Anti-bcma single domain antibodies and uses thereof

Abstract

Provided are an anti-BCMA single-domain antibody and applications thereof. Specifically, provided is a single-domain antibody specifically targeting BCMA, which has high affinity to BCMA and is capable of binding to BCMA of human or monkey origin. Further provided are CAR cells comprising the single-domain antibody and therapeutic methods.

Description

Anti-BCMA single-domain antibodies and their applications Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to anti-BCMA single-domain antibodies and applications thereof. Background Art

[0002] Multiple Myeloma (MM) is a malignant tumor derived from plasma cells of B lymphocytes in the bone marrow. MM symptoms may include bone pain (often occurring in the back or ribs), fever, fatigue, anemia, frequent infections, and easy bruising or bleeding. Treatment may include a variety of chemotherapy and targeted therapy drugs, including proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies, radiotherapy and stem cell transplantation, etc. However, it is still a non-curable disease, and most patients will eventually relapse, and its treatment still faces great challenges. The recent FDA approval of two CART cell therapies and one approved by China for the treatment of MM have made breakthrough progress in the treatment of MM. However, this field is still limited by treatment costs and production in the treatment of MM, and new treatment products and treatment plans are still needed.

[0003] Therefore, there is a need in the art to develop multiple myeloma therapeutic antibodies with improved therapeutic effects and their applications.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-BCMA single domain antibody and its application.

[0006] In a first aspect of the present invention, an anti-BCMA single-domain antibody is provided, wherein the VHH chain of the single-domain antibody has complementary determining regions CDR1, CDR2 and CDR3 derived from the heavy chain variable region shown in SEQ ID NO: 1, and the complementary determining regions CDR1, CDR2 and CDR3 are defined by Chothia, Abm, Kabat, or IMGT rules.

[0007] In another preferred embodiment, the CDR1, CDR2 and CDR3 are selected from the following groups:

[0008] (1) Based on the definition of Chothia rules:

[0009] CDR1 shown in SEQ ID NO: 2,

[0010] CDR2 shown in SEQ ID NO: 3, and

[0011] CDR3 shown in SEQ ID NO: 4; or

[0012] (2) Based on the definition of Kabat rules:

[0013] CDR1 shown in SEQ ID NO:5,

[0014] CDR2 shown in SEQ ID NO:6, and

[0015] CDR3 shown in SEQ ID NO: 4; or

[0016] (3) Based on IMGT rules:

[0017] CDR1 shown in SEQ ID NO:7,

[0018] CDR2 shown in SEQ ID NO:8, and

[0019] CDR3 shown in SEQ ID NO:9;

[0020] Furthermore, any one of the amino acid sequences in the above-mentioned CDR sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid, and enables the derivative antibody composed of the heavy chain and light chain containing the derived CDR sequence to retain BCMA binding affinity.

[0021] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, preferably 1-2, and more preferably 1.

[0022] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0023] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0024] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0025] In another preferred example, the VHH chain of the single-domain antibody has an amino acid sequence that is ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0026] In another preferred example, the VHH chain amino acid sequence of the single-domain antibody is as shown in SEQ ID NO: 1.

[0027] In the second aspect of the present invention, an anti-BCMA antibody is provided, wherein the antibody comprises one or more VHH chains of the single-domain antibody according to the first aspect of the present invention.

[0028] In another preferred example, the VHH chain amino acid sequence of the single-domain antibody is as shown in SEQ ID NO: 1.

[0029] In another preferred embodiment, the antibody is a monovalent antibody, a bivalent antibody, and / or a multivalent antibody.

[0030] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).

[0031] In another preferred embodiment, the CDR region of the humanized antibody contains 0, 1, 2, or 3 amino acid changes, and the humanized antibody retains ≥70%, preferably ≥80%, and more preferably ≥90% of the BCMA binding activity compared to the animal-derived antibody before humanization.

[0032] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0033] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0034] In another preferred embodiment, the antibody is a monoclonal antibody.

[0035] In another preferred embodiment, the antibody is a partially or fully humanized antibody.

[0036] In another preferred embodiment, the antibody further comprises a heavy chain constant region.

[0037] In another preferred embodiment, the heavy chain constant region is derived from IgG, preferably human IgG.

[0038] In another preferred embodiment, the antibody is a heavy chain antibody.

[0039] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain BCMA binding affinity.

[0040] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably ≤20%, more preferably ≤10%.

[0041] In the third aspect of the present invention, a multispecific antibody is provided, wherein the multispecific antibody comprises: the single domain antibody according to the first aspect of the present invention or the anti-BCMA antibody according to the second aspect of the present invention.

[0042] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions that target additional antigen targets.

[0043] In another preferred embodiment, the second antigen binding region is an antibody or antibody fragment, and the antibody fragment includes: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; (vi) dAb fragment.

[0044] In another preferred embodiment, the second antigen binding region is a single domain antibody.

[0045] In a fourth aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0046] (i) the single domain antibody according to the first aspect of the present invention, or the anti-BCMA antibody according to the second aspect of the present invention; and

[0047] (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or

[0048] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0049] In another preferred embodiment, the improving the physicochemical properties or drugability of the protein includes prolonging the half-life of the anti-BCMA single domain antibody.

[0050] In another preferred embodiment, the recombinant protein further comprises: (iv) an optional tag sequence for facilitating expression and / or purification.

[0051] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0052] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0053] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, and cytokines.

[0054] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0055] In another preferred embodiment, the fusion protein includes a multispecific antibody or a chimeric antibody.

[0056] In another preferred embodiment, the functional domain for improving the physicochemical properties or drugability of a protein includes an Fc segment and human serum albumin (HSA).

[0057] In another preferred example, the amino acid sequence of the VHH chain of the anti-BCMA single-domain antibody is shown in SEQ ID NO: 1.

[0058] In the fifth aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the extracellular antigen binding domain nucleotide sequence of the CAR encodes the VHH chain of the single-domain antibody as described in the first aspect of the present invention.

[0059] In another preferred example, the amino acid sequence of the VHH chain of the single-domain antibody has an amino acid sequence that is ≥65%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identical to the amino acid sequence shown in SEQ ID NO: 1, or is as shown in SEQ ID NO: 1.

[0060] In another preferred embodiment, the structure of the CAR is shown in the following formula I: L-EB-H-TM-C n -IS (I)

[0061] Where,

[0062] Each "-" is independently a connecting peptide or a peptide bond;

[0063] L is none or a signal peptide sequence;

[0064] EB is the extracellular antigen binding domain;

[0065] H is the absence or hinge region;

[0066] TM is the transmembrane domain;

[0067] C n is n costimulatory signal molecules connected in series, wherein C represents a costimulatory signal molecule, and n is an integer selected from 1, 2, 3 or 4;

[0068] IS is a cytoplasmic signaling sequence.

[0069] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, SECTM1 or a combination thereof.

[0070] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0071] In another preferred embodiment, the L amino acid sequence is shown in SEQ ID NO: 14.

[0072] In another preferred embodiment, the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0073] In another preferred embodiment, the H is a hinge region derived from CD8.

[0074] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0075] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0076] In another preferred embodiment, the amino acid sequence of the H-TM fragment is shown in SEQ ID NO: 16.

[0077] In another preferred embodiment, the n costimulatory signal molecules are each independently a costimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0078] In another preferred embodiment, the n is 1.

[0079] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from 4-1BB.

[0080] In another preferred embodiment, the cytoplasmic signaling sequence is the CD3ζ cytoplasmic signaling sequence

[0081] In another preferred embodiment, the C n The amino acid sequence of the -IS fragment is shown in SEQ ID NO: 18.

[0082] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO: 25.

[0083] In a sixth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes a polypeptide selected from the group consisting of:

[0084] (1) the single-domain antibody according to the first aspect of the present invention, the anti-BCMA antibody according to the second aspect of the present invention, or the multispecific antibody according to the third aspect of the present invention;

[0085] (2) the recombinant protein according to the fourth aspect of the present invention; or

[0086] (3) The CAR as described in the fifth aspect of the present invention.

[0087] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0088] In another preferred embodiment, the polynucleotide further includes a sequence encoding an additional functional protein selected from the following group: IL-21 or a functionally active fragment thereof, IL-15 or a functionally active fragment thereof, IL-2 or a functionally active fragment thereof, IL-7 or a functionally active fragment thereof, IL-10 or a functional fragment thereof, cytokines, immune blockers PD1, TIGIT, VGEF, CTLA-4, CD73, antibodies, bispecific T cell engagers (BiTE), or a combination thereof.

[0089] In another preferred embodiment, the polynucleotide includes sequences encoding IL-15 and / or IL-21.

[0090] In another preferred embodiment, the polynucleotide comprises a left homology arm and a right homology arm, and the homology arms are used to integrate the polynucleotide into the host cell.

[0091] In another preferred embodiment, the nucleic acid molecule comprises a tandem expression unit, wherein the expression unit comprises:

[0092] (E1) a first expression unit for expressing the chimeric antigen receptor;

[0093] (E2) a second expression unit for expressing the IL-15, or a functionally active fragment thereof, or a fusion protein thereof;

[0094] (E3) a third expression unit for expressing the IL-21, or a functionally active fragment thereof, or a fusion protein thereof; and

[0095] (E4) optionally a fourth expression unit for expressing the additional functional protein,

[0096] The positions of the first, second, third and fourth expression units can be interchanged arbitrarily.

[0097] In the nucleic acid molecule, each independent expression unit is driven by an operably linked exogenous promoter or endogenous promoter, or driven by a further upstream promoter with a cleavable nucleic acid sequence at its 5' end (eg, 2A nucleic acid sequence, IRES nucleic acid sequence).

[0098] In another preferred embodiment, the number of the first expression unit, the second expression unit and the third expression unit are each independently 1, 2, or 3.

[0099] In another preferred embodiment, the number of the fourth expression units is 0, 1, 2, 3, 4, or 5.

[0100] In another preferred embodiment, the nucleic acid molecule encodes 1, 2, or 3 identical or different CAR molecules.

[0101] In another preferred embodiment, the nucleic acid molecule has the following structure:

[0102] ARM5—P1—CAR1—P2 / L1—Z1—P3 / L2—Z2—(P4 / L3—Z3) m —ARM3(Ia)

[0103] Where,

[0104] ARM5 is none or 5′ homology arm;

[0105] ARM3 is none or 3′ homology arm;

[0106] P1 is a promoterless, splicing acceptor, or the first exogenous promoter;

[0107] CAR1 is a nucleic acid sequence encoding the chimeric antigen receptor (CAR) according to claim 7;

[0108] P2 / L1 is the second exogenous promoter P2 or the nucleic acid sequence L1 encoding the cleavable part;

[0109] Z1 is absent, or a nucleotide sequence encoding a second CAR, or IL-15 or a functional protein fragment thereof;

[0110] P3 / L2 is none or a third exogenous promoter P3 or a nucleic acid sequence L2 encoding a cleavable portion;

[0111] Z2 is absent or contains a nucleotide sequence encoding IL-21 or a functional protein fragment thereof;

[0112] P4 / L3 are each independently a fourth exogenous promoter P4 or a nucleic acid sequence L3 encoding a cleavable portion;

[0113] Z3 is a nucleic acid sequence encoding an additional functional protein;

[0114] m is 0, 1, 2, 3, 4 or 5.

[0115] In another preferred embodiment, the promoters P1, P2, P3, and each P4 are the same or different exogenous promoters, and the cleavable parts L1, L2, and each L3 are nucleic acid sequences encoding the same or different cleavable parts.

[0116] In another preferred embodiment, the m Z3s are nucleic acid sequences encoding the same or different additional functional proteins.

[0117] In another preferred embodiment, the ARM5 is the 5' homology arm, and the ARM3 is the 3' homology arm.

[0118] In another preferred embodiment, the amino acid sequence of IL-15 is shown in SEQ ID NO: 20.

[0119] In another preferred embodiment, the amino acid sequence of IL-21 is shown in SEQ ID NO: 21.

[0120] In another preferred embodiment, the exogenous promoter is PGK promoter or EF1a promoter.

[0121] In another preferred embodiment, the PGK promoter nucleotide sequence is shown in SEQ ID NO: 23.

[0122] In another preferred embodiment, the EF1a promoter nucleotide sequence is shown in SEQ ID NO: 26

[0123] In another preferred embodiment, the functional protein is selected from the following group: IL-21 or a functionally active fragment thereof, IL-15 or a functionally active fragment thereof, IL-2 or a functionally active fragment thereof, IL-7 or a functionally active fragment thereof, IL-10 or a functional fragment thereof, cytokines, immune blockers PD1, TIGIT, VGEF, CTLA-4, CD73, antibodies, BiTE, or a combination thereof.

[0124] In another preferred embodiment, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO: 10, or has ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identity with SEQ ID NO: 10.

[0125] In the seventh aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the sixth aspect of the present invention.

[0126] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0127] In another preferred embodiment, the vector is a plasmid.

[0128] In another preferred embodiment, the plasmid is used to be transduced into host cells by electroporation.

[0129] In the eighth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector according to the seventh aspect of the present invention or the polynucleotide according to the sixth aspect of the present invention is integrated into its genome.

[0130] In the ninth aspect of the present invention, a recombinant immune cell is provided, wherein the immune cell expresses the exogenous CAR as described in the fifth aspect of the present invention, contains the vector as described in the seventh aspect of the present invention, or has the polynucleotide as described in the sixth aspect of the present invention integrated into its genome.

[0131] In another preferred embodiment, the immune cells are selected from NK cells or T cells.

[0132] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0133] In another preferred embodiment, the immune cells also express functional proteins selected from the following groups: IL-21 or its functionally active fragment, IL-15 or its functionally active fragment, IL-2 or its functionally active fragment, IL-7 or its functionally active fragment, IL-10 or its functional fragment, cytokines, immune blockers PD1, TIGIT, VGEF, CTLA-4, CD73, antibodies, BiTE, or a combination thereof.

[0134] In another preferred embodiment, the polynucleotide is introduced into the immune cells by electro-transfection of plasmids.

[0135] In another preferred embodiment, the polynucleotide is positioned and integrated into the DNA of the immune cell by electroporation of plasmids and targeting nuclease.

[0136] In another preferred embodiment, the positioning nuclease includes CRISPR-related protein and sgRNA.

[0137] In another preferred embodiment, the positioning nuclease includes sgRNA targeting the TRAC gene.

[0138] In another preferred embodiment, the polynucleotide is inserted into the TRAC gene of the immune cell.

[0139] In another preferred embodiment, one or more endogenous genes of the immune cells are knocked out.

[0140] In another preferred embodiment, the endogenous gene is TRAC.

[0141] In another preferred embodiment, the immune cell contains a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 10, or a polynucleotide having a nucleotide sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identical to SEQ ID NO: 10.

[0142] In the tenth aspect of the present invention, an immunoconjugate is provided, wherein the immunoconjugate comprises:

[0143] (a) an antibody portion, wherein the antibody portion is the single domain antibody according to the first aspect of the present invention or the anti-BCMA antibody according to the second aspect of the present invention; and

[0144] (b) a conjugated moiety conjugated to the single-domain antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

[0145] In another preferred embodiment, the immunoconjugate is a single domain antibody drug conjugate.

[0146] In another preferred embodiment, the single domain antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0147] In another preferred embodiment, the coupling moiety is a chemical label or a biological label.

[0148] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0149] In another preferred embodiment, the biomarker is biotin, avidin or an enzyme label.

[0150] In another preferred embodiment, the drug is a cytotoxic drug.

[0151] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0152] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0153] (ii) therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.

[0154] In another preferred embodiment, the immunoconjugate contains: a multivalent (such as bivalent) single domain antibody as described in the first aspect of the present invention or an anti-BCMA antibody as described in the second aspect of the present invention.

[0155] In another preferred embodiment, the multivalency refers to the amino acid sequence of the immunoconjugate containing multiple repeats of the single domain antibody as described in the first aspect of the present invention or the anti-BCMA antibody as described in the second aspect of the present invention.

[0156] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0157] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing BCMA protein.

[0158] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0159] in:

[0160] nAbs are single-domain antibodies against BCMA, antibodies against BCMA, or multispecific antibodies;

[0161] LU is the linker (also called connector);

[0162] D is for medicine;

[0163] The subscript p is a value selected from 1-10.

[0164] In the eleventh aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0165] (i) the single domain antibody according to the first aspect of the present invention, the anti-BCMA antibody according to the second aspect of the present invention, the multispecific antibody according to the third aspect of the present invention, the recombinant protein according to the fourth aspect of the present invention, the recombinant immune cell according to the ninth aspect of the present invention, or the immunoconjugate according to the tenth aspect of the present invention;

[0166] (ii) a pharmaceutically acceptable carrier.

[0167] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0168] 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.

[0169] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0170] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0171] In another preferred embodiment, the pharmaceutical composition is an injection.

[0172] In the twelfth aspect of the present invention, a use of an active ingredient is provided, wherein the active ingredient is selected from the following group: the single-domain antibody as described in the first aspect of the present invention, the anti-BCMA antibody as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, the recombinant immune cell as described in the ninth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention, or a combination thereof, and the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a drug for preventing and / or treating BCMA-related diseases.

[0173] In another preferred embodiment, the detection reagent, detection plate or kit is used for:

[0174] (1) detecting BCMA protein in a sample; and / or

[0175] (2) detecting endogenous BCMA protein in tumor cells; and / or

[0176] (3) Detect tumor cells expressing BCMA protein.

[0177] In another preferred embodiment, the detection types include but are not limited to flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay, immunoblotting detection, etc.

[0178] In another preferred embodiment, the detection reagent, detection plate or kit is used to diagnose BCMA-related diseases.

[0179] In another preferred embodiment, the BCMA-related disease is selected from the following group: cancer.

[0180] In another preferred embodiment, the cancer includes myeloma, such as multiple myeloma.

[0181] In a thirteenth aspect of the present invention, a method for detecting BCMA in a sample (including diagnostic or non-diagnostic) in vitro is provided, the method comprising the steps of:

[0182] (1) contacting the sample in vitro with the single domain antibody described in the first aspect of the present invention, the anti-BCMA antibody described in the second aspect of the present invention, or the immunoconjugate described in the tenth aspect of the present invention;

[0183] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of BCMA in the sample.

[0184] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0185] In a fourteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0186] (a) culturing the host cell according to the eighth aspect of the present invention under conditions suitable for expression;

[0187] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the single domain antibody described in the first aspect of the present invention, the anti-BCMA antibody described in the second aspect of the present invention, the multispecific antibody described in the third aspect of the present invention, or the recombinant protein described in the fourth aspect of the present invention.

[0188] In the fifteenth aspect of the present invention, a method for treating BCMA-related diseases is provided, the method comprising: administering to a subject in need thereof the single-domain antibody as described in the first aspect of the present invention, the anti-BCMA antibody as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, the recombinant immune cell as described in the ninth aspect of the present invention, the immunoconjugate as described in the tenth aspect of the present invention, or the pharmaceutical composition as described in the eleventh aspect of the present invention, or a combination thereof.

[0189] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0190] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0191] 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

[0192] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0193] FIG1 shows the results of nested PCR amplification of heavy chain antibody (VHH3).

[0194] Figure 2 shows the SDS-PAGE electrophoresis of nanobody BC-HC-15 transfected into 293F cells and transiently expressed.

[0195] FIG3 shows the binding ability of nanobody BC-HC-15 to BCMA.

[0196] Figure 4 shows the test results of the binding of Nanobody BC-HC-15 to BCMA-negative cells K562 and BCMA-positive cells NCI-H929.

[0197] Figure 5 shows the CAR molecular structure constructed by nanobody BC-HC-15 and the CAR construct expression cassette structure.

[0198] FIG6 shows the expression level of BC-HC-15 CAR molecules.

[0199] Figure 7 shows the specific killing of BCMA-positive cells by BC-HC-15 CAR-T.

[0200] Figure 8 shows the expansion of BC-HC-15 CAR-T cells under RPMI-8226 stimulation of target cells.

[0201] Figure 9 shows the results of BC-HC-15 CAR-T transplantation into the peripheral blood of NCG mice.

[0202] Figure 10 shows the therapeutic effect of BC-HC-15 CAR-T on subcutaneous tumors in NCG mice.

[0203] Figure 11 shows the therapeutic effect of G5 CAR-T on the RPMI-1640 subcutaneous tumor animal model. DETAILED DESCRIPTION

[0204] After extensive and in-depth research, the inventors have developed for the first time a single-domain antibody that specifically targets BCMA. The single-domain antibody of the present invention has a high affinity for BCMA and can bind to human or monkey BCMA. CAR cells containing the single-domain antibody of the present invention as the extracellular antigen binding domain have significant tumor-killing ability in animals. This is based on the present invention.

[0205] the term

[0206] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0207] Unless otherwise defined, 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. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0208] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0209] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including antibodies against respiratory syncytial virus fusion protein (preferably pre-fusion F protein) and compositions thereof, to a patient experiencing one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

[0210] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0211] As used herein, "sequence identity" may refer to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0212] Antibody

[0213] As used herein, the terms "single domain antibody of the present invention", "anti-BCMA nanobody of the present invention", and "BCMA single domain antibody of the present invention" are used interchangeably and refer to the single domain antibody of the first aspect of the present invention that specifically recognizes and binds to BCMA (including human BCMA), and particularly preferably the single domain antibody whose VHH chain amino acid sequence is as shown in SEQ ID NO: 1.

[0214] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0215] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to the variable region of the heavy chain of a monoclonal antibody. A single-domain antibody (VHH) consisting solely of a single heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0216] As used herein, the term "heavy chain antibody" refers to an antibody containing only a heavy chain. A portion of antibodies found in the blood of camelids are "heavy chain antibodies" that lack light chains. The heavy chain antibodies of the present invention comprise a heavy chain variable region (VHH) and heavy chain constant regions CH2 and CH3. The heavy chain antibodies of the present invention may be antibodies isolated from animals (such as camel-derived) that naturally lack light chains and heavy chain constant region 1 (CH1); or they may be recombinant antibodies obtained by recombining a single domain antibody (VHH) of the present invention with a heavy chain constant region. The heavy chain antibodies of the present invention may comprise a constant region derived from, for example, IgG1, IgG2, IgG3 or IgG4, preferably a constant region derived from IgG1.

[0217] 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 complementarity 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-dependent cytotoxicity of the antibody.

[0218] As known to those skilled in the art, immunoconjugates and fusion products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the anti-BCMA protein antibodies or fragments thereof.

[0219] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0220] As used herein, the terms "hypervariable region" and "complementarity determining region (CDR)" are used interchangeably.

[0221] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0222] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0223] In the present invention, the terms "recombinant protein of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to the BCMA protein, such as proteins or polypeptides comprising the VHH chains of the single-domain antibodies of the present invention. These may or may not contain an initial methionine.

[0224] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0225] Generally, an antibody's antigen-binding properties are described by three specific regions within the heavy chain variable region, known as hypervariable regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a looped structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0226] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0227] As used herein, "Chothia", "Kabat", "IMGT", and "AbM" refer to the determination of complementary binding determining regions (CDRs) under the above-mentioned different assignment systems. The assignment systems include, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International Immuno GeneTics database (IMGT), and Chothia definitions based on loop structural positions.

[0228] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0229] Preferably, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is capable of retaining binding affinity to BCMA.

[0230] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0231] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a human-animal chimeric antibody, and preferably a humanized antibody.

[0232] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0233] Wherein, the animal is preferably a mammal, such as a mouse or a camel.

[0234] In a preferred embodiment, the present invention discloses a variety of camel-derived and humanized nanobodies with high specificity and high affinity targeting BCMA, which only include heavy chains, wherein the heavy chains contain the amino acid sequence of the heavy chain variable region (VHH) and optional constant regions CH2 and CH3.

[0235] Recombinant protein (or fusion protein)

[0236] The present invention also includes a recombinant protein (or fusion protein) containing the BCMA single-domain antibody of the present invention. A preferred fusion protein is a multispecific antibody (e.g., a bispecific antibody). Preferably, the multispecific antibody includes one or more second antigen-binding regions, or further includes a third antigen-binding region.

[0237] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0238] 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. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide 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) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0239] The antibodies of the present invention refer to polypeptides that have BCMA protein binding activity and include the aforementioned CDR regions. The term also includes variant forms of polypeptides that include the aforementioned CDR regions and have the same function 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 (generally 1-10, preferably 1-5, more preferably 1-3, and most preferably 1) amino acids, and addition of one or more (generally within 10, preferably within 5, and more preferably within 3) 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 alter 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 alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0240] Variant forms of the polypeptide 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.

[0241] 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.

[0242] Table A

[0243] Chimeric Antigen Receptor (CAR)

[0244] The design of CARs has evolved through the following process: First-generation CARs contain only a single intracellular signaling component, CD3ζ or FcγRI. Because they only have a single activation domain, they can only induce transient T cell proliferation and minimal cytokine secretion, but cannot provide long-term T cell proliferation signals or sustained in vivo anti-tumor effects, resulting in poor clinical efficacy. Second-generation CARs incorporate a co-stimulatory molecule, such as CD28, 4-1BB, OX40, or ICOS, into the existing structure. Compared to first-generation CARs, these molecules significantly enhance the persistence of CAR-T cells and their ability to kill tumor cells. New immune co-stimulatory molecules, such as CD27 and CD134, are added in series to these second-generation CARs, resulting in the development of third- and fourth-generation CARs.

[0245] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain or antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including costimulatory molecules. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0246] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0247] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting BCMA. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to not grow, be caused to die or be affected in other ways, and causing the patient's tumor load to shrink or be eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of ICOS and / or 4-1BB signaling domains and CD3ζ signaling domains.

[0248] In the present invention, the antigen-binding domain comprises the anti-BCMA single-domain antibody of the present invention.

[0249] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid such a domain being bound to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0250] The intracellular domain in the CAR of the present invention includes the signaling domain of ICOS and / or 4-1BB and the signaling domain of CD3ζ.

[0251] CAR-immune cells

[0252] The present invention provides a recombinant immune cell comprising the chimeric antigen receptor specifically targeting BCMA of the present invention.

[0253] The chimeric antigen receptor immune cell of the present invention can be a CAR-T cell, or a CAR-NK cell, or a CAR-macrophage. Preferably, the chimeric antigen receptor immune cell of the present invention is a CAR-T cell.

[0254] The CAR-immune cells of the present invention may additionally express one or more functional proteins. The functional proteins include, but are not limited to, IL-21 or its functionally active fragment, IL-15 or its functionally active fragment, IL-2 or its functionally active fragment, IL-7 or its functionally active fragment, IL-10 or its functional fragment, cytokine, immune blocker PD1, TIGIT, VGEF, CTLA-4, CD73, antibody, BiTE, or a combination thereof. The functional protein may be a fusion protein of the above-mentioned protein. In one embodiment, the functional protein is exogenous relative to the CAR-immune cell of the present invention.

[0255] The multiple functional proteins can be expressed by the same promoter or different promoters. For example, the functional protein and the CAR of the present invention can be expressed by the same promoter. The multiple functional proteins or the functional protein and the CAR can be separated by a cleavable portion. The cleavable portion can include a 2A peptide. The 2A peptide can include P2A, T2A, F2A or E2A.

[0256] In the CAR-immune cells of the present invention, the expression of CAR and one or more functional proteins can be regulated by an endogenous or exogenous promoter. The exogenous promoter can be a PGK promoter or an EF1a promoter.

[0257] The CAR of the present invention can be prepared using a non-viral vector, for example, using a plasmid. A method for introducing the CAR of the present invention into immune cells includes transducing a plasmid containing the CAR into immune cells, for example, using electroporation. The transduction can be combined with a gene editing system, for example, using a positioning nuclease to locate and integrate the nucleic acid molecule containing the CAR into a specific position of the immune cell. The gene editing system that can be used includes CRISPR / sgRNA. For example, a plasmid containing the CAR encoding nucleic acid of the present invention and a positioning nuclease (such as CRISPR / sgRNA) can be co-electrotransfected into immune cells.

[0258] The nucleic acid encoding the CAR of the present invention can be inserted into the specific gene of the immune cell by homologous recombination.The polynucleotide can include a left homology arm and a right homology arm, and the homology arm is used to integrate the polynucleotide into the host cell.

[0259] One or more endogenous genes in the CAR immune cells of the present invention can be knocked out or knocked down. The nucleic acid sequence containing the CAR of the present invention can be knocked into the gene that is desired to be knocked out. The gene editing system may include a guide RNA (sgRNA) targeting the gene to be knocked out. In one embodiment, using Cas protein and sgRNA targeting the TRAC gene, the nucleic acid sequence containing the CAR of the present invention is knocked into the TRAC gene position of the immune cell in a one-step electroporation method, and the TRAC gene of the immune cell is knocked out at the same time.

[0260] In some embodiments, the CAR-immune cells express a chimeric antigen receptor (CAR) of the present invention, IL-15 or a functionally active fragment thereof, and IL-21 or a functionally active fragment thereof. In some embodiments, the immune cells contain a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 10, or a polynucleotide having a nucleotide sequence with SEQ ID NO: 10 having ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identity.

[0261] Nucleic Acids

[0262] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof, or fusion proteins thereof, or chimeric antigen receptors (CARs). 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.

[0263] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0264] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0265] 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.

[0266] Antibody preparation

[0267] The DNA sequence of the antibody or fragment thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. This is generally done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells using conventional methods.

[0268] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0269] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through 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 the protein sequences of the present invention through chemical synthesis.

[0270] 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.

[0271] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0272] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0273] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0274] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated 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 shock, ultrasonic treatment, 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.

[0275] Antibody-drug conjugates (ADCs)

[0276] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0277] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0278] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0279] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0280] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0281] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0282] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0283] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0284] In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0285] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0286] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0287] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0288] application

[0289] The present invention also provides uses of the antibodies, antibody conjugates ADCs, recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for preparing diagnostic preparations or preparing drugs.

[0290] Preferably, the drug is a drug for preventing and / or treating diseases associated with abnormal BCMA expression or function. The BCMA-related diseases include tumors, such as myeloma.

[0291] Detection Uses and Kits

[0292] The antibodies or antibody conjugates thereof of the present invention can be used in detection applications, such as for detecting samples, thereby providing diagnostic information.

[0293] In the present invention, the samples used include cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tumor resection samples, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.

[0294] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0295] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0296] Pharmaceutical composition

[0297] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned single domain antibody, antibody, or fusion protein thereof, or ADC or CAR-immune cell thereof, 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 nature of the formulated substance and the condition to be treated.

[0298] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0299] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) 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, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. 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 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0300] 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 20 mg / kg body weight. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0301] The main advantages of the present invention include:

[0302] 1) The single-domain antibody of the present invention has a specific high affinity for BCMA and can bind to human or monkey BCMA, especially has a high affinity for binding to cell surface BCMA.

[0303] 2) CAR cells containing the single-domain antibody of the present invention as the extracellular antigen binding domain have significant tumor killing ability both in vivo and in vitro.

[0304] 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 specific conditions are not specified, are 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 by weight.

[0305] Experimental methods

[0306] 1. Camel Immunity

[0307] Camels were immunized five times using recombinant antigen. Human BCMA-hFC was provided by Chengdu Shengshi Junlian Biotechnology Co., Ltd.

[0308] Table 1 Immunization method

[0309] Table 2 Immunization steps

[0310] Camel serum titer test:

[0311] 1) Coating: Use 2 μg / mL Human BCMA-His antigen, 100 μL / well, and coat overnight at 4°C.

[0312] 2) Blocking: Use 1% PVA and block at room temperature (RT) for 2 h.

[0313] 3) Sample incubation: Camel plasma was diluted at a ratio of 1:4000-1:128000, added at 100 μL / well, and incubated at RT for 2 h.

[0314] 4) Secondary antibody incubation: Add 100 μL / well of HRP-anti-alpaca IgG Fc and incubate at RT for 1 h.

[0315] 5) Color development: Add 100 μL / well of TMB and develop color at 37°C for 5-15 min.

[0316] 6) Termination: Add 50 mL / well of 1 M phosphoric acid to terminate the reaction.

[0317] 7) Reading: Measure the absorbance at 450 nm using a microplate reader.

[0318] 2. Antibody Library Construction

[0319] The condition for library construction is that the serum titer of camel after five immunizations reaches ELISA titer>32000. VHH3 immune library was constructed using PBMC of camel after five immunizations:

[0320] 1) PBMCs were isolated using Ficoll density gradient centrifugation.

[0321] 2) Total RNA was extracted using an RNA extraction kit (FORGENE, catalog number: RE-03111).

[0322] 3) cDNA was synthesized using a cDNA reverse transcription kit (TAKARA, Cat. No. 6210A).

[0323] 4) VHH amplification: A two-step method (nested PCR) was used for amplification.

[0324] 5) Phagemid construction: The VHH fragment was inserted into the New Lib VHH XE phagemid by Gioson Assembly reaction.

[0325] 6) Phage amplification: Electroporation of phagemids into super competent cells SR320+Helper Phage (cells were prepared by Chengdu Shengshi Junlian Biotechnology Co., Ltd.).

[0326] 7) Antibody library harvesting: Phages were separated and purified by PEG8000 / NaCl precipitation to obtain an antibody library.

[0327] 3. Bio-panning

[0328] A protein panning screening strategy was used. Human BCMA-His (provided by Chengdu Shengshi Junlian Biotechnology Co., Ltd., Catalog No. BCA-H522y) was first used for solid-phase screening for 1-5 rounds to obtain an enriched phage library. Cyno BCMA-His antigen (provided by Chengdu Shengshi Junlian Biotechnology Co., Ltd., Catalog No. BCA-C52H7) was then used for another 1-5 rounds of screening to obtain a human-monkey double-positive phage library.

[0329] 3.1 Protein screening

[0330] 1) On the first day, the antigen was plated at 500 ng / well onto a Maxi-Sorp plate and incubated at 4°C overnight.

[0331] 2) On the second day, the ELISA plate was blocked with 1% PVA at room temperature for 2 h.

[0332] 3) Add the antibody library and mix at room temperature for 2 hours.

[0333] 4) Clean the Maxi-sorp board with PT.

[0334] 5) Add 100 mL of 100 mM HCl to the Maxi-Sorp plate to elute the bound phage.

[0335] 6) Neutralize with 1 M Tris-HCl.

[0336] 7) Infect 1 mL of NEBalpha5F' cells (NEB, Cat. No. C2992I) at OD600 = 0.8 with 100 mL of neutralization solution and shake at 37°C for 1 hour. Add M13K07 helper phage and shake at 37°C for 1 hour. Transfer the cells to 40 mL of 2YT / Carb / Kan medium and expand the culture overnight at 37°C.

[0337] 8) Enrichment Identification: Plate by drop method (conducted simultaneously with step 7) and calculate the degree of enrichment on day 3, using PVA wells as negative controls. Enrichment is considered successful if the phage clones enriched in the antigen wells are more than 10 times the phage clones enriched in the PVA wells.

[0338] 9) Harvest: On the third day, the expanded phage culture was isolated and purified using the PEG8000 / NaCl precipitation method, and then entered the next round of panning until successful enrichment.

[0339] 10) After successful enrichment, randomly pick single clones for Phage ELISA.

[0340] Phage ELISA method:

[0341] 1) Coating: Day 1: Coat the ELISA plate with 100 ng of recombinant antigen protein per well at 4°C overnight.

[0342] 2) Shake the cells: On Day 1, pick the positive phage clones and shake them in a deep-well plate at 37°C overnight.

[0343] 3) Blocking: On Day 2, the ELISA plate was blocked with 1% PVA at room temperature for 2 h.

[0344] 4) Binding: On Day 2, centrifuge the deep-well plate and the supernatant is the phage; transfer 50 mL of the supernatant to an ELISA plate and incubate at room temperature for 2 h.

[0345] 5) Washing: Wash the ELISA plate with PT, add 100 mL of Anti-M13 HRP antibody, and incubate at room temperature for 1 hour.

[0346] 6) Color development: Wash the ELISA plate with PT and then with PBS, add 100 mL of TMB, and incubate at 37°C for 5-10 minutes for color development.

[0347] 7) Termination: Add 50 mL of 1 M phosphoric acid to terminate the reaction.

[0348] 8) Measurement: Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0349] 4. Antibody Expression Verification

[0350] The positive clone BC-HC-15 VHH gene fragment was amplified by PCR and inserted into a eukaryotic expression vector to construct a VHH-hFC structure recombinant expression plasmid. The recombinant antibody protein was obtained by transfecting 293F cells for transient expression and purification.

[0351] 4.1 293F eukaryotic transient expression

[0352] 1) Construction of recombinant expression plasmid: PCR amplified the VHH antibody sequence and inserted it into the pVHH-hIgG1Fc expression plasmid to construct the VHH-hIgG1 Fc antibody recombinant expression plasmid. The clones with correct insertion were identified by sequencing.

[0353] 2) Plasmid extraction: Expand the competent cells containing the plasmid and use an endotoxin-free plasmid extraction kit to extract the recombinant plasmid.

[0354] 3) Transfection: Use transfection reagent to transfer the recombinant plasmid into 293F cells.

[0355] 4) Expression: 50 mL of 293F cells were cultured for 5 days, the cell supernatant was collected, and the target protein was purified using protein A dextran.

[0356] 5) SDS-PAGE: Run the purified protein on SDS-PAGE gel to determine the protein purity and size.

[0357] 4.2 ELISA affinity verification

[0358] Verify the affinity of nanoantibodies to antigen proteins by ELISA:

[0359] 1) Coating: Antigen protein was coated on ELISA plates at 200 ng / well at 4°C overnight.

[0360] 2) Blocking: Block the ELISA plate with 1% PVA at room temperature for 1.5 h.

[0361] 3) Sample incubation: After serial dilution of the antibody, 100 mL / well was added to the ELISA plate and incubated at room temperature for 2 h.

[0362] 4) Secondary antibody incubation: Wash the ELISA plate with PT, add 100 mL / well of anti-human Fc HRP antibody, and incubate at room temperature for 1 hour.

[0363] 5) Color development: Wash the ELISA plate with PT and then with PBS, add 100 mL of TMB, and incubate at 37°C for 5-10 minutes for color development.

[0364] 6) Termination: Add 50 mL / well of 1 M phosphoric acid to terminate the reaction.

[0365] 7) Measurement: Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0366] 4.3 FACS affinity verification

[0367] The affinity of the nanobody to BCMC-positive cells was verified by FACS.

[0368] 1) Cell preparation: K562 and NCI-H929 cells were grouped at 4×10E5 cells / group, centrifuged at 300 g for 3 min, and washed three times with PBS.

[0369] 2) Sample incubation: Add 100 μL of nanoantibodies with a final concentration of 10 μg / mL and incubate at 4°C for 45 min.

[0370] 3) Secondary antibody incubation: Centrifuge the cells at 300 g for 3 min, wash three times with PBS, add 100 μL of PE-labeled Anti-Human Fc antibody, and incubate at 4°C for 45 min.

[0371] 4) Washing: Centrifuge the cells at 300 g for 3 min, wash three times with PBS, and resuspend in PBS at a volume of 300 mL per tube.

[0372] 5) Flow cytometry: Flow cytometry is used to detect the fluorescence value of cells.

[0373] Example 1 Preparation of anti-BCMA nanobody

[0374] 1. Antibody Library Construction

[0375] The titer of camel serum after five immunizations met the ELISA titer of >32,000, meeting the library construction requirements. VHH3 immune library was constructed using PBMC from camels after five immunizations.

[0376] 1.1. Immune library construction

[0377] Peripheral blood collected from camels after 5 immunizations was used to isolate PBMCs, extract total RNA, and then reverse transcribe it into cDNA. Nested PCR (two-step method) was used to amplify the VHH3 gene. In the first step, PCR amplified the gene fragment of the Leader-CH2 region. The DNA electrophoresis results (Figure 1A) showed that two gene fragments were obtained, of which the 700bp fragment was the Leader-VHH-Hinge-CH2 region of the IgG2 antibody subtype; and the 900bp fragment was the Leader-VH-CH1-Hinge-CH2 region of the IgG1 antibody subtype. The 700bp IgG2 gene fragment was recovered by gel excision, and the second step PCR amplification of the VHH gene fragment was performed using specific primers. The DNA electrophoresis results (Figure 1B) showed that a gene fragment of approximately 400bp in size was obtained, which was the correct length of the VHH3 gene fragment.

[0378] 1.2. Antibody library quality

[0379] Using the GA reaction kit, clone the VHH3 gene fragment into the New Lib VHH XE vector. Electrophoretically transfer the GA reaction product into the super-state SR320 cells containing helper phage. After expansion and culture, harvest the phage to obtain the immune library. The quality of the immune library: library capacity 1.40×10 10 The recombination efficiency (number of clones positive by PCR / total number of clones) was 90%, which met the requirements of the immune library. The culture supernatant was collected to obtain the antibody library (Phage), which was frozen at -80°C.

[0380] 2. Bio-screening

[0381] Protein screening

[0382] The antibody library immunized with Human BCMA-His solid-phase screening was screened, and significant enrichment was achieved in round R4. The Human BCMA-His antigen-enriched library was then screened using Cyno BCMA-His antigen, and significant enrichment was achieved in round R2, at which point screening was stopped. (Note: Enrichment was considered successful if the number of clones enriched with the antigen protein (pro) / the number of clones enriched with the negative control (PVA, blocking solution) > 5.)

[0383] 2.2.phage ELISA

[0384] A total of 96 single clones were randomly selected from the Cyno BCMA-His antigen-enriched R2 clones for phage ELISA. Clones with an OD value of ≥2 for binding to human-P-ECD-his and mouse-P-ECD-his recombinant proteins were defined as positive clones. These clones were sequenced to obtain their gene sequences.

[0385] Table 3 BCMA camel library human and monkey double positive monoclonal sequences

[0386] Table 4 BC-HC-15 CDR sequences

[0387] Example 2 Expression and Verification of Nanobodies

[0388] 1. Expression of Nanobodies

[0389] The positive cloned BC-HC-15 VHH gene fragment was amplified by PCR, inserted into a eukaryotic expression vector, and transfected into 293F cells for transient expression and purification to obtain the recombinant antibody. The results of SDS-PAGE electrophoresis are shown in Figure 2. The SDS-PAGE purity was >90%.

[0390] 2. ELISA verification of nanobodies

[0391] Human-BCMA-His antigen protein was plated at 200 ng / well, and the affinity of BC-HC-15 nanobody was tested by ELISA. The results showed that the affinity EC50 of BC-HC-15 nanobody for human-BCMA-His antigen protein was less than 10 nM (Figure 3).

[0392] Table 5. Eukaryotic transient expression data of preferred antibodies

[0393] 3. FACS Validation of Nanobodies

[0394] The affinity of the BC-HC-15 nanobody (10 μg / mL, 100 μL / test) was measured by FACS using BCMA-negative K562 cells and BCMA-positive NCI-H929 cells. The results showed that the binding affinity of the BC-HC-15 nanobody to NCI-H929 cells was >95% (Figure 4), indicating that the BCMA nanobody of the present invention has high affinity for cell surface BCMA.

[0395] Table 6. Eukaryotic transient expression data of preferred antibodies

[0396] Example 3 BC-HC-15 CAR molecule construction and expression

[0397] The BC-HC-15 CAR molecule of this embodiment includes, from N-terminus to C-terminus:

[0398] ① Signal peptide (SEQ ID NO: 14);

[0399] ②BC-HC-15 nanobody;

[0400] ③ hinge and transmembrane region (SEQ ID NO: 16);

[0401] ④ Intracellular 41BB co-stimulatory signal region and CD3ζ signal transduction sequence (SEQ ID NO: 18).

[0402] To enhance the killing effect of CAR cells, the following are sequentially connected to the C-terminus of the BC-HC-15 CAR molecule through the T2A sequence:

[0403] 5. IL-15 (SEQ ID NO: 20) and

[0404] ⑥IL-21 (SEQ ID NO: 22).

[0405] The coding sequence of the above molecule was constructed, and the 5' homology arm (SEQ ID NO: 11) targeting the T cell TRAC gene and the PGK promoter (SEQ ID NO: 23) were connected to its 5' end, and the poly A sequence and the 3' homology arm (SEQ ID NO: 12) were connected to the 3' end. The encoding nucleic acid molecule was transferred into the minipUC-57 plasmid to obtain a plasmid encoding BCMA-CAR.

[0406] A chemically synthesized sgRNA targeting the human TRAC gene (5'TCAGGGTTCTGGATATC TGT (SEQ ID NO: 27) was designed, GenScript Biotech, Nanjing. Cas9 protein was purchased from Sino Biological (Cat. No. 40572-A08B).

[0407] The BCMA-CAR plasmid was electroporated into human T cells that had been activated with Dynal beads for 2-3D. The plasmid was then knocked into the TRAC gene and expressed from either the endogenous or exogenous promoter of the TRAC gene. Control cells were the same human T cells that had not been electroporated but had been activated with Dynal beads for 2-3D.

[0408] Fluorescently labeled BCMA protein was used to detect BCMA-CAR expression in T cells transfected with the BCMA-CAR plasmid using FACS. The results are shown in Figure 6. The gate for BCMA-CAR-positive cells was established by ensuring that the positive rate of live cells in the control group was ≤1%.

[0409] Example 4 Detection of BCMA-CART's specific killing ability on cells

[0410] To detect the specific killing ability of BCMA-CART, target cells expressing different amounts of BCMA (BCMA-positive cells H929, BCMA-negative cells SNU-16, and Huh-7) were stained with Calcein-AM and co-cultured overnight at the effector-target ratio shown in Figure 7. The number of Calcein-positive target cells was detected by constant-speed and timed flow cytometry, and the target cell killing rate was calculated (killing rate = 100% * (NC-N transfer) / NC, where NC = the number of Calcein-positive target cells under control T cells + target cells; N transfer = the number of Calcein-positive target cells under transfected BCMA-CART + target cells). The results showed that BCMA-CART can specifically kill BCMA-positive target cells.

[0411] Example 5 Detection of BCMA-CART amplification under target cell stimulation

[0412] Two batches of BCMA-CART were prepared and cultured. At 0, 7, and 14 days (arrows) shown in Figure 8A, RPMI-8226 target cells were added at an effector-target ratio of 1: 1 or 1: 2 to activate BCMA-CART, and NC-200 was used to count at the time points shown in Figure 8 to obtain the expansion of CART cells after each round of activation. Before each cell activation, CART was counted and adjusted to 1E6, and the amount of target cells added was 1E6 or 2E6. Figure 8B is the total number of cells after activation calculated based on the data in Figure 8A. The results show that BCMA-CART can be activated and amplified by BCMA-positive target cells.

[0413] Example 6 BCMA-CART in vivo transplantation and in vivo tumor inhibition effect detection

[0414] NCG mice were subcutaneously implanted with RPMI-8226 multiple myeloma cells. The tumors grew to approximately 100 mm. 3 At the same time, the mice were randomly divided into three groups and injected intravenously with approximately 2e6 resuscitated BCMA-CART cells.

[0415] The results are shown in Figures 9 and 10. Figure 9 is the flow cytometry analysis of the peripheral blood of three mice about 20 days after CART injection, showing the proportion of hCD45-positive human cells in the peripheral blood of the three mice and the expression rate of BCMA-CAR in the hCD45-positive population. The results show that the CAR of the present invention has a high expression rate and is expressed in most hCD45-positive cell populations.

[0416] Figure 10A shows the changes in mouse body weight over time after CART injection, indicating that the CART cells of the present invention have no significant effect on the body weight of NCG mice. Figure 10B shows the killing ability of CART on tumors in vivo, wherein, about 24 days after grouping, the tumor volume of the control group without CART injection rapidly increased to about 3000 mm 3 The tumor volume in the CART-injected group decreased to zero. Figure 10C shows the survival rate of mice in the control and CART-injected groups. The CART-injected group survived until the end of the experiment at day 90.

[0417] The results show that the T cells described in this application can effectively kill tumors in the body through intravenous injection and have excellent anti-tumor effects.

[0418] Example 7 Comparison of BC-HC VHH CAR-T and Existing Targeted BC CAR-T

[0419] The G5 CAR molecule of this embodiment includes, from N-terminus to C-terminus:

[0420] ① Signal peptide (SEQ ID NO: 14);

[0421] ②BC-HC-15 nanobody;

[0422] ③ hinge and transmembrane region (SEQ ID NO: 16);

[0423] ④ Intracellular 41BB co-stimulatory signal region and CD3ζ signal transduction sequence (SEQ ID NO: 18).

[0424] The 2A peptide is sequentially connected to the C-terminus of the BC-HC-15 CAR molecule:

[0425] ⑤CD34t sequence

[0426] ⑥IL-15 (SEQ ID NO: 20) and

[0427] ⑦IL-21 (SEQ ID NO: 22).

[0428] Among them, CD34t (CD34 Truncated) is a truncated form of CD34, with only 16 amino acids remaining in the intracellular portion. It lacks CD34 function, but expresses the extracellular region of CD34 on the membrane surface for enrichment and detection. The positive control G2 CAR structure is identical to the G5 CAR, except that the BC-HC-15 nanobody is replaced with the scFv of the existing antibody BCMA-50. Its heavy chain variable region and light chain variable region sequences are described in SEQ ID NOs: 11 and 12 of US2017 / 0183418A1, respectively. The negative control G7 is an empty vector.

[0429] An exogenous EF1a promoter (SEQ ID NO: 26) was connected to the 5' end of the CAR molecule coding sequence, and the plasmid was constructed by the method described in the above embodiment and transferred into T cells to obtain CAR-T cells expressing G5 CAR. According to the method described in Example 6, an anti-tumor in vivo test was performed on an RPMI-1640 subcutaneous tumor-bearing animal model. The number of CARTs injected intravenously per animal was 2.5E6.

[0430] The results are shown in Figure 11. Both the positive control G2 CAR and the G5 CAR of the present invention showed significant anti-tumor efficacy, resulting in almost complete suppression of subcutaneous tumors. Among them, G5 CAR was able to suppress tumors for a long time and reach the 120D endpoint of the experimental plan, while all animals of the positive control G2 CAR died before the end of the experimental plan at about 42D. The animal weight also showed that the weight of animals in the G2 CAR group began to drop sharply from about 20D, which coincided with the death of animals at about 42D, while the weight of animals in the G5 CAR group increased steadily during the 120D planned experiment. The results of peripheral blood flow cytometric analysis on the 21st day after CART injection showed that the proportion of CART in the humanized hCD45 population of G5 and G2 was between 20-40%, but G5 had better cell engraftment than G2. This result suggests that the CAR-T cells of the present invention have both efficient tumor suppression effects and good safety.

[0431] The nucleotide sequence encoding the BC-HC-15 CAR-IL15-IL21 molecule is shown below (including homology arms):

[0432] Left homology arm sequence (SEQ ID NO: 11):

[0433] Right homology arm sequence (SEQ ID NO: 12):

[0434] Signal peptide nucleic acid sequence (SEQ ID NO: 13):

[0435] Signal peptide amino acid sequence (SEQ ID NO: 14):

[0436] Hinge-transmembrane nucleic acid sequence (SEQ ID NO: 15):

[0437] Hinge-transmembrane amino acid sequence (SEQ ID NO: 16):

[0438] 41BBz nucleic acid sequence (SEQ ID NO: 17):

[0439] 41BBz amino acid sequence (SEQ ID NO: 18):

[0440] IL-15 nucleic acid sequence (SEQ ID NO: 19):

[0441] IL-15 amino acid sequence (SEQ ID NO: 20):

[0442] IL-21 nucleic acid sequence (SEQ ID NO: 21):

[0443] IL-21 amino acid sequence (SEQ ID NO: 22):

[0444] PGK sequence (SEQ ID NO: 23):

[0445] BC-HC-15 CAR (BC-HC-15 VHH-Hinge-TM-41BBz) nucleic acid sequence (SEQ ID NO: 24):

[0446] BC-HC-15 CAR (BC-HC-15 VHH-Hinge-TM-41BBz) amino acid sequence (SEQ ID NO: 25):

[0447] EF1a promoter sequence (SEQ ID NO: 26):

[0448] 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. An anti-BCMA single domain antibody, characterized in that: The VHH chain of the single-domain antibody has complementary determining regions CDR1, CDR2 and CDR3 derived from the heavy chain variable region shown in SEQ ID NO: 1, and the complementary determining regions CDR1, CDR2 and CDR3 are defined by Chothia, Abm, Kabat, or IMGT rules.

2. The single domain antibody according to claim 1, characterized in that The CDR1, CDR2 and CDR3 are selected from the following groups: (1) Based on the definition of Chothia rules: CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4; or, (2) Based on the definition of Kabat rules: CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:4; or, (3) Based on the definition of IMGT rules: CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:

9.

3. The single domain antibody according to claim 1, characterized in that The amino acid sequence of the VHH chain of the single-domain antibody has an amino acid sequence that is ≥65%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identical to the amino acid sequence shown in SEQ ID NO:1, or is as shown in SEQ ID NO:

1.

4. An anti-BCMA antibody, characterized in that The antibody comprises one or more VHH chains of the single domain antibody of claim 1.

5. A multispecific antibody, characterized in that: The multispecific antibody comprises: the single domain antibody according to claim 1 or the anti-BCMA antibody according to claim 4.

6. A recombinant protein, characterized in that The recombinant protein has: (i) the single domain antibody of claim 1, or the anti-BCMA antibody of claim 4; and (ii) optional polypeptide molecules or fragments having therapeutic functions; and / or (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

7. A chimeric antigen receptor (CAR), characterized in that The nucleotide sequence of the extracellular antigen binding domain of the CAR encodes the VHH chain of the single-domain antibody as described in claim 1.

8. The CAR according to claim 7, wherein: The amino acid sequence of the VHH chain of the single-domain antibody has an amino acid sequence that is ≥65%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identical to the amino acid sequence shown in SEQ ID NO:1, or is as shown in SEQ ID NO:

1.

9. The CAR according to claim 7, wherein: The structure of the protein sequence encoded by the CAR nucleotide sequence is shown in Formula I below: L-EB-H-TM-C n -IS (I) In the formula, Each "-" is independently a connecting peptide or a peptide bond; L is none or a signal peptide sequence; EB is the extracellular antigen binding domain; H is no or hinge region; TM is the transmembrane domain; C n is n costimulatory signal molecules connected in series, wherein C represents a costimulatory signal molecule, and n is an integer selected from 1, 2, 3 or 4; IS is a cytoplasmic signaling sequence.

10. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the single domain antibody according to claim 1, the anti-BCMA antibody according to claim 4, or the multispecific antibody according to claim 5; (2) the recombinant protein according to claim 6; or (3) The CAR as described in claim 7.

11. The polynucleotide according to claim 10, wherein The nucleic acid molecule encoding CAR has the following formula Ia structure: ARM5—P1—CAR1—P2 / L1—Z1—P3 / L2—Z2—(P4 / L3—Z3) m —ARM3 (Ia) In the formula, ARM5 is none or 5′ homology arm; ARM3 is none or 3′ homology arm; P1 is promoterless, splicing acceptor, or the first exogenous promoter; CAR1 is a nucleic acid sequence encoding the chimeric antigen receptor (CAR) according to claim 7; P2 / L1 is the second exogenous promoter P2 or the nucleic acid sequence L1 encoding the cleavable part; Z1 is none, or a nucleotide sequence encoding a second CAR, or IL-15 or a functional protein fragment thereof; P3 / L2 is none or a third exogenous promoter P3 or a nucleic acid sequence L2 encoding a cleavable portion; Z2 is none, or a nucleotide sequence encoding IL-21 or a functional protein fragment thereof; P4 / L3 are each independently a fourth exogenous promoter P4 or a nucleic acid sequence L3 encoding a cleavable portion; Z3 is a nucleic acid sequence encoding an additional functional protein; m is 0, 1, 2, 3, 4 or 5.

12. The polynucleotide according to claim 11, wherein The promoters P1, P2, P3, and each P4 are the same or different exogenous promoters, and the cleavable moieties L1, L2, and L3 are nucleic acid sequences encoding the same or different cleavable moieties.

13. The polynucleotide according to claim 11, wherein The nucleotide sequence of the polynucleotide is shown in SEQ ID NO:

10.

14. The polynucleotide according to claim 11, wherein The functional protein is selected from the following group: IL-21 or its functionally active fragment, IL-15 or its functionally active fragment, IL-2 or its functionally active fragment, IL-7 or its functionally active fragment, IL-10 or its functional fragment, cytokine, immune blocker PD1, TIGIT, VGEF, CTLA-4, CD73, antibody, BiTE, or a combination thereof.

15. A carrier, characterized in that The vector contains the polynucleotide according to claim 10.

16. The vector according to claim 15, characterized in that The vector is a plasmid.

17. A recombinant immune cell, characterized in that: The immune cell expresses the exogenous CAR as described in claim 7, contains the vector as described in claim 15, or has the polynucleotide as described in claim 10 integrated into its genome.

18. The immune cell according to claim 17, characterized in that The polynucleotide is introduced into the immune cells by electro-transfection plasmid method.

19. The immune cell according to claim 18, characterized in that The polynucleotide is positioned and integrated into the DNA of the immune cell by electro-transfection of plasmid and positioning of nuclease.

20. The immune cell according to claim 19, wherein The positioning nuclease includes CRISPR-related proteins and sgRNA.

21. The immune cell according to claim 20, characterized in that The localized nuclease includes a sgRNA targeting the TRAC gene.

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