 70H1.IgG1.Y61C+20K demonstrated significant tumor growth inhibition in a subcutaneous RAJI xenograft mouse model. After 31 days of treatment, the TGI inhibition rates for the 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) groups were 60.91%, 6.45%, 69.46%, 98.97%, and 74.55%, respectively. The T / C ratios were 41.81%, 93.73%, 33.06%, 4.51%, and 28.36%, respectively. No significant changes in body weight were observed during treatment.

[0350] As shown in Figure 11, the results demonstrate that 70H1.IgG1.Y61C+20K is capable of being activated in the tumor microenvironment and stimulating an anti-tumor immune response. It exhibits similar in vivo efficacy to 70H1.IgG1.Y61C while increasing the administered dose. Specific data for tumor volume at different days (Day, abbreviated as D) are shown in Table 8.1 or Table 8.2 below.

[0351] Table 8.1. Detailed data of tumor volume on different days

[0352] Table 8.2. Detailed data of tumor volume on different days

[0353] Example 11. In vivo efficacy of CD47 TMEAbody in a mouse tumor (SHP-77) model

[0354] Human small cell lung cancer (SHP-77) cells were subcutaneously transplanted into CB17-SCID mice to establish an in vivo model. SHP-77 cells were cultured according to the culture conditions, and cells were harvested and counted during the exponential growth phase. Each mouse was inoculated with 2.5×10 6 Human small cell lung cancer 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 body weight and tumor volume. The experiment was divided into:

[0355] PBS group,

[0356] 70H1.IgG1.Y61C+20K (0.3 mg / kg) group,

[0357] 70H1.IgG1.Y61C+20K (1 mg / kg) group,

[0358] 70H1.IgG1.Y61C+20K (3 mg / kg) group,

[0359] Among them, 70H1.IgG1.Y61C+20K is a CD47TMEAbody coupled with PEG with a molecular weight of approximately 20K (i.e., PEG20K).

[0360] 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 during each measurement.

[0361] As shown in Figure 12, 70H1.IgG1.Y61C+20K demonstrated significant tumor growth inhibition in a subcutaneous SHP77 xenograft mouse tumor model. After 38 days of treatment, the TGI inhibition rates for the 70H1.IgG1.Y61C+20K (0.3 mg / kg), 70H1.IgG1.Y61C+20K (1 mg / kg), and 70H1.IgG1.Y61C+20K (3 mg / kg) groups were 75.38%, 105.46%, and 105.53%, respectively, and the T / C ratios were 29.52%, 1.13%, and 1.02%, respectively. There was no significant change in body weight during treatment.

[0362] As shown in Figure 12, the results indicate that 70H1.IgG1.Y61C+20K can be activated in the tumor microenvironment and stimulate anti-tumor immune responses. Specific data on tumor volume at different days are shown in Tables 9.1 and 9.2 below.

[0363] Table 9.1. Detailed data of tumor volume on different days

[0364] Table 9.2. Detailed data of tumor volume on different days

[0365] Example 12. In vivo efficacy of CD47 TMEAbody in a mouse tumor (SHP-77) model

[0366] Human small cell lung cancer (SHP-77) cells were subcutaneously transplanted into CB17-SCID mice to establish an in vivo model. SHP-77 cells were cultured according to the culture conditions, and cells were harvested and counted during the exponential growth phase. Each mouse was inoculated with 2.5×10 6 Human small cell lung cancer SHP-77 cells (suspended in 0.1 ml of basal culture medium). After inoculation, wait until the tumor grows to 60-140 mm 3 At 18 days after tumor loading, mice were randomly divided into groups according to their body weight and tumor volume. The experiment was divided into:

[0367] PBS group,

[0368] 70H1.IgG1.Y61C+40K (3 mg / kg) group,

[0369] 70H1.IgG1.Y61C+40K (6 mg / kg) group,

[0370] 70H1.IgG1.Y61C+40K (9 mg / kg) group,

[0371] Among them, 70H1.IgG1.Y61C+40K is a CD47TMEAbody coupled with PEG with a molecular weight of approximately 40K (i.e., PEG40K).

[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 3Tumor 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 during each measurement.

[0373] As shown in Figure 13, 70H1.IgG1.Y61C+40K demonstrated significant tumor growth inhibition in a subcutaneous SHP77 xenograft mouse tumor model. After 24 days of treatment, the TGI inhibition rates for the 70H1.IgG1.Y61C+40K (3 mg / kg), 70H1.IgG1.Y61C+40K (6 mg / kg), and 70H1.IgG1.Y61C+40K (9 mg / kg) groups were -5.23%, 86.99%, and 101.20%, respectively, and the T / C ratios were 105.11%, 18.43%, and 5.12%, respectively. Body weight did not change significantly during treatment.

[0374] As shown in Figure 13, the results indicate that 70H1.IgG1.Y61C+40K can be activated in the tumor microenvironment and stimulate anti-tumor immune responses. Specific data on tumor volume at different days are shown in Tables 10.1 and 10.2 below.

[0375] Table 10.1. Detailed data of tumor volume on different days

[0376] Table 10.2. Detailed data of tumor volume on different days

[0377] Example 13. In vivo toxicity characterization in hSIRPA / hCD47 (C57 background) mice

[0378] hSIRPA / hCD47 (C57 background) mice were randomly divided into 4 groups according to body weight, with 3 mice in each group. The hematological toxicity of 70H1.IgG1.Y61C+20K and 70H1.IgG1.Y61C+40K was studied by intraperitoneal injection.

[0379] The test is divided into:

[0380] 70H1.IgG1.Y61C+20K (5 mg / kg) group,

[0381] 70H1.IgG1.Y61C+40K (10 mg / kg) group,

[0382] 70H1.IgG1.Y61C+40K (20 mg / kg) group,

[0383] 70H1.IgG1.Y61C+40K (30 mg / kg) group,

[0384] After the experimental groups were formed, the mice were injected with a single dose of the antibody into the abdominal cavity. The blood cell counts of the mice were performed on DO (before administration), D2, D5, D7, D9, and D12, and the changes in red blood cells and platelets were observed to evaluate the effect of the antibody on the blood routine of the mice.

[0385] As shown in Figure 14: 70H1.IgG1.Y61C+20K 5 mg / kg showed a reversible decrease in red blood cells on D2 after administration, and 70H1.IgG1.Y61C+40K various dose groups showed a slight decrease in red blood cells, and there was no significant difference between the dose groups compared with PBS; 70H1.IgG1.Y61C+20K 5 mg / kg showed a reversible decrease in platelets on D5 after administration, and no obvious decrease in platelets was seen in the 70H1.IgG1.Y61C+40K various dose groups.

[0386] The specific data of mouse red blood cells and platelets on different days are shown in Tables 11.1 and 11.2.

[0387] Table 11.1. Specific data of mouse red blood cells (RBC) at different days (unit: 10^12 / L):

[0388] Note: Change rate (%) refers to the percentage change of mouse red blood cells on different days compared with D0 (before administration).

[0389] Table 11.2. Specific data of mouse platelets (PLT) at different days (unit: 10^9 / L):

[0390] Note: Change rate (%) refers to the percentage change of platelets in mice on different days compared with D0 (before administration).

[0391] The sequence involved in the present invention is as follows:

[0392] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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. The mutant CD47 antibody according to claim 1, characterized in that: The mutant CD47 antibody includes a modified mutant CD47 antibody having the following structure: Ab-(P-R1)n in, Ab is a mutant CD47 antibody; R1 is each independently a blocker; P is a cleavable connecting peptide; n is a positive integer from 1 to 5; "-" is a chemical bond, linker or connector.

4. The mutant CD47 antibody according to claim 3, characterized in that: The masking body is selected from polyethylene glycol-C 1-5 -Alkylcarbonyl group: 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; The wavy line indicates where R2 is connected.

5. The mutant CD47 antibody according to claim 3, characterized in that: The masking agent is selected from the group consisting of PEG5K, PEG10K, PEG20K, or PEG40K.

6. 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.

7. A recombinant protein, characterized in that The recombinant protein comprises: (1) the mutant CD47 antibody according to any one of claims 1 to 5 or the CD47 antibody or antigen-binding fragment thereof according to claim 6; (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.

8. An isolated polynucleotide, characterized in that The polynucleotide encodes the mutant CD47 antibody according to any one of claims 1 to 5 or the CD47 antibody or antigen-binding fragment thereof according to claim 6.

9. A carrier, characterized in that The vector contains the polynucleotide according to claim 8.

10. A genetically engineered host cell, characterized in that The host cell contains the vector of claim 9 or the polynucleotide of claim 8 is integrated into its genome.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (I) the mutant CD47 antibody according to any one of claims 1 to 5 or the CD47 antibody or antigen-binding fragment thereof according to claim 6; and (II) a pharmaceutically acceptable carrier.

12. An immunoconjugate, characterized in that: The immunoconjugate comprises: (a) the mutant CD47 antibody according to any one of claims 1 to 5 or the CD47 antibody or antigen-binding fragment thereof according to claim 6; 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.

13. Use of the mutant CD47 antibody according to any one of claims 1 to 5, the CD47 antibody or antigen-binding fragment thereof according to claim 6, or the immunoconjugate according to claim 12, 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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