A nanobody against BCMA, antibody and application thereof

Abstract

The present invention discloses an anti-BCMA nanobody, an antibody and an application thereof, and relates to the field of biomedical technology. The nanobody and antibody provided by the present invention can specifically bind to the BCMA antigen, and have good affinity. In addition, the present invention uses knob-into-hole technology to construct a nanobody targeting BCMA and an anti-CD3 antibody OKT3 into a heterodimeric bispecific antibody, which has good affinity for BCMA and still maintains good antigen binding activity. It has been confirmed by in vivo and in vitro experiments that the heterodimeric bispecific antibody prepared by the present invention has a strong killing activity against multiple myeloma cells, effectively improves the therapeutic effect, and provides a key material for the subsequent development of immunotherapy with high efficiency and lasting anti-tumor activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-BCMA nanobody, an antibody and applications thereof. Background Art

[0002] Multiple myeloma (MM) is a blood tumor characterized by abnormal proliferation of plasma cells (PCs) and the production of a large amount of pathological immunoglobulins in the body. Among blood tumors, its incidence is second only to lymphoma, accounting for about 10% of blood system malignancies, with a male-female ratio of 1.6:1, and most patients are >40 years old. In the past two decades, with the introduction of immunomodulatory drugs (lenalidomide, pomalidomide), proteasome inhibitors (bortezomib, carfilzomib, ixazomib) and histone deacetylase inhibitors, the treatment of MM has made significant progress. Since the late 1980s, these advances have almost doubled the 5-year survival rate of MM patients. Although the diagnosis and treatment of MM in my country has made significant progress and gradually caught up with the international level, MM is still a non-radical disease, and most patients will eventually relapse, and its treatment still faces great challenges. Therefore, in order to improve the problem of reduced efficacy during treatment, it is necessary to develop safer, more effective and more diverse new drugs.

[0003] BCMA (B-cell maturation antigen; CD269; TNFRSF17) is a member of the tumor necrosis factor receptor superfamily and was first discovered in the early 1990s. It is composed of a glycoprotein of 184 amino acids. Structurally, BCMA consists of three main domains: the extracellular segment (amino acids 1-54), the transmembrane region (amino acids 55-77), and the intracellular segment (amino acids 78-184). In normal human tissues, BCMA mRNA and protein are almost exclusively found on plasma cells, and are selectively overexpressed during the malignant transformation of plasma cells, promoting tumor cell growth, survival, and drug resistance. It mainly inhibits the immune system by activating intracellular NF-κB, AKT, PI3K, STAT3, and MAPK signaling pathways. The consistent upregulation and uniqueness of BCMA on the surface of MM cells, whether in cell lines or patient samples, makes BCMA an attractive target for MM drug discovery and development.

[0004] Nanobody (Nb) is a type of antibody that exists in camelids and has the ability to bind to antigens and is naturally missing the first constant region (CH1) of the light chain and heavy chain. It has a small molecular weight (15kDa), which is only 1 / 10 of the traditional monoclonal antibody. It has good stability, solubility, affinity, specificity, flexibility in coupling with different protein molecules, and easy modification. It has broad development and application value in the fields of disease treatment and detection.

[0005] Bispecific antibodies (BsAb) are artificial antibodies that can simultaneously target two antigens or different epitopes of the same antigen. Bispecific antibodies (BiTE) targeting CD3 molecules mediate the formation of immune synapses to redirect T cells, thereby activating T cells and releasing cytotoxins and cytokines to exert killing functions. The above characteristics provide theoretical support for targeted immunotherapy of tumors. Bispecific antibody-mediated T cell redirection to kill tumor cells is considered to be the most promising cancer treatment strategy.

[0006] The IgG subtype in BsAb also affects its functional activity, because different subtypes have different variable region compositions, hinge region sequence lengths and flexibility, and disulfide bond structures. Studies have shown that the flexibility of the hinge region of IgG1 is the most ideal, followed by IgG4. Kapelski S et al. used CD19×CD3 bispecific antibodies as an example, and used Fc mutations of different subtypes to construct multiple combinations to evaluate the effects of antibody subtypes on T cell redirection and activity. The results showed that when the concentration of the bispecific antibody was 20 μg / mL, the binding to the two target cells with high and low expression of CD19 did not differ due to different subtypes, but CD19×CD3 IgG2σ1 was 25% weaker than other subtypes in binding to T cells. In the experiment of observing the inhibitory effect of CD19×CD3 bispecific antibodies on target cells, it was found that the IgG4 subtype bispecific antibody had a 100% inhibitory effect on the target cell HBL-1RFP, the inhibition rate of the IgG1 FES subtype was 75%, and the inhibition rate of the IgG2σ1 subtype was between the intermediate level of 90%, but its half-inhibitory concentration was 10 times lower than that of other subtypes. Therefore, the IgG2σ1 subtype is less suitable for the redirection effect of dual antibodies compared to IgG1 or IgG4.

[0007] Knob-in-hole (KiH) is a technology invented by the Genentech team to prevent heavy chain mispairing. It uses genetic engineering technology to engineer heavy chains to obtain heterodimers. That is, a small amino acid is used to replace a large amino acid in one heavy chain of the antibody to form a "hole", and a large amino acid is used to replace a small amino acid in the other heavy chain to form a "knob", and finally a heterodimer is formed instead of a homodimer according to the electrostatic guidance theory.

[0008] On October 25, 2022, the "first-in-class" bispecific antibody targeting BCMA and CD3 molecules (trade name Tecvayli) developed by Janssen, a subsidiary of Johnson & Johnson, was approved by the FDA for marketing for the treatment of adult patients with relapsed or refractory multiple myeloma who have received four or more treatments (proteasome inhibitors, immunomodulatory drugs and anti-CD38 monoclonal antibodies). The overall response rate (ORR) reached 63%, and 58.8% of patients achieved very good partial remission or better remission (≥VGPR), and 39.4% of patients achieved complete remission (CR) or above.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The object of the present invention is to provide an anti-BCMA nanobody for the treatment of multiple myeloma.

[0011] The present invention is achieved in that:

[0012] In a first aspect, the present invention provides an anti-BCMA nanobody, the nanobody comprising a heavy chain variable region of any of the following:

[0013] (1) the heavy chain variable region CDR1, the heavy chain variable region CDR2 and the heavy chain variable region CDR3 as shown in the amino acid sequences of SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3;

[0014] (2) the heavy chain variable region CDR1, the heavy chain variable region CDR2 and the heavy chain variable region CDR3 as shown in the amino acid sequences of SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7;

[0015] (3) The heavy chain variable region CDR1, heavy chain variable region CDR2 and heavy chain variable region CDR3 as shown in the amino acid sequences of SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 11.

[0016] The nanobodies with the above amino acid sequences have good reactivity with multiple myeloma cell lines and have strong killing activity against natural multiple myeloma cell lines. In particular, the nanobodies with heavy chain variable region CDR1, heavy chain variable region CDR2 and heavy chain variable region CDR3 as shown in the amino acid sequences of SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7 have higher killing activity against multiple myeloma cells than the other two nanobodies.

[0017] In a preferred embodiment of the present invention, the nanobody further comprises a framework region, and the structure of the heavy chain variable region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0018] In an optional embodiment, the nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific antibody and a fusion nanobody.

[0019] The "multivalent nanoantibodies" in the present invention are polymers of monovalent antibodies that recognize the same epitope, and have higher antigen affinity than monovalent antibodies; "multispecific antibodies" are polymers of monovalent antibodies that recognize different epitopes, and can bind to different targets or different epitopes of the same target, and have higher antigen recognition ability than monovalent antibodies.

[0020] Fusion-type nanobodies include but are not limited to new fusion molecules formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering technology, such as enzymes, antimicrobial peptides or imaging substances that can prolong their half-life.

[0021] In an alternative embodiment, when the Nanobody is a monovalent Nanobody, the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID Nos: 4, 8, 12, 13, 14 and 15.

[0022] Among them, SEQ ID No: 13, 14 and 15 are humanized nanobodies after replacing the amino acids in certain specific sites of the antibody framework region (FR region) of the camel-derived nanobodies shown in SEQ ID No: 4, 8, 12 and keeping the amino acid sequence of the CDR region unchanged.

[0023] In a second aspect, the present invention also provides an antibody comprising the above-mentioned anti-BCMA Nanobody or the heavy chain variable region of the anti-BCMA Nanobody.

[0024] The antibody can be any one of a full-length antibody, a heavy chain antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, a tetraspecific antibody, etc.), a mouse antibody, a humanized antibody, or an antigen-binding fragment. The antigen-binding fragment includes any one selected from F(ab')2, Fab', Fab, Fv, and scFv of an antibody, as long as they exhibit the desired antigen-binding activity.

[0025] The above antigen-binding fragments, i.e., functional fragments of antibodies, generally have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0026] The "chimeric antibody" described in the present invention is an antibody formed by fusing the variable region of a non-human antibody with the constant region or framework region of a human antibody, which can reduce the immune response induced by the non-human antibody.

[0027] The above antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0028] In a preferred embodiment of the present invention, the above-mentioned antibody is a bispecific antibody, which includes a heavy chain a and a heavy chain b, wherein the heavy chain a includes a first protein functional region targeting BCMA and a second protein functional region targeting CD3; the heavy chain b includes a first protein functional region targeting BCMA; and the first protein functional region includes the heavy chain variable region of the above-mentioned nanoantibody.

[0029] In a preferred embodiment of the present invention, the heavy chain a also includes a heavy chain constant region.

[0030] In an optional embodiment, the heavy chain constant region is selected from a human heavy chain constant region. In other embodiments, the heavy chain constant region may be a mouse heavy chain constant region, a rabbit heavy chain constant region, a sheep heavy chain constant region or a monkey heavy chain constant region. The heavy chain constant region selected from a human has a stronger binding ability to T cells.

[0031] In an alternative embodiment, the human heavy chain constant region is selected from the heavy chain constant region of hIgG1, hIgG2, hIgG3, hIgG4 or a mutation thereof.

[0032] In an alternative embodiment, the human heavy chain constant region is selected from the heavy chain constant region of hIgG4 or a mutation thereof.

[0033] The heavy chain constant region of IgG4 is used as the constant region of the bispecific antibody, which has a very high inhibition rate on target cells and maintains good binding activity with antigens. The bispecific antibody of the present invention carries a human Fc fragment and retains the binding effect with FcRn, thereby having a longer half-life.

[0034] In an optional embodiment, the mutations in the human heavy chain constant region are selected from the following group: (1) knob mutations S228P, F234A, L235A and T366W; (2) hole mutations S228P, F234A, L235A, T366S, L368A and Y407V. The mutation positions of the above amino acids refer to the numbering of the antibody sequence amino acids in the IMGT database.

[0035] In an alternative embodiment, the heavy chain b also includes the heavy chain constant region of hIgG4 or a mutation thereof.

[0036] In an alternative embodiment, the mutations in the human heavy chain constant region are selected from: (1) knob mutations S228P, F234A, L235A and T366W; (2) hole mutations S228P, F234A, L235A, T366S, L368A and Y407V.

[0037] In an optional embodiment, the heavy chain constant region on the heavy chain a is a knob chain, the knob chain is arranged at the C-terminus of the second protein functional region, and the amino acid sequence of the knob chain is shown in SEQ ID No: 16; the heavy chain b also includes a hole chain, the hole chain is arranged at the C-terminus of the first protein functional region on the heavy chain b, and the amino acid sequence of the hole chain is shown in SEQ ID No: 17.

[0038] The amino acid sequence shown in SEQ ID No: 16 is the knob chain after the knob mutations S228P, F234A, L235A and T366W. The amino acid sequence shown in SEQ ID No: 17 is the hole chain after the hole mutations S228P, F234A, L235A, T366S, L368A and Y407V.

[0039] In an optional embodiment, the amino acid sequence of the second protein functional region is as shown in SEQ ID No: 20; that is, the scFv amino acids of CKT3.

[0040] In an alternative embodiment, on the heavy chain a, the first protein functional region and the second protein functional region are separated by (G 4 S) n Connection, n is a non-zero natural number.

[0041] In an alternative embodiment, n is 1-20; in another alternative embodiment, n is 3 or 4.

[0042] In an alternative embodiment, the amino acid sequence of heavy chain a is shown in SEQ ID No: 18, and the amino acid sequence of heavy chain b is shown in SEQ ID No: 19.

[0043] The heavy chain a shown in SEQ ID No: 18 is the humanized anti-BCMA nanobody shown in SEQ ID No: 14 + (G 4 S) 3 + scFv amino acids of OKT3 + knob chain shown in SEQ ID No: 16, referred to as k2 chain in the present invention.

[0044] The heavy chain b shown in SEQ ID No: 19 is the humanized anti-BCMA nanobody shown in SEQ ID No: 14 + the hole chain shown in SEQ ID No: 17, referred to as the h1 chain in the present invention.

[0045] The combination of K2 and H1 chains forms a heterodimeric bispecific antibody (K2H1) with an affinity for BCMA of 10 - 10 M, maintains good antigen binding activity and has higher affinity than the combination of K1 and H1 chains (K1H1), and K2H1 exhibits relatively stronger killing activity at low concentrations.

[0046] In a preferred embodiment of the present invention, the present invention also provides another bispecific antibody, which includes a heavy chain c and a heavy chain d, wherein the heavy chain c includes a first protein functional region targeting BCMA, and the heavy chain d includes a second protein functional region targeting CD3.

[0047] The first protein functional region includes the heavy chain variable region of the above-mentioned nanobody.

[0048] In a preferred embodiment of the present invention, the heavy chain c also includes a heavy chain constant region.

[0049] In an alternative embodiment, the heavy chain constant region is selected from a human heavy chain constant region.

[0050] In an alternative embodiment, the human heavy chain constant region is selected from the heavy chain constant region of hIgG1, hIgG2, hIgG3, hIgG4 or a mutation thereof.

[0051] In an alternative embodiment, the human heavy chain constant region is selected from the heavy chain constant region of hIgG4 or a mutation thereof.

[0052] In an alternative embodiment, the mutations in the human heavy chain constant region are the following group: (1) knob mutations S228P, F234A, L235A and T366W; (2) hole mutations S228P, F234A, L235A, T366S, L368A and Y407V.

[0053] In an alternative embodiment, the heavy chain d also includes the heavy chain constant region of hIgG4 or a mutation thereof.

[0054] In an alternative embodiment, the mutations in the human heavy chain constant region on heavy chain d are the following group: (1) knob mutations S228P, F234A, L235A and T366W; (2) hole mutations S228P, F234A, L235A, T366S, L368A and Y407V;

[0055] In an optional embodiment, the heavy chain constant region on the heavy chain c is a hole chain, the hole chain is arranged at the C-terminus of the first protein functional region, and the amino acid sequence of the hole chain is shown in SEQ ID No: 17.

[0056] The heavy chain constant region on the heavy chain d is a knob chain. The knob chain is arranged at the C-terminus of the second protein functional region on the heavy chain d. The amino acid sequence of the knob chain is shown in SEQ ID No:16.

[0057] In an optional embodiment, the amino acid sequence of the second protein functional region is as shown in SEQ ID No: 20, which is the scFv amino acid of OKT3.

[0058] In an optional embodiment, the amino acid sequence of the heavy chain c is as shown in SEQ ID No: 19, which is composed of the humanized anti-BCMA nanobody shown in SEQ ID No: 14 + the hole chain shown in SEQ ID No: 17, which is referred to as the h1 chain in the present invention.

[0059] The amino acid sequence of heavy chain d is shown in SEQ ID No: 21, which is composed of the scFv amino acids of OKT3 + the knob chain shown in SEQ ID No: 16, and is referred to as k1 chain in the present invention.

[0060] The combination of H1 chain and K1 chain forms a heterodimeric bispecific antibody (K1H1), which has an affinity for BCMA of 10 -10 M, can maintain good antigen binding activity.

[0061] In a third aspect, the present invention also provides an isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, wherein the isolated nucleic acid encodes the above-mentioned anti-BCMA nanobody or the above-mentioned antibody.

[0062] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0063] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can directly introduce the target DNA fragment into the host cell by transformation, transfection or transduction to express the target gene.

[0064] In a fourth aspect, the present invention also provides a host cell containing the above-mentioned recombinant vector. Specifically, the host cell includes 293 cells, 293T cells, 293FT cells, CHO cells, and Per6 cells. 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

[0065] The embodiment of the present invention also provides a method for preparing an antibody, which comprises: culturing the host cell as described above to obtain the antibody. Specifically, the present invention does not specifically limit the culturing conditions of the host cell, and the culturing conditions that enable the host cell to express and produce the antibody can be obtained based on conventional technical knowledge.

[0066] The embodiment of the present invention also provides a method for preparing a bispecific antibody, wherein the bispecific antibody is prepared using the knob-into-hole technology, wherein the Fc used in the bispecific antibody is of human IgG4 subtype, and the hinge region, CH2 and CH3 regions thereof are mutated and modified, wherein the mutations corresponding to the knob chain in the modification are: S228P, F234A, L235A and T366W, and the corresponding amino acid sequence is shown in SEQ ID No: 16; the mutations corresponding to the hole chain in the modification are: S228P, F234A, L235A, T366S, L368A and Y407V, and the corresponding amino acid sequence is shown in SEQ ID No: 17.

[0067] In a fifth aspect, the present invention also provides an immunoconjugate or a pharmaceutical composition, which comprises the above-mentioned anti-BCMA nanobody or the above-mentioned antibody.

[0068] In an alternative embodiment, the immunoconjugate further comprises a therapeutic agent.

[0069] In an optional embodiment, the therapeutic agent includes at least one of: a chemotherapeutic drug, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody to an inhibitory second signal molecule, a PD-L1 inhibitor, and a PD-1 / PD-L1 monoclonal antibody drug.

[0070] Relevant biomarkers for immune checkpoint inhibitor treatment include PD-L1, MSI / bMSI, TMB / bTMB, TNB, EGFR mutation, ALK fusion, TP53 mutation, and KRAS mutation.

[0071] In an optional embodiment, the above-mentioned chemotherapy drug is selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, flavopiridol, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, danucetinib, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecin and saquinavir.

[0072] The photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or its derivatives; (2) photosensitizer compounds containing a tetrapyrrole ring; (3) traditional Chinese medicine photosensitizers; or (4) a combination of ALA or its derivatives and the compounds in (2) or (3).

[0073] The photothermal agent is selected from IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061 and IR-26.

[0074] The inhibitory second signal molecule can be PD-1; CTLA-4; PD-1 and CTLA-4.

[0075] In a preferred embodiment of the present invention, the PD-1 / PD-L1 monoclonal antibody drug is selected from at least one of the following groups: Nivolumab, Pembrolizumab, Pidilizumab, Lambrolizumab, BMS-936559, Atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROOl, REGN2810, SHR1210, STIAl lOX, STIAl lO, TSR042, BMS-936558, BGB-A317, BCD-100 and JS001.

[0076] In other embodiments, the above therapeutic agent further comprises a cytotoxic agent.

[0077] In an alternative embodiment, the pharmaceutical composition comprises at least one of a pharmaceutically acceptable excipient, a carrier and a diluent.

[0078] The above-mentioned pharmaceutically acceptable carriers include, but are not limited to, fillers, lubricants, disintegrants, binders, glidants and the like.

[0079] In the preferred technical scheme of the present invention, the pharmaceutically acceptable carrier includes but is not limited to polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0080] In a sixth aspect, the present invention also provides the use of the above-mentioned anti-BCMA nanobodies, antibodies, isolated nucleic acids, expression cassettes containing isolated nucleic acids, or recombinant vectors containing isolated nucleic acids, host cells, or immunoconjugates or pharmaceutical compositions in the preparation of products for preventing or treating tumors.

[0081] In an optional embodiment, the product includes at least one of: immune cells, reagents, kits, drugs and pharmaceutical compositions.

[0082] In an alternative embodiment, the product is used to treat multiple myeloma.

[0083] The present invention has the following beneficial effects:

[0084] The anti-BCMA nanobody screened from the Bactrian camel VHH immune library can specifically bind to the BCMA antigen with good affinity. In addition, the present invention uses the knob-into-hole technology to construct a heterodimeric bispecific antibody targeting BCMA and the anti-CD3 antibody OKT3, which has an affinity for BCMA of 10 -10 M, and still maintains good antigen binding activity. In vivo and in vitro experiments have confirmed that the heterodimeric bispecific antibody prepared by the present invention has strong killing activity against multiple myeloma cells, effectively improves the therapeutic effect, and provides a key material for the subsequent development of immunotherapy with high efficiency and lasting anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0086] Figure 1 In the embodiment of the present invention, IFA is used to detect recombinant nano antibodies and BCMA + -Hela cell binding effect diagram;

[0087] Figure 2 This is a diagram showing the binding effect of recombinant nano antibodies and multiple myeloma cell lines detected by flow cytometry in an embodiment of the present invention;

[0088] Figure 3 This is a graph showing the killing efficiency of Nbs-OKT3 bispecific antibody (BiTE) on target cells in an embodiment of the present invention;

[0089] Figure 4 is a schematic diagram of the construction of heterodimeric bispecific antibodies K1H1 and K2H1 in an embodiment of the present invention;

[0090] Figure 5 The expression detection of heterodimeric bispecific antibodies K1H1 and K2H1 by SDS-PAGE in the embodiment of the present invention;

[0091] Figure 6 is the purification of the bispecific antibody K2H1 in the embodiment of the present invention; wherein M is a marker, 1 is unpurified reduced, 2 is unpurified non-reduced, 3 is purified reduced, and 4 is purified non-reduced;

[0092] Figure 7 The bispecific antibody of the present invention is against BCMA + - In vitro killing activity analysis of Hela cells;

[0093] Figure 8 The preferred heterodimeric bispecific antibody K2H1 in the embodiments of the present invention is the survival analysis of the MM mouse transplantation model. DETAILED DESCRIPTION

[0094] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0095] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0096] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0097] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0098] Example 1

[0099] Preparation of anti-BCMA protein specific nanobody

[0100] The present invention first mixed BCMA protein (1 mg / time) and an equal volume of Freund's adjuvant, and then immunized Bactrian camels 4 times continuously and collected peripheral blood to separate lymphocytes, and then used an RNA extraction kit to extract total RNA of lymphocytes, and amplified VHH genes by RT-PCR, and cloned the amplified VHH genes into a phage display vector pMECS, and successfully constructed a VHH phage antibody library. Anti-BCMA nanoantibodies were obtained by continuous screening for 3 rounds using phage display technology, and VHH genes were amplified using them as templates and constructed into a eukaryotic expression vector pcDNA3.1-hFc-His by homologous recombination, and then transfected into HEK293T cells in the logarithmic growth phase using a transfection reagent PEI, and the supernatant was collected after 5 days of expression, and purified by affinity chromatography using an NTA-Ni column to obtain a high-purity recombinant nanoantibody.

[0101] Example 2

[0102] IFA detection of recombinant nanoantibodies and BCMA + -HeLa cell binding

[0103] First, Hela cells were infected with a lentivirus containing the full-length BCMA gene, and high-purity BCMA cells that stably expressed the BCMA gene were obtained by flow cytometry. + -Hela cells, and then the recombinant nanobody prepared in Example 1 and the positive control antibody 11D5.3 (2.5 μg / mL) were mixed with BCMA + -Hela cells were incubated at 37°C for 40 min, washed 3 times with PBS, and then incubated with 594@goatanti-human secondary antibody. After washing 3 times with PBS, the images were imaged by fluorescence microscopy. The results are shown in Figure 1 As shown, the recombinant nanoantibodies Nb381, Nb388 and Nb394 and the positive control antibody 11D5.3 can all bind well to the above cells.

[0104] Example 3

[0105] Flow cytometry detection of the binding of recombinant nanobodies to multiple myeloma cell lines

[0106] The recombinant nanobodies Nb381, Nb388 and Nb394 prepared in Example 1, the positive control antibody 11D5.3 and the commercial anti-BCMA antibody (2.5 μg / mL) were incubated with multiple myeloma cells RPMI-8226 and MM.1S at 37°C for 40 min, washed 3 times with PBS, and then incubated with APC@goat anti-human secondary antibody. After washing 3 times with PBS, the cells were analyzed by flow cytometry. The results are shown in FIG. Figure 2As shown, the recombinant nanobodies Nb381, Nb388 and Nb394 had good reactivity with RPMI-8226 and MM.1S cells.

[0107] Example 4

[0108] Humanization, expression and purification of nanobodies

[0109] Referring to the sequence characteristics of the fully human antibody framework region, the amino acids at certain specific sites of the camel-derived nanoantibody framework region (FR region) were replaced while keeping the amino acid sequence of the CDR region unchanged, the corresponding humanized nanoantibody gene was designed and fully synthesized, and the vector was constructed, expressed and purified with reference to the method of Example 1.

[0110] Example 5

[0111] Preparation of anti-BCMA and CD3 bispecific antibodies

[0112] 5.1 Preparation and activity detection of anti-BCMA and CD3 bispecific antibodies (BiTE)

[0113] The three humanized nanobodies of the present invention were respectively combined with the anti-CD3 antibody OKT3 using (G 4 S) 3 The linker was constructed in series into a eukaryotic expression vector, transfected into HEK293T cells for expression, and purified using a Ni column. The purified bispecific antibodies of different concentrations were co-incubated with multiple myeloma cell line MM.1S-luciferase and activated T cells at an effector-target ratio of 3:1 for 24 hours, and the killing activity was evaluated by detecting the fluorescence intensity. The results are shown in Figure 2. Figure 3 As shown, the above three bispecific antibodies all had strong killing activity against natural MM cell lines, and hu388-OKT3 had relatively strong activity, so hu388 was selected for subsequent experiments.

[0114] 5.2 Design of anti-BCMA and CD3 heterodimeric bispecific antibodies

[0115] The present invention refers to the knob-into-hole technology, selects human IgG4-Fc as the bispecific antibody construction framework and performs mutation modification on it, specifically as follows: the mutations corresponding to the knob chain in the mutation modification are: S228P, F234A, L235A and T366W, and the corresponding amino acid sequence is shown in SEQ ID No.16; the mutations corresponding to the hole chain in the modification are: S228P, F234A, L235A, T366S, L368A and Y407V, and the corresponding amino acid sequence is shown in SEQ ID No.17. Based on the above design, the anti-BCMA humanized nanobody hu388 and the anti-CD3 antibody OKT3 were fused to the above framework to construct bispecific antibodies and named k1h1 and k2h1, respectively, where k1 is OKT3-knob, and the corresponding amino acid sequence is shown in SEQ ID No.21; h1 is hu388-hole, and the corresponding amino acid sequence is shown in SEQ ID No.19; k2 is hu388-OKT3-knob, and the corresponding amino acid sequence is shown in SEQ ID No.18. The structural diagram of the above two heterodimeric bispecific antibodies is shown in Figure 1. Figure 4 shown.

[0116] 5.3 Preparation of heterodimeric bispecific antibodies K1H1 and K2H1

[0117] After k1, h1 and k2, h1 were mixed with transfection reagent PEI (mass-to-volume ratio 3:1) according to a certain mass ratio, left to stand at room temperature for 20 minutes, and slowly added to HEK293T cells in the logarithmic growth phase. After transfection for 6-8 hours, the medium was replaced with Freestyle medium and cultured for 5 days. The supernatant was collected and purified with protein G filler according to the instructions. The results are shown in Figure 5 As shown, the heterologous dimeric bispecific antibodies K1H1 and K2H1 were successfully expressed and the assembly effect was best when the ratio of knob chain to hole chain was 1:1.

[0118] 5.4 Further purification of heterodimeric bispecific antibodies

[0119] The bispecific antibody prepared in Example (2) was purified using a HiTrapTM Heparin HP (GE) purification column, wherein the buffer used in the purification process was 20 mM Tris, 250 mM NaCl. The results are as follows: Figure 6 a, 6b, where 6a is a real-time monitoring diagram of the purification process, SDS-PAGE ( Figure 6b) shows that the highly pure bispecific antibody K2H1 was obtained after purification. At the same time, the affinity detection of the above antibodies to BCMA using Biacore 8k showed that the affinities of K1H1 and K2H1 to BCMA were 8.28E-10M and 1.59E-10M, respectively.

[0120] Example 6

[0121] Bispecific Antibodies Against BCMA + -In vitro killing activity analysis of Hela cells

[0122] First, BCMA + -Hela cells were added to a 96-well plate dedicated to a label-free killing detector (RTCA) and a common 96-well cell culture plate at a rate of 5000 cells per well and 100 μL / well (duplicate wells), respectively. The RTCA-dedicated 96-well plate was placed in the instrument and cultured until the Cell Index value was between 1.0 and 2.0. Then, the bispecific antibodies prepared in Example 5 at different concentrations (0.2 nM and 0.02 nM) and the negative control antibody hu248-OKT3 were co-incubated with activated T cells from the peripheral blood of healthy donors at an effector-target ratio of 3:1, and the RTCA monitoring time was 72 h. At 24 h and 48 h of co-culture, the cell state of the common 96-well plate was observed by fluorescence microscopy to evaluate the killing activity.

[0123] The results are as follows Figure 7 As shown, the above bispecific antibodies, except for the control antibody hu248-OKT3, are + -HeLa cells all have strong killing activity and K2H1 shows relatively stronger killing activity at low concentrations, so K2H1 was selected for subsequent in vivo activity verification.

[0124] Example 7

[0125] In vivo testing of the antitumor activity of the bispecific antibody K2H1 in a MM mouse xenograft model

[0126] MM.1S-luciferase and RPMI-8226-luciferase cells were cultured at 2×10 6 The cells were inoculated into 6-10 week old NCG mice through the tail vein. Ten days after inoculation, the tumor growth was detected in a live imaging instrument and the mice were randomly divided into three groups, with 5 mice in each group. The control group was set up with PBS of the same volume as the drug administration. The bispecific antibody was administered intraperitoneally at 30 μg / mouse. At the same time as the first administration, 2×10 6The bispecific antibody was administered once every 7 days for a total of 3 times. The survival status of each mouse in the experimental group (k2h1), control group (hu248-OKT3) and blank group (PBS) was observed and statistically analyzed for 60 days. The Kaplan-Meier method was used to draw the mouse survival curve and the log-rank (Mantel-Cox) test was used to statistically compare the differences in the survival of mice in each group.

[0127] The results are as follows Figure 8 As shown, the survival period of the bispecific antibody K2H1 group was significantly longer than that of the negative control antibody HU248-OKT3 and PBS groups, indicating that the bispecific antibody K2H1 has persistent anti-tumor activity against multiple myeloma cells.

[0128] The antibody sequences involved in the aforementioned embodiments and the CDR sequences summarized based on the analysis and comparison of antibody sequences in the IMGT database are shown in Table 1 below.

[0129] Table 1 Antibody sequence information

[0130]

[0131]

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bispecific antibody, characterized in that: It comprises heavy chain a and heavy chain b, the amino acid sequence of the heavy chain a is shown in SEQ ID No: 18, and the amino acid sequence of the heavy chain b is shown in SEQ ID No:

19.

2. An isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, characterized in that: The isolated nucleic acid encodes the bispecific antibody of claim 1.

3. A host cell, characterized in that It contains the recombinant vector as claimed in claim 2.

4. An immunoconjugate or pharmaceutical composition, characterized in that It comprises the bispecific antibody according to claim 1.

5. The immunoconjugate or pharmaceutical composition according to claim 4, characterized in that The immunoconjugate also includes: at least one of a chemotherapeutic drug, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, and an antibody to an inhibitory second signal molecule; the inhibitory second signal molecule is selected from PD-1 or CTLA-4; and the pharmaceutical composition includes at least one of a pharmaceutical excipient, a carrier, and a diluent.

6. The immunoconjugate or pharmaceutical composition according to claim 5, characterized in that The chemotherapeutic drug is selected from kinase inhibitors.

7. The immunoconjugate or pharmaceutical composition according to claim 5, characterized in that The immune checkpoint inhibitor is selected from a PD-L1 inhibitor or a PD-1 / PD-L1 monoclonal antibody drug.

8. Use of the bispecific antibody according to claim 1, the isolated nucleic acid according to claim 2, an expression cassette containing the isolated nucleic acid or a recombinant vector containing the isolated nucleic acid, the host cell according to claim 3, or the immunoconjugate or pharmaceutical composition according to any one of claims 4 to 7 in the preparation of a product for preventing or treating multiple myeloma.

9. The use according to claim 8, characterized in that: The product includes at least one of immune cells, reagents, kits and drugs.

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