BCMA-targeting single-domain antibody, chimeric antigen receptor and use thereof

Abstract

Provided are a BCMA-targeting single-domain antibody, a chimeric antigen receptor, and a use thereof. Specifically provided are a BCMA-targeting single-domain antibody BCMA-VHH#81, a dual-epitope chimeric antigen receptor containing two different single-domain antibodies, a BCMA CAR-T cell, and a use thereof. The single-domain antibody BCMA-VHH#81 has high affinity. The BCMA CAR-T cell developed on the basis of the single-domain antibody BCMA-VHH#81 can well secrete T cell-specific effector molecule IFN-γ, can effectively specifically kill BCMA+ target cells, has good in-vivo tumor killing activity, and can not only significantly inhibit the proliferation of tumor cells in mice but also significantly prolong the survival time of the mice. The BCMA CAR-T cell has good anti-tumor ability, can be used in immunotherapy for BCMA-target-related diseases, and has wide clinical application prospects.

Description

Single-domain antibodies and chimeric antigen receptors targeting BCMA and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410312112.4 filed with the Patent Office of China on March 19, 2024, entitled “Single-domain antibodies, chimeric antigen receptors targeting BCMA and their applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application belongs to the field of cellular immunotherapy technology, and specifically relates to single-domain antibodies targeting BCMA, chimeric antigen receptors, and their applications. Background Art

[0004] Multiple myeloma (MM) is a malignant disease characterized by the abnormal proliferation of monoclonal plasma cells. It is characterized by the abnormal proliferation of bone marrow plasma cells and the secretion of monoclonal immunoglobulins or their fragments (M proteins). It is a common hematological malignancy, accounting for 10% of hematological malignancies. Common symptoms of MM include myeloma-related organ dysfunction and related manifestations such as secondary amyloidosis. Excessive secretion of immunoglobulins and uncontrolled plasma cells cause damage to the patient's end organs, ultimately leading to the patient's death. Currently, several CAR-T therapies for the treatment of multiple myeloma have been approved both domestically and internationally. MM patients have achieved early, deep and lasting remission, but most will eventually relapse. Therefore, how to enhance the safety and efficacy of CAR-T in multiple myeloma is the current research focus.

[0005] BCMA stands for B cell maturation antigen, also known as CD269. It is only expressed on the surface of mature B cells and is an important B cell biomarker. In normal human tissues, BCMA protein and mRNA are almost only found on plasma cells, and are selectively overexpressed during the malignant transformation of plasma cells, promoting tumor cell growth, survival and drug resistance. In multiple myeloma, BCMA expression increases, pro-proliferation signals are enhanced, and eventually cancerization occurs. Therefore, the expression level of BCMA on multiple myeloma cells is significantly higher than that on healthy plasma cells. Whether it is a cell line or a patient sample, the consistent upregulation and uniqueness of BCMA on the surface of MM cells makes BCMA an attractive new target for MM drug discovery and development.

[0006] CAR is a synthetic protein with a modular design, including an antigen binding domain (the specificity of the antibody used for CAR molecule design is crucial), a connecting region (the hinge region and transmembrane region are usually derived from CD8 or CD28, and the differences in their length and composition will affect the expression, flexibility, signal transduction, etc. of the CAR molecule) and an intracellular co-stimulatory domain and a signal transduction domain. The CAR structure causes cellular cascade signal transduction, inducing effector T cell functions such as proliferation, cytokine release and cytotoxicity. Different designs will result in different activation dynamics and intensities of signal transduction molecules. For example, the most common co-stimulatory domains are CD28 and 4-1BB. The CD28 co-stimulatory factor can induce a stronger immune response, but the duration of its effect is limited, while the 4-1BB factor has a lower activation ability but a higher long-term persistence.

[0007] In view of this, the continued development of new therapies for multiple myeloma and improving the therapeutic effect, safety, and efficacy of BCMA-targeted CAR-T cells (BCMA CAR-T) are of great clinical significance for the prevention and treatment of multiple myeloma (MM).

[0008] SUMMARY OF THE INVENTION

[0009] The technical problem to be solved in this application is how to improve the therapeutic effect of immunotherapy targeting BCMA. In order to solve this technical problem, this application uses BCMA recombinant protein to immunize alpacas and then screens a large number of high-affinity anti-BCMA single-domain antibodies through phage display technology, and further designs and develops dual-epitope CAR-T cells (BCMA CAR-T cells) targeting BCMA with a novel structure based on the single-domain antibody. The BCMA CAR-T cells of this application can secrete IFN-γ well, effectively and specifically kill BCMA+ target cells, have good in vivo tumoricidal activity, can not only significantly inhibit the proliferation of tumor cells in mice, but also significantly prolong the survival time of mice. Technical issues

[0010] The technical problem to be solved by this application is to provide a single-domain antibody targeting BCMA (BCMA-VHH#81), a (dual-epitope) chimeric antigen receptor containing the single-domain antibody, and / or a modified immune effector cell containing the chimeric antigen receptor, and applications thereof. The technical problem to be solved is not limited to the technical subject matter described above, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description. Technical Solutions

[0011] To solve the above technical problems, the present application first provides a single-domain antibody targeting BCMA, which may be named BCMA-VHH#81.

[0012] The single-domain antibody comprises three complementarity determining regions, CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 includes positions 179-186 of SEQ ID NO: 1, the amino acid sequence of CDR2 includes positions 204-211 of SEQ ID NO: 1, and the amino acid sequence of CDR3 includes positions 250-260 of SEQ ID NO: 1.

[0013] Specifically, the single-domain antibody comprises the complementary determining region CDR1 with amino acid sequences of positions 179-186 of SEQ ID NO: 1, the complementary determining region CDR2 with amino acid sequences of positions 204-211 of SEQ ID NO: 1, and the complementary determining region CDR3 with amino acid sequences of positions 250-260 of SEQ ID NO: 1.

[0014] The single domain antibody further comprises four framework regions named FR1, FR2, FR3 and FR4.

[0015] FR1 comprises an amino acid sequence selected from the group consisting of:

[0016] a1) positions 154-178 of SEQ ID NO: 1;

[0017] a2) a sequence that is more than 75% identical to a1);

[0018] FR2 comprises an amino acid sequence selected from the group consisting of:

[0019] a3) positions 187-203 of SEQ ID NO: 1;

[0020] a4) a sequence that is more than 75% identical to a3);

[0021] FR3 comprises an amino acid sequence selected from the group consisting of:

[0022] a5) positions 212-249 of SEQ ID NO: 1;

[0023] a6) a sequence that is more than 75% identical to a5);

[0024] FR4 comprises an amino acid sequence selected from the group consisting of:

[0025] a7) positions 261-271 of SEQ ID NO: 1;

[0026] a8) A sequence having 75% or more identity with a7).

[0027] The sequences of the complementarity determining regions are defined according to the Kabat numbering system.

[0028] Furthermore, the single-domain antibody BCMA-VHH#81 may be any of the following:

[0029] A1) a protein whose amino acid sequence is positions 154-271 of SEQ ID NO: 1;

[0030] A2) a protein having at least 80% identity with the protein of A1) and having the same function as the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1;

[0031] A3) A fusion protein having the same function obtained by connecting a tag or signal peptide to the N-terminus and / or C-terminus of A1) or A2);

[0032] A4) A protein whose amino acid sequence includes positions 154-271 of SEQ ID NO: 1.

[0033] The present application also provides a chimeric antigen receptor, which includes the single-domain antibody BCMA-VHH#81.

[0034] Furthermore, the chimeric antigen receptor further includes a second single-domain antibody, which may be named BCMA-VHH#54. The second single-domain antibody BCMA-VHH#54 may be any of the following:

[0035] B1) a protein whose amino acid sequence is positions 22-138 of SEQ ID NO: 1;

[0036] B2) a protein having at least 80% identity with the protein of B1) and having the same function as the protein of B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1 from positions 22 to 138;

[0037] B3) A fusion protein having the same function obtained by connecting a tag or signal peptide to the N-terminus and / or C-terminus of B1) or B2);

[0038] B4) A protein whose amino acid sequence includes positions 22 to 138 of SEQ ID NO: 1.

[0039] The single-domain antibody BCMA-VHH#81 and the second single-domain antibody BCMA-VHH#54 can be connected by a linker.

[0040] The single-domain antibody BCMA-VHH#81 may be located at the N-terminus or C-terminus of the second single-domain antibody BCMA-VHH#54. In one embodiment of the present application, the single-domain antibody BCMA-VHH#81 is located at the C-terminus of the second single-domain antibody BCMA-VHH#54.

[0041] The linker may be a flexible peptide linker, for example a peptide linker comprising glycine, serine, proline and / or lysine residues. The peptide linker may be composed of 1-40 amino acids.

[0042] Furthermore, the linker includes, but is not limited to: (G)n, (S)n, (GxS)n, (SxG)n, (GSSGG)n (SEQ ID NO: 11), (GGSGG)n (SEQ ID NO: 12), (GSGGSG)n (SEQ ID NO: 13), (GSGSGS)n (SEQ ID NO: 14), (GGQGG)n (SEQ ID NO: 15), (EAAAK)n (SEQ ID NO: 16) and IEGRMD (SEQ ID NO: 17), and various combinations thereof. Wherein: n can be any integer between 1-10; x can be any integer between 1-6. In one embodiment of the present application, the linker is (GGGGS)3 (SEQ ID NO: 18).

[0043] The above-mentioned single-domain antibody BCMA-VHH#81 and the second single-domain antibody BCMA-VHH#54 jointly constitute the antigen-binding region of the chimeric antigen receptor.

[0044] Furthermore, the antigen binding region of the chimeric antigen receptor can be a single-domain antibody BCMA-VHH#81, a linker, and a second single-domain antibody BCMA-VHH#54 from the N-terminus to the C-terminus, which contains two different anti-BCMA single-domain antibodies and can target and recognize two different epitopes of the tumor antigen BCMA. Therefore, it can also be called a dual-epitope single-domain antibody (BCMA-VHH#54-VHH#81). The amino acid sequence of the dual-epitope single-domain antibody can be as shown in positions 22-271 of SEQ ID NO: 1. The chimeric antigen receptor containing the dual-epitope single-domain antibody can also be called a dual-epitope chimeric antigen receptor (dual-epitope CAR).

[0045] Furthermore, the chimeric antigen receptor also includes a hinge region, a transmembrane region, an intracellular signaling region and CD27.

[0046] The hinge region is located at the N-terminus of the transmembrane region and the C-terminus of the antigen binding region, and can be derived from the hinge region of CD8a (CD8α), CD4, CD5, CD7, CD28, CD134, CD137, ICOS, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM.

[0047] The transmembrane region may be derived from the α, β or ζ chain of the T cell receptor, CD8a (CD8α), CD4, CD9, CD45, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152 or the transmembrane domain of CD154.

[0048] The intracellular signaling region may include an activation domain and / or a costimulatory domain. The activation domain may be derived from an intracellular signaling domain of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66d. The costimulatory domain may be an intracellular signaling domain from a costimulatory molecule, for example, the costimulatory domain may be derived from an intracellular signaling domain of CARD11, CD2, CD7, CD27, CD28, CD30, ICAM-1 (CD54), LFA-1 (CD11a), CD134 (OX40), CD137 (4-1BB), 2B4, CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10.

[0049] The intracellular signaling region may comprise one, two or more costimulatory domains. The costimulatory domain may comprise the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell.

[0050] Furthermore, the intracellular signaling region may include an activation domain CD3ζ and a costimulatory domain 4-1BB.

[0051] Furthermore, the amino acid sequence of the activation domain CD3ζ may be as shown at positions 388-499 of SEQ ID NO: 1. The amino acid sequence of the costimulatory domain 4-1BB may be as shown at positions 341-387 of SEQ ID NO: 1.

[0052] Furthermore, the hinge region and transmembrane region may be derived from CD8a or CD28.

[0053] Furthermore, the hinge region may be a CD8a hinge region or a CD28 hinge region.

[0054] Furthermore, the transmembrane region may be a CD8a transmembrane region or a CD28 transmembrane region.

[0055] Furthermore, the amino acid sequence of the CD8a hinge region may be as shown at positions 272-316 of SEQ ID NO: 1. The amino acid sequence of the CD8a transmembrane region may be as shown at positions 317-340 of SEQ ID NO: 1. The amino acid sequence of the CD28 hinge region may be as shown at positions 272-310 of SEQ ID NO: 3. The amino acid sequence of the CD28 transmembrane region may be as shown at positions 311-337 of SEQ ID NO: 3.

[0056] Furthermore, in the chimeric antigen receptor, the CD27 may be located at the C-terminus of the intracellular signaling region of the chimeric antigen receptor. The intracellular signaling region of the chimeric antigen receptor and the CD27 may be connected via a cleavable linker.

[0057] Furthermore, the CD27 may be human full-length CD27.

[0058] The amino acid sequence of the human full-length CD27 may be shown as positions 522-781 of SEQ ID NO: 1.

[0059] Furthermore, the cleavable linker includes P2A, F2A, T2A and E2A, or any combination thereof, but is not limited thereto.

[0060] Furthermore, the amino acid sequence of P2A may be as shown in SEQ ID NO: 7. The amino acid sequence of F2A may be as shown in SEQ ID NO: 8. The amino acid sequence of T2A may be as shown in SEQ ID NO: 9. The amino acid sequence of E2A may be as shown in SEQ ID NO: 10.

[0061] Furthermore, the cleavable linker may be P2A.

[0062] Furthermore, the antigen binding region of the chimeric antigen receptor may also be preceded (N-terminus) by a signal peptide.

[0063] The signal peptide can be derived from the signal peptides of CD4, CD8, CD8a, CD28, CD137 and GM-CSF.

[0064] Furthermore, the signal peptide may be a human CD8 signal peptide. The amino acid sequence of the human CD8 signal peptide may be as shown in positions 1 to 21 of SEQ ID NO: 1.

[0065] In one embodiment of the present application, the chimeric antigen receptor may be composed of a signal peptide (Signal), a second single-domain antibody BCMA-VHH#54, a linker, a single-domain antibody BCMA-VHH#81, a CD8a hinge region, a CD8a transmembrane region, a co-stimulatory domain 4-1BB, an activation domain CD3ζ, a cleavable linker, and CD27 from N-terminus to C-terminus.

[0066] In another embodiment of the present application, the chimeric antigen receptor may be composed of a signal peptide (Signal), a second single-domain antibody BCMA-VHH#54, a linker, a single-domain antibody BCMA-VHH#81, a CD28 hinge region, a CD28 transmembrane region, a co-stimulatory domain 4-1BB, an activation domain CD3ζ, a cleavable linker, and CD27 from N-terminus to C-terminus.

[0067] Furthermore, the chimeric antigen receptor may be any of the following:

[0068] C1) a protein whose amino acid sequence is positions 22-781 of SEQ ID NO: 1;

[0069] C2) a protein whose amino acid sequence is positions 22-778 of SEQ ID NO: 3;

[0070] C3) a protein having at least 80% identity with the protein of C1) and having the same function as the protein of C1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1;

[0071] C4) a protein having at least 80% identity with the protein of C2) and having the same function as the protein of C2) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of positions 22-778 of SEQ ID NO: 3;

[0072] C5) A fusion protein with the same function obtained by connecting a tag or signal peptide to the N-terminus and / or C-terminus of C1), C2), C3) or C4);

[0073] C6) a protein whose amino acid sequence includes positions 22-781 of SEQ ID NO: 1;

[0074] C7) A protein whose amino acid sequence includes positions 22-778 of SEQ ID NO: 3.

[0075] Furthermore, the amino acid sequence of the fusion protein in C5) may be as shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0076] The protein whose amino acid sequence is positions 22-781 of SEQ ID NO: 1 can be named BCMA CAR1. BCMA CAR1 can further contain a signal peptide at its N-terminus. The complete amino acid sequence of BCMA CAR1 can be shown in SEQ ID NO: 1.

[0077] The protein whose amino acid sequence is positions 22-778 of SEQ ID NO:3 can be named BCMA CAR2. BCMA CAR2 can further contain a signal peptide at the N-terminus. The complete amino acid sequence of BCMA CAR2 can be shown in SEQ ID NO:3.

[0078] Tags described herein include, but are not limited to, GST (glutathione sulfhydryl transferase) tag protein, His tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein. Those skilled in the art know how to select a suitable tag protein according to the desired purpose (e.g., purification, detection or tracing).

[0079] As used herein, linkage refers to the association of two or more molecules. The linkage can be covalent or non-covalent.

[0080] Furthermore, the connection described herein can be directly connected via a peptide bond, or connected via a linker.

[0081] The present application also provides a biomaterial, which may be any of the following:

[0082] D1) a nucleic acid molecule encoding the single-domain antibody BCMA-VHH#81;

[0083] D2) a nucleic acid molecule encoding any one of the chimeric antigen receptors described herein;

[0084] D3) an expression cassette containing the nucleic acid molecule described in D1) or D2);

[0085] D4) a recombinant vector containing the nucleic acid molecule described in D1) or D2);

[0086] D5) a recombinant microorganism containing the nucleic acid molecule described in D1) or D2);

[0087] D6) A recombinant host cell containing the nucleic acid molecule described in D1) or D2).

[0088] In the above-mentioned biological material, the nucleic acid molecule may be any of the following:

[0089] E1) the coding sequence or nucleotide sequence is the DNA molecule shown in positions 460-813 of SEQ ID NO: 2;

[0090] E2) the coding sequence or nucleotide sequence is positions 64-2343 of SEQ ID NO: 2 or the DNA molecule set forth in SEQ ID NO: 2;

[0091] E3) The coding sequence or nucleotide sequence is positions 64-2334 of SEQ ID NO: 4 or the DNA molecule shown in SEQ ID NO: 4.

[0092] The DNA molecule represented by positions 460-813 of SEQ ID NO: 2 encodes the single-domain antibody BCMA-VHH#81.

[0093] The DNA molecule shown in SEQ ID NO: 2 encodes the chimeric antigen receptor BCMA CAR1; the DNA molecule shown in positions 64-2343 of SEQ ID NO: 2 encodes the chimeric antigen receptor BCMA CAR1 without a signal peptide.

[0094] The DNA molecule shown in SEQ ID NO:4 encodes the chimeric antigen receptor BCMA CAR2; the DNA molecule shown at positions 64-2334 of SEQ ID NO:4 encodes the chimeric antigen receptor BCMA CAR2 without a signal peptide.

[0095] The present application also provides a modified immune effector cell, which comprises the single-domain antibody BCMA-VHH#81, the chimeric antigen receptor described herein, or the nucleic acid molecule.

[0096] The modified immune effector cell may be any chimeric antigen receptor-modified immune effector cell described herein, which expresses any chimeric antigen receptor described herein.

[0097] Furthermore, the immune effector cells include but are not limited to T cells, NK cells, NKT cells, γδT cells, αβT cells, regulatory T cells (Treg), MAIT cells, B cells, macrophages, dendritic cells (DC), peripheral blood mononuclear cells (PBMC) and monocytes (MC).

[0098] The immune effector cells may also include stem cells that can be induced to differentiate into immune effector cells in vivo or in vitro, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells or hematopoietic stem cells (HSCs).

[0099] The immune effector cells may be autologous or non-autologous (allogeneic, isogenic or xenogeneic). Further, the immune effector cells are autologous or allogenic.

[0100] Furthermore, the modified immune effector cells may include CAR-T cells, CAR-NK cells, CAR-NKT cells, CAR-γδT cells, CAR-αβT cells, CAR-regulatory T cells (CAR-Treg cells), CAR-MAIT cells, CAR-B cells, CAR-macrophages (CAR-M cells), CAR-dendritic cells (CAR-DC cells), CAR-peripheral blood mononuclear cells (CAR-PBMC cells) and CAR-monocytes (CAR-MC cells) but are not limited thereto.

[0101] Furthermore, the immune effector cells may be T cells.

[0102] Furthermore, the modified immune effector cells may be CAR-T cells.

[0103] Furthermore, the CAR-T cells may be BCMA CAR1-T cells or BCMA CAR2-T cells.

[0104] The BCMA CAR1-T cells are CAR-T cells obtained by transferring the chimeric antigen receptor BCMA CAR1 constructed in the present application into T cells through a genetic engineering in vitro recombination method for stable expression.

[0105] The BCMA CAR2-T cells are CAR-T cells obtained by transferring the chimeric antigen receptor BCMA CAR2 constructed in the present application into T cells through a genetic engineering in vitro recombination method for stable expression.

[0106] Furthermore, the BCMA CAR1-T cells contain and / or express the DNA molecule shown in SEQ ID NO: 2 or positions 64-2343 of SEQ ID NO: 2. The BCMA CAR2-T cells contain and / or express the DNA molecule shown in SEQ ID NO: 4 or positions 64-2334 of SEQ ID NO: 4.

[0107] The BCMA CAR1-T cell or BCMA CAR2-T cell may have at least any one of the following characteristics:

[0108] (1) Secretion of T cell-specific effector molecule IFN-γ;

[0109] (2) Specific killing of BCMA-positive tumor cells;

[0110] (3) Inhibit the growth of BCMA-positive tumors.

[0111] The present application also provides use of the single-domain antibody BCMA-VHH#81, any chimeric antigen receptor described herein, the biomaterial, or the modified immune effector cell in preparing a product having any of the following functions:

[0112] F1) Prevention or treatment of tumors;

[0113] F2) Prevent or treat BCMA target-related diseases;

[0114] F3) Killing BCMA-positive tumor cells;

[0115] F4) inhibit the proliferation of BCMA-positive tumor cells;

[0116] F5) inhibiting the growth of BCMA-positive tumors;

[0117] F6) Detection of BCMA-expressing tumor cells or BCMA protein.

[0118] The present application also provides any of the following uses of the single-domain antibody BCMA-VHH#81 or a nucleic acid molecule encoding the single-domain antibody BCMA-VHH#81:

[0119] G1) Use in the preparation of products for screening, diagnosis or auxiliary diagnosis of BCMA target-related diseases;

[0120] G2) Use in the preparation of a product for binding BCMA protein;

[0121] G3) Use in the preparation of a product that mediates drug-specific recognition of tumors expressing BCMA antigen;

[0122] G4) Use in the preparation of a product for in vivo imaging of BCMA protein;

[0123] G5) Use in preparing CAR cells targeting BCMA.

[0124] The product for mediating the drug to specifically recognize tumors expressing the BCMA antigen in G3) may be a drug delivery system that specifically targets the BCMA protein, and the drug delivery system contains the single-domain antibody BCMA-VHH#81.

[0125] The drug delivery system can be a liposome drug delivery system, a polymer micelle drug delivery system, a polymer disc drug delivery system or a nanoparticle drug delivery system.

[0126] The drug-carrying system can be used for targeted transport and / or site-specific release of drugs.

[0127] The tumor described herein may be a BCMA-positive tumor (a tumor that expresses BCMA protein).

[0128] The BCMA target-related disease described herein may be BCMA-positive cancer.

[0129] The BCMA target-related diseases or BCMA-positive cancers described herein may include multiple myeloma (MM), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma, but are not limited thereto.

[0130] The products described herein may include reagents, kits, chips, test strips, drugs, and pharmaceutical compositions.

[0131] The present application also provides a pharmaceutical composition for preventing or treating BCMA target-related diseases, which comprises the single-domain antibody BCMA-VHH#81 or the modified immune effector cells, and one or more pharmaceutically acceptable carriers.

[0132] The pharmaceutically acceptable carrier is selected from diluents, excipients, fillers, binders, wetting agents, disintegrants, preservatives, stabilizers, absorption promoters, adsorption carriers, surfactants, lubricants, aerosolizers, suspending agents, plasticizers and dispersants.

[0133] In order to prepare the pharmaceutical composition into an injectable preparation, such as a solution, emulsion, lyophilized powder injection, and suspension, all diluents (carriers) commonly used in the art can be used, for example, water, saline, phosphate-buffered saline, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose, or glycerol can be added to the injectable preparation. In addition, conventional carriers such as cosolvents, buffers, and pH adjusters can also be added.

[0134] The pharmaceutical composition may further include a cell freezing solution (containing dimethyl sulfoxide, sodium chloride, human serum albumin, etc.).

[0135] The pharmaceutical composition may have at least one of the following uses: (1) for preventing or treating BCMA target-related diseases; (2) for killing BCMA-positive tumor cells; (3) for inhibiting the proliferation of BCMA-positive tumor cells; and (4) for inhibiting the growth of BCMA-positive tumors.

[0136] The present application also provides a kit containing the single-domain antibody BCMA-VHH#81, any one of the chimeric antigen receptors described herein, or the modified immune effector cells.

[0137] The kit can have any of the following uses: (1) screening, diagnosis or auxiliary diagnosis of BCMA target-related diseases; (2) detection of BCMA protein; (3) in vivo imaging of BCMA protein.

[0138] Furthermore, the kit can be used to detect cancer cells expressing BCMA, or to detect whether BCMA-positive cancer exists in a subject.

[0139] The kit may be a chemiluminescence immunoassay kit, an enzyme-linked immunosorbent assay kit, a colloidal gold immunoassay kit, or a fluorescence immunoassay kit, but is not limited thereto.

[0140] The test sample of the kit can be a blood sample (such as whole blood, plasma, serum), a tissue sample, a cell sample, etc., but is not limited thereto.

[0141] The detection of BCMA protein described herein may be detecting whether the sample to be tested contains BCMA protein and / or detecting the content of BCMA protein in the sample to be tested.

[0142] The present application also provides an antibody-drug conjugate (ADC), comprising an antibody portion and a conjugated portion, wherein the antibody portion comprises the single-domain antibody BCMA-VHH#81.

[0143] Furthermore, the conjugated moiety may include a detectable label, a chemical drug or a cytotoxin.

[0144] The antibody moiety and the conjugated moiety may be covalently linked directly or through a linker.

[0145] The detectable label may be selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

[0146] The chemical drugs may be chemotherapeutic drugs or other drugs that can kill tumor cells, such as apoptosis inducers (Bcl-xL inhibitors), nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, anti-tumor antibiotics, immunomodulators, anti-angiogenic agents, antimetabolites, boron drugs, alkylating agents, hormones and anti-hormonal drugs, corticosteroids, photodynamic therapy drugs, etc.

[0147] The cytotoxin may be a tubulin inhibitor (such as maytansine, auristatin), a DNA damaging agent (such as calicheamicin, dukamicin, camptothecin) or an RNA synthesis inhibitor (such as amatoxin, tylanstatin and its analogs).

[0148] The present invention also provides the single-domain antibody BCMA-VHH#81 for use as a drug.

[0149] Furthermore, the single-domain antibody BCMA-VHH#81 may be any of the following:

[0150] M1) Single-domain antibody BCMA-VHH#81 for preventing or treating tumors;

[0151] M2) BCMA-VHH#81, a single-domain antibody for the prevention or treatment of BCMA-related diseases;

[0152] M3) BCMA-VHH#81, a single-domain antibody for killing BCMA-positive tumor cells;

[0153] M4) single-domain antibody BCMA-VHH#81 for inhibiting the proliferation of BCMA-positive tumor cells;

[0154] M5) Single-domain antibody BCMA-VHH#81 for inhibiting the growth of BCMA-positive tumors.

[0155] The present invention also provides any modified immune effector cell described herein for use as a medicament.

[0156] Furthermore, the modified immune effector cells may be any of the following:

[0157] N1) Modified immune effector cells for preventing or treating tumors;

[0158] N2) Modified immune effector cells for preventing or treating BCMA target-related diseases;

[0159] N3) Modified immune effector cells for killing BCMA-positive tumor cells;

[0160] N4) Modified immune effector cells for inhibiting the proliferation of BCMA-positive tumor cells;

[0161] N5) Modified immune effector cells for inhibiting BCMA-positive tumor growth.

[0162] The present application also provides a method for preventing or treating a BCMA target-related disease, comprising administering the single-domain antibody BCMA-VHH#81, the modified immune effector cell, or the pharmaceutical composition to a subject suffering from a BCMA target-related disease.

[0163] Furthermore, the modified immune effector cells may be the CAR-T cells described herein (including BCMA CAR1-T cells and BCMA CAR2-T cells). The CAR-T cells may be autologous or allogeneic.

[0164] Furthermore, the amount administered may be a therapeutically effective amount. The therapeutically effective amount may refer to the amount of the single-domain antibody BCMA-VHH#81, the modified immune effector cell, or the pharmaceutical composition that (i) treats or prevents a specific disease, condition, or disorder; (ii) alleviates, improves, or eliminates one or more symptoms of a specific disease, condition, or disorder; or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The therapeutically effective amount can be determined by testing in a known in vitro or in vivo (e.g., animal model) system.

[0165] Furthermore, the administration can be performed by injection or continuous infusion (including but not limited to intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and intraportal).

[0166] Furthermore, the BCMA target-related disease may be BCMA-positive cancer.

[0167] Furthermore, the BCMA-positive cancer may include multiple myeloma, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma but is not limited thereto.

[0168] The present application also provides a method for screening, diagnosing or assisting in the diagnosis of BCMA target-related diseases, which comprises isolating a sample from a subject, then using the single-domain antibody BCMA-VHH#81 to detect the content of BCMA protein in the sample, and performing screening, diagnosis or assisting in the diagnosis of BCMA target-related diseases based on the content of the BCMA protein.

[0169] Furthermore, the detection can be performed by conventional assays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or immunohistochemistry.

[0170] The present application also provides a method for preparing the modified immune effector cell, which comprises: introducing a nucleic acid molecule encoding any chimeric antigen receptor described herein into an immune effector cell for expression, thereby obtaining the modified immune effector cell.

[0171] Furthermore, the immune effector cells may be T cells, and the modified immune effector cells may be CAR-T cells.

[0172] Furthermore, the method for preparing the CAR-T cells may include the following steps:

[0173] (1) cloning the BCMA CAR1 gene (SEQ ID NO: 2) or the BCMA CAR2 gene (SEQ ID NO: 4) into a viral vector to obtain a recombinant viral vector;

[0174] (2) introducing the recombinant viral vector into packaging cells for packaging to obtain a recombinant virus;

[0175] (3) Infecting (transfecting) human T cells with the recombinant virus to obtain the CAR-T cells.

[0176] Furthermore, the viral vector may include, but is not limited to, baculovirus, retrovirus, lentivirus, adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus).

[0177] Furthermore, the viral vector may be a retroviral vector (such as retroviral vector MP71).

[0178] Furthermore, the packaging cells are used to package viral vector plasmids that lack at least one gene encoding a protein required for viral packaging, which can provide a comprehensive range of viral proteins for viral packaging into recombinant viruses, and cannot produce any form of viral particles themselves. Any cell that provides proteins or polypeptides lacking in viral packaging can be considered as a packaging cell. Suitable packaging cells are well known to those skilled in the art, such as ECO cells, PG13 cells, HEK293 cells, CHO cells, 293T cells, 293 cells, MDCK cells, NIH3T3 cells, PA317 cells, COS cells, HeLa cells, Vero cells, PsiCRIP cells, etc.

[0179] The present application also provides a method for preparing the single-domain antibody BCMA-VHH#81, which comprises: constructing a recombinant expression vector containing a nucleic acid molecule encoding the single-domain antibody BCMA-VHH#81; introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the single-domain antibody BCMA-VHH#81; culturing the recombinant cell, and obtaining the single-domain antibody BCMA-VHH#81 through separation and purification.

[0180] Furthermore, the nucleic acid molecule encoding the single-domain antibody BCMA-VHH#81 may be any of the following:

[0181] (1) The coding sequence or nucleotide sequence is the DNA molecule shown in positions 460-813 of SEQ ID NO: 2;

[0182] (2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in (1) and having the same function.

[0183] Furthermore, the introduction can be carried out by transforming the host cell with the vector carrying the single-domain antibody BCMA-VHH#81 gene of the present application through any known transfection method such as calcium phosphate co-precipitation, liposome-mediated method, electroporation method or viral vector method.

[0184] Other variants of the sequence of the single-domain antibody BCMA-VHH#81 described herein with improved affinity and / or potency can be obtained by using methods known in the art and are included in the scope of protection of this application. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can also be used to improve translation efficiency in expression systems for producing antibodies. In addition, polynucleotides comprising sequences that optimize antibody specificity by applying directed evolution methods to any nucleic acid sequence of this application are also within the scope of this application.

[0185] In certain embodiments, substitutions, insertions, or deletions may occur in one or more complementary determining regions or framework regions of the single-domain antibody BCMA-VHH#81 described herein, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as is well known to those skilled in the art, where conservative substitutions of amino acids do not change the properties and functions of the protein) may be made to the complementary determining regions and / or framework regions without substantially reducing binding affinity. For example, such changes may be outside the antigen contact residues in the complementary determining regions.

[0186] BCMA (B-cell maturation antigen, CD269, TNFRSF17), described herein, is a type III transmembrane glycoprotein that belongs to the human tumor necrosis factor receptor (TNFR) superfamily and plays a major role in B cell maturation and differentiation into plasma cells. BAFF and APRIL are two B cell activation and expansion-inducing ligands that bind to BCMA and jointly regulate B cell function. BCMA is primarily expressed in mature B cells, where its expression gradually increases with B cell maturation and is selectively overexpressed during the malignant transformation of plasma cells, promoting tumor cell growth, survival, and the development of drug resistance. BCMA is expressed on tumor cells such as multiple myeloma, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

[0187] Definition of terms

[0188] In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand this application, the definitions and explanations of relevant terms are provided below.

[0189] The term "identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Identity as described herein may refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies of identical sequences have 100% identity. Those skilled in the art will appreciate that sequence identity can be determined using an identity search site or sequence analysis software on the Internet, such as the BLAST page on the NCBI homepage website. Herein, 75% or more identity may be 80% or more identity. Herein, 80% or more identity may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0190] The term "vector" refers to a vector that can carry exogenous DNA or a target gene into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are amplified and / or expressed in the host cell. Suitable vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages (such as lambda phage or M13 phage), cosmids (i.e., cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC) or Ti plasmid artificial chromosomes (TAC), etc.), viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), herpes viruses (such as herpes simplex virus), etc.).

[0191] The term "host cell" refers to any type of cell that can be used to introduce a vector. The host cell can be a eukaryotic cell (e.g., a plant cell, an animal cell, a fungus, or an algae), or can be a prokaryotic cell (e.g., a bacterium or a protozoan). The host cell is understood to refer not only to a specific host cell, but also to the progeny of such a cell, and due to natural, accidental, or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell.

[0192] The term “chimeric antigen receptor (CAR)” is a transmembrane molecule encoded by an artificially constructed fusion gene that can guide immune cells to specifically track, identify and eliminate tumor cells expressing relevant target ligands, with high targeting. The basic structure of CAR can be divided into four parts, from the extracellular to the intracellular direction, namely the extracellular tumor antigen binding region (abbreviated as antigen binding region), the hinge region, the transmembrane region and the intracellular signaling region. Methods for preparing chimeric antigen receptors and immune effector cells (such as T cells) comprising the chimeric antigen receptor are known in the art and may include transfecting cells with at least one nucleic acid molecule encoding CAR and expressing the nucleic acid molecule in the cell. For example, the nucleic acid molecule encoding the CAR of the present application can be constructed into an expression vector (such as a retroviral vector), which can be expressed in a host cell such as a T cell to prepare the CAR. T cells expressing CAR are referred to herein as CAR-T cells or CAR-modified T cells. “Dual epitope CAR” described herein refers to a CAR having an antigen binding region specific for two different epitopes of a tumor antigen (such as BCMA).

[0193] The term "linked" refers to the association of two or more molecules. The link can be covalent or non-covalent. The link described herein can be directly connected by a peptide bond or connected by a linker.

[0194] The term "linker" may also be referred to as a connecting peptide, peptide linker or connector, which is used to fuse or connect two proteins or polypeptides to prevent steric hindrance. As an indispensable component of fusion protein recombinant, linker plays an important role in constructing stable, biologically active fusion proteins. It should be understood that the presence of a linker is optional and the length of the flexible linker can be adjusted to allow the correct folding of the fusion protein or to achieve optimal biological activity. The characteristics of the linker and its suitability for specific purposes are known in the art, and those skilled in the art can independently select and optimize each peptide linker.

[0195] The term "cleavable linker (also known as cleavable linker)" refers to a linker comprising a cleavage site such that when expressed, it can be selectively cleaved to produce two or more products.

[0196] The term "antigen binding region", also known as the extracellular tumor antigen binding region or extracellular antigen binding domain, refers to the part of the chimeric antigen receptor that specifically binds to the target antigen and is responsible for recognizing tumor antigens. It can include the antibody light chain variable region (VL), antibody heavy chain variable region (VH), antibody Fab fragment, antibody Fab' fragment, single-chain antibody variable region (scFv), single-domain antibody (sdAb), etc.

[0197] The term "hinge," also known as the hinge domain, refers to any oligopeptide or polypeptide that connects the extracellular antigen-binding region and the transmembrane region. It is the peptide chain of the CAR located outside the cell. Selecting an appropriate hinge region can increase the flexibility of the antigen recognition region and reduce the spatial constraints between the antigen and the CAR, thereby promoting synaptic formation between the CAR-T cell and the target cell.

[0198] The term "transmembrane domain (TD)," also known as the transmembrane domain, is used to connect the antigen-binding region and the intracellular signaling region of the chimeric antigen receptor, primarily serving to anchor the antibody to the cell membrane. It can be natural or synthetic, and can be derived from any membrane-bound or transmembrane protein, typically consisting of homo- or heterodimeric membrane proteins.

[0199] The term "intracellular signaling region (intracellular signaling domain)" refers to a protein portion that transduces effector function signals and instructs cells to perform specialized functions. The intracellular signaling region may include an activation domain and / or a costimulatory domain. The intracellular signaling region may include one, two or more costimulatory domains. The costimulatory domain may be an intracellular signaling domain from a costimulatory molecule, which may include the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. "Costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell.

[0200] The term "signal peptide" refers to a short peptide chain (generally 5-30 amino acids in length) that guides the transfer of newly synthesized proteins to the secretory pathway. The antigen binding region of the chimeric antigen receptor described in this application may be preceded by a signal peptide (Signal) (N-terminus). The signal peptide of the CAR structure can guide the transfer of the antigen binding region peptide chain to the extracellular space, thereby recognizing tumor cell surface antigens. The signal peptide can be a signal peptide derived from CD4, CD8, CD8a, CD28, CD137 and GM-CSF.

[0201] The term "immune effector cell" refers to an immune cell that participates in an immune response, performs immune effector functions (such as cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Immune effector cells described herein can be derived from stem cell differentiation, such as immune effector cells differentiated from adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSC), totipotent stem cells or hematopoietic stem cells (HSC). In one embodiment of the application, the immune effector cell is a T cell. T cells are present in a variety of sources, including but not limited to peripheral blood, mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, infection site tissue, ascites, pleural effusion, spleen tissue and tumor. T cells can be obtained by separation using a variety of techniques well known to those skilled in the art (such as density gradient separation technology, adhesion separation method, magnetic bead separation technology, flow cytometry sorting technology, etc.).

[0202] The term "autologous" refers to cells derived from the same subject.

[0203] The term "allogeneic" refers to cells of the same species that are genetically different from the cells being compared.

[0204] The term "isogenic" refers to cells from a different subject that are genetically identical to the cells being compared.

[0205] The term "xenogeneic" refers to cells that are of a different species than the cells being compared.

[0206] The term "antibody-drug conjugate (ADC)" may refer to a drug with highly effective cytotoxic activity coupled to an antibody through chemical, biological or other methods. The specific affinity between the antibody and the antigen is utilized to deliver the linked drug to the tumor cell site, thereby enhancing the therapeutic activity of the antibody, improving the targeting of the cytotoxic drug in killing tumor cells, and reducing its toxic side effects on normal tissues.

[0207] The term "treatment" refers to therapeutic treatment, wherein the subject is to slow down (mitigate) an undesirable physiological change or disease, or to provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, weakening of the extent of the disease, a stable (i.e., non-exacerbated) state of the disease, a delay or slowing down of disease progression, an improvement or alleviation of the disease state, and / or relief (whether partially or completely), whether detectable or undetectable. "Treatment" may also mean extending survival compared to the expected survival when the subject is not receiving treatment. Those subjects in need of treatment include those subjects who have suffered from undesirable physiological changes or diseases and those subjects who tend to suffer from physiological changes or diseases. Beneficial effects

[0208] After in-depth research and creative work, the inventors of this application first used BCMA recombinant protein to immunize alpacas and then screened a large number of high-affinity anti-BCMA single-domain antibodies through phage display technology. After sequencing the candidate antibody gene sequence, the antibody was prepared by genetic engineering methods and affinity testing was performed. Finally, a single-domain antibody with high affinity was obtained, named BCMA-VHH#81. This single-domain antibody can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system. It can be further developed into an anti-tumor drug as an anti-tumor antibody with clinical application prospects by targeting and binding to the tumor antigen BCMA with high affinity. At the same time, it can be used as an antibody to detect BCMA protein in applications such as ELISA, Western blot, and immunohistochemistry and developed into a detection product, which can greatly save scientific research costs and time. The single-domain antibody drug of this application has a stable structure, small molecules, easy recombinant expression, low production cost, can be used alone or as a drug delivery system to carry related drugs, and has very broad prospects and important significance in the fields of drug application and clinical diagnosis.

[0209] This application further designs and develops BCMA-targeting CAR-T cells (BCMA CAR-T cells) with a novel structure based on the above-mentioned single-domain antibody BCMA-VHH#81. In order to optimize the efficacy of CAR-T cell therapy, for the BCMA target, this application adopts a dual-epitope CAR (a chimeric antigen receptor comprising two anti-BCMA single-domain antibodies) design for antigen loss, which improves the antigen binding affinity. At the same time, 4-1BB and CD27 are expressed in a non-coupling manner. The CD70-CD27 signaling pathway can provide additional stimulation signals, increase the proportion of memory T cells in CAR-T products, enhance the anti-tumor ability of CAR-T cells, and thus improve the efficacy of CAR-T. Experiments have shown that the BCMA CAR-T cells (BCMA CAR1-T cells and BCMA CAR2-T cells) of this application can well secrete the T cell-specific effector molecule IFN-γ, effectively and specifically kill BCMA+ target cells, have good in vivo tumoricidal activity, and can not only significantly inhibit the proliferation of tumor cells in mice, but also significantly prolong the survival time of mice. The BCMA CAR-T cells of this application have good anti-tumor ability and can be used for immunotherapy of BCMA target-related diseases, with broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0210] Figure 1 is a schematic diagram of the BCMA CAR molecular structure.

[0211] Figure 2 shows the BCMA-VHH#81 antibody clone screening.

[0212] Figure 3 shows the detection of CAR expression in BCMA CAR-T cells.

[0213] Figure 4 shows the cell function detection of BCMA CAR-T cells secreting IFN-γ.

[0214] Figure 5 shows the in vitro tumoricidal activity detection of BCMA CAR-T cells.

[0215] Figure 6 shows the in vivo tumor inhibition activity detection of BCMA CAR-T cells. Modes for Carrying Out the Invention

[0216] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0217] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0218] The retroviral vector MP71 in the following examples is described in the following literature: Engels B, Cam H, et al. Retroviral Vectors for High-Level Transgene Expression in TLymphocytes[J]. Human Gene Therapy, 2003, 14(12): 1155-1168. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this application and cannot be used for other purposes.

[0219] The MM1.S cells (BCMA-positive cells) in the following examples were obtained from Kyowa Cell Bank, resource number: 1101HUM-PUMC000680.

[0220] The MM1.S-LUC (also known as MM1.S-Luc) cells in the following examples were obtained from Kyowa Cell Bank, resource number: 1101HUM-PUMC000934.

[0221] The Phoenix-ECO cells in the following examples are strains of the American Type Culture Collection (ATCC) with the collection number CRL-3214.

[0222] The PG13 cells in the following examples are strains of the American Type Culture Collection (ATCC) with the accession number CRL-3597.

[0223] The hBCMA-his antigen in the following examples is a product of Shanghai Baiying Biotechnology Co., Ltd., product number: BCA-H522Y.

[0224] The Recombinant Human BCMA / hFc Protein (BCMA(rp)) in the following examples is a product of Shanghai Baiying Biotechnology Co., Ltd., catalog number: B528001

[0225] The vector pcDNA3.1 in the following examples is a product of Changsha Abiwei Biotechnology Co., Ltd., product number: HG-VPI0001.

[0226] In the following examples, BCMA-positive K562-BCMA cells were prepared by inserting the BCMA gene between the BamHI and EcoRI sites of pcDNA3.1. The prepared recombinant vector was then transfected into K562 cells to generate BCMA-positive K562-BCMA cells. The nucleotide sequence of the BCMA gene is from positions 1-3802 of GenBank Accession No. AB052772 (Update Date AB052772.1).

[0227] Pierce in the following examples TM NHS-Fluorescein Antibody Labeling Kit is a product of Thermo Scientific, catalog number 53029.

[0228] The human peripheral blood mononuclear cells (PBMCs) in the following examples were derived from the peripheral blood of healthy volunteers.

[0229] The following examples were analyzed using Graphpad statistical software. The experimental results are expressed as mean ± standard deviation. The log-rank test was used. P < 0.05 (*) indicates a statistically significant difference, P < 0.01 (**) indicates a statistically significant difference, and P < 0.001 (***) indicates a very significant difference. The quantitative experiments in the following examples were performed in triplicate unless otherwise specified, and the results were averaged.

[0230] Example 1. Structure and sequence of chimeric antigen receptors

[0231] The chimeric antigen receptors (CARs) designed and constructed in this embodiment are named BCMA CAR1 and BCMA CAR2. From N-terminus to C-terminus, they are signal peptide (Signal), dual-epitope single-domain antibody (BCMA-VHH#54-VHH#81), CD8a / CD28 hinge region (Hinge) and transmembrane region, costimulatory domain 4-1BB and activation domain CD3ζ, which can cleave linker P2A and CD27 protein. The structural diagram is shown in Figure 1. The chimeric antigen receptor of the present application is also called dual-epitope chimeric antigen receptor (dual-epitope CAR) because it contains a dual-epitope single-domain antibody.

[0232] Among them, the bivalent single-domain antibody is two single-domain antibodies (BCMA-VHH#54-VHH#81) that specifically bind to the BCMA protein. The single-domain antibody is the extracellular antigen binding region of the CAR, responsible for recognizing and binding to the tumor surface antigen BCMA. The dual-epitope antibody can improve the antigen binding affinity; the front of the antigen binding region (N-terminus) is accompanied by a signal peptide (Signal), which is used to guide the peptide chain of the antigen binding region to transfer to the extracellular space, thereby recognizing the tumor cell surface antigen BCMA; the CD8a / CD28 hinge region (Hinge) is a peptide chain located outside the cell of the CAR, connecting the extracellular antigen binding region and the transmembrane region; the CD8a / CD28 transmembrane region is used to anchor the single-domain antibody (BCMA-VHH) on the cell membrane; the intracellular signaling region includes the co-stimulatory domain 4-1BB and the activation domain CD3ζ, which are used to co-stimulate T cells and activate intracellular signals. CD27 is a co-stimulatory molecule that is co-expressed with CAR through a cleavable linker. It binds to its ligand CD70 on the cell surface and can promote T cell proliferation and anti-tumor ability.

[0233] The chimeric antigen receptor targeting BCMA (BCMA-VHH CAR) designed above can specifically be BCMA CAR1 and BCMA CAR2. In the BCMA-VHH CAR designed above, the cleavable linker P2A (GSGATNFSLLK QAGDVEENPGP, SEQ ID NO: 7) is a "self-cleaving" peptide. The "self-cleavage" function of the P2A peptide forms two proteins, the upstream product and the downstream product, by self-cleavage. Those skilled in the art can replace P2A in the BCMA-VHH CAR molecule, for example, using F2A (VKQTLNFDLLKLAGCVESNPG, SEQ ID NO: 8), T2A (EGRGSLLTCGDVEENPG, SEQ ID NO: 9), E2A (QCTNYALLKLAGDVESNPG, SEQ ID NO: 10), etc., which have the same "self-cleavage" function as P2A.

[0234] The amino acid sequence of BCMA CAR1 is shown in SEQ ID NO: 1, where positions 1-21 of SEQ ID NO: 1 are the amino acid sequence of the signal peptide (Signal); positions 22-138 of SEQ ID NO: 1 are the amino acid sequence of the single-domain antibody (BCMA-VHH#54); positions 139-153 of SEQ ID NO: 1 are the amino acid sequence of the G4S linker; positions 154-271 of SEQ ID NO: 1 are the amino acid sequence of the single-domain antibody (BCMA-VHH#81); positions 272-340 of SEQ ID NO: 1 are the amino acid sequences of the CD8a hinge region (Hinge) and transmembrane region (TM); positions 341-387 of SEQ ID NO: 1 are the amino acid sequence of the costimulatory domain 4-1BB; positions 388-499 of SEQ ID NO: 1 are the amino acid sequence of the activation domain CD3ζ; positions 500-521 of SEQ ID NO: 1 are the amino acid sequence of P2A; Positions 522-781 of SEQ ID NO: 1 represent the amino acid sequence of the full-length CD27. In the single-domain antibody (BCMA-VHH#81), positions 179-186 of SEQ ID NO: 1 represent the complementarity determining region (CDR1); positions 204-211 of SEQ ID NO: 1 represent the complementarity determining region (CDR2); and positions 250-260 of SEQ ID NO: 1 represent the complementarity determining region (CDR3).

[0235] The nucleotide sequence of BCMA CAR1 is shown in SEQ ID NO: 2. The DNA molecule shown in SEQ ID NO: 2 (also referred to as the BCMA CAR1 gene) encodes the chimeric antigen receptor BCMA CAR1 having the amino acid sequence of SEQ ID NO: 1.

[0236] The amino acid sequence of BCMA CAR2 is shown in SEQ ID NO: 3, where positions 1-21 of SEQ ID NO: 3 are the amino acid sequence of the signal peptide (Signal); positions 22-138 of SEQ ID NO: 3 are the amino acid sequence of the single-domain antibody (BCMA-VHH#54); positions 139-153 of SEQ ID NO: 3 are the amino acid sequence of the G4S linker; positions 154-271 of SEQ ID NO: 3 are the amino acid sequence of the single-domain antibody (BCMA-VHH#81); positions 272-337 of SEQ ID NO: 3 are the amino acid sequences of the CD28 hinge region (Hinge) and transmembrane region (TM); positions 338-384 of SEQ ID NO: 3 are the amino acid sequence of the costimulatory domain 4-1BB; positions 385-496 of SEQ ID NO: 3 are the amino acid sequence of the activation domain CD3ζ; positions 497-518 of SEQ ID NO: 3 are the amino acid sequence of P2A; Positions 519-778 of NO:3 are the amino acid sequence of the full-length CD27.

[0237] The nucleotide sequence of BCMA CAR2 is shown in SEQ ID NO: 4. The DNA molecule shown in SEQ ID NO: 4 (also referred to as the BCMA CAR2 gene) encodes the chimeric antigen receptor BCMA CAR2 having an amino acid sequence of SEQ ID NO: 3.

[0238] Example 2. BCMA-VHH#81 Antibody Affinity Determination

[0239] 1. ELISA detection of antibody clones

[0240] The single-domain antibody BCMA-VHH#81 in Example 1 was obtained by immunizing alpacas with BCMA recombinant protein and screening using phage display technology. After three rounds of phage screening, BCMA-VHH antibodies with high absorbance values ​​were identified using ELISA. The specific steps are as follows:

[0241] 1) Dilute the target molecule hBCMA-his antigen with carbonate buffer (pH 9.6) to a final concentration of 2 μg / mL. Add 100 μL / well to the enzyme-labeled wells and coat overnight at 4°C.

[0242] 2) Discard the coating solution, wash three times with PBST, add 300 μL of 5% skim milk to each well, and block at 37°C for 1 h;

[0243] 3) Wash once with PBST, add 50 μL of phage culture supernatant and 50 μL of 5% skim milk to each well, and incubate at 37°C for 1 h;

[0244] 4) Wash with PBST five times, add horseradish peroxidase-labeled anti-M13 antibody (diluted 1:10,000 in PBS) at 100 μL / well and incubate at 37°C for 1 h.

[0245] 5) Wash the plate six times with PBST. Add TMB colorimetric solution (100 μL / well) for color development at 37°C for 7 min. Add stop solution (50 μL / well) to terminate the reaction and measure the optical density at 450 nm.

[0246] The test results are shown in Figure 2. The ELISA value of the BCMA-VHH#81 antibody is higher than 2.5, and the ratio of the values ​​of the experimental group and the negative control group is 46.1, indicating that the antibody has a high affinity.

[0247] 2. Flow cytometry detection of antibodies

[0248] 2-1. Preparation of Antibodies

[0249] The BCMA-VHH#81 antibody was prepared using genetic engineering methods. The specific steps are as follows:

[0250] 1) To facilitate purification, a human IgG1 Fc gene was added to the 3' end of the gene encoding the BCMA-VHH antibody (the nucleotide sequence of the gene encoding BCMA-VHH#81 is positions 460-813 of SEQ ID NO:2) to generate a BCMA-VHH-hFc fusion gene. A BamHI restriction site was then added to the 5' end of the fusion gene before the start codon and an EcoRI restriction site was added after the 3' end of the stop codon. This fusion gene was then cloned into the eukaryotic expression vector pcDNA3.1 between the BamHI and EcoRI recognition sites to generate a recombinant eukaryotic expression vector expressing the single-domain antibody BCMA-VHH#81-hFc fused to the C-terminus with the hFc gene. The nucleotide sequence of the hFc gene is SEQ ID NO:5, and the amino acid sequence it encodes is SEQ ID NO:6.

[0251] 2) The eukaryotic expression vectors obtained in step 1) were transfected into HEK293 cells using conventional electroporation methods for expression.

[0252] 3) Protein A has the activity of binding to the Fc fragment of IgG, so the anti-BCMA single-domain antibody expressed in HEK293 cells was purified using a Protein A column (conventional purification step).

[0253] Finally, the purified anti-BCMA single-domain antibody BCMA-VHH#81-hFc fused with the hFc gene was prepared.

[0254] 2-2. Antibody affinity testing

[0255] The affinity of BCMA-VHH antibodies was detected by flow cytometry. The specific steps are as follows:

[0256] 1) The test antibody BCMA-VHH#81-hFc was TM The FITC-labeled antibody FITC-BCMA-VHH#81-hFc was obtained using the NHS-Fluorescein Antibody Labeling Kit for flow cytometry detection.

[0257] 2) Resuspend BCMA-positive K562-BCMA cells to a density of 2×10 6 cells / mL, 100 μL was placed in 96-well V-bottom plates, centrifuged, and the supernatant was discarded to collect the cells (K562 was used as a BCMA-negative cell control);

[0258] 3) Mix 40 μL of FITC-BCMA-VHH#81-hFc diluted in different gradients with the above cells and incubate at room temperature in the dark for 10 minutes;

[0259] 4) Wash twice by centrifugation with 200 μL FACS buffer, resuspend in 200 μL FACS buffer, and immediately perform flow cytometry to measure the mean fluorescence intensity (MFI);

[0260] 5) Calculate the equilibrium dissociation constant (KD) between the BCMA VHH antibody and the target cell to determine the affinity. The smaller the KD value, the stronger the affinity.

[0261] 1 / (MFI-Con)=1 / Fmax+(KD / Fmax)(1 / [BCMA IgG Fc]).

[0262] Among them, MFI-Con represents the relative mean fluorescence intensity, which is the value of the mean fluorescence intensity of the experimental group minus the mean fluorescence intensity of the background; BCMA IgG Fc represents the antibody dosage (unit: nM).

[0263] A standard curve was plotted with the reciprocal of the antibody dosage as the abscissa and the reciprocal of the relative mean fluorescence intensity as the ordinate, generating a linear regression equation. According to the linear regression equation, the intersection of the standard curve and the ordinate is 1 / Fmax, and the KD is the slope of the line multiplied by Fmax. The calculated equilibrium dissociation constant is shown in Table 1.

[0264] Table 1. Equilibrium dissociation constant (KD) of BCMA-VHH#81 and K562-BCMA cells

[0265] Example 3. Preparation and Expression Detection of BCMA CAR-T Cells

[0266] The chimeric antigen receptor T cells specifically targeting BCMA (BCMA CAR-T cells) prepared in this example were prepared by transferring the BCMA CAR1 gene (SEQ ID NO: 2) and the BCMA CAR2 gene (SEQ ID NO: 4) into T cells for stable expression. The resulting BCMA CAR-T cells were named BCMA CAR1-T cells and BCMA CAR2-T cells, respectively. The specific steps are as follows:

[0267] 1. Construction of recombinant retroviral vector

[0268] (1) The recombinant vectors pUC57-BCMA CAR1 and pUC57-BCMA CAR2 were double-digested with NotI (NEB) and EcoRI (NEB), and the target gene fragments (BCMA CAR1 gene and BCMA CAR2 gene) were recovered by gel excision. The recombinant vector pUC57-BCMA CAR1 is a recombinant expression vector obtained by cloning the BCMA CAR1 gene (SEQ ID NO: 2) into the pUC57 vector, and the recombinant vector pUC57-BCMA CAR2 is a recombinant expression vector obtained by cloning the BCMA CAR2 gene (SEQ ID NO: 4) into the pUC57 vector. The above recombinant expression vectors were synthesized and provided by Qingke Biotechnology Co., Ltd.

[0269] (2) The retroviral vector MP71 was double-digested with NotI and EcoRI, and the large vector fragment was recovered by gel cutting.

[0270] (3) Use T4 ligase (NEB) to connect the target gene fragments BCMA CAR1 gene and BCMA CAR2 gene with the large vector fragment, thereby obtaining recombinant retroviral vectors MP71-BCMA CAR1 and MP71-BCMA CAR2 carrying the BCMA CAR1 gene and BCMA CAR2 gene, respectively.

[0271] (4) The recombinant retroviral vectors MP71-BCMA CAR1 and MP71-BCMA CAR2 were transformed into competent Escherichia coli DH5α, and the plasmids were extracted and purified using the Qiagen plasmid purification kit to obtain MP71-BCMA CAR1 plasmid and MP71-BCMA CAR2 plasmid.

[0272] The recombinant retroviral vector MP71-BCMA CAR1 (i.e., MP71-BCMA CAR1 plasmid) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment with the nucleotide sequence of SEQ ID NO: 2 in the sequence listing, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0273] The recombinant retroviral vector MP71-BCMA CAR2 (i.e., MP71-BCMA CAR2 plasmid) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment with the nucleotide sequence of SEQ ID NO: 4 in the sequence listing, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0274] 2. Retroviral packaging

[0275] The MP71-BCMA CAR1 plasmid and the MP71-BCMA CAR2 plasmid prepared in step 1 are respectively introduced into packaging cells for packaging to complete virus assembly and obtain retrovirus. The specific steps of virus packaging are as follows:

[0276] a) Day 1: Phoenix-ECO cells should be less than 20 generations old and not overgrown. 6 Cells were plated at a cell density of 1 / ml, and 10 mL of DMEM medium (10% Fetal Bovine Serum, 2 mM L-glutamine, 1% Penicillin / Streptomycin) was added to a 10 cm cell culture dish. The cells were thoroughly mixed and cultured at 37°C overnight.

[0277] b) Day 2: Transfect ECO cells when they reach approximately 90% confluency (usually 14-18 hours after plating). Add chloroquine half an hour before transfection. Prepare 15 μg of plasmid, 250 μL of 1.5 M CaCl2, and 1 mL of H2O to a total volume of 1.25 mL. Add an equal volume of 2× HBS to the plasmid complex, mix thoroughly, and let stand at room temperature for 15 minutes. Then, add the mixture to the ECO cell dish. Incubate at 37°C for 6 hours, remove the culture medium, wash once with PBS, and add 10 mL of prewarmed fresh culture medium.

[0278] c) Day 4: 48 hours after transfection, collect the supernatant, filter through a 0.45 μm filter to obtain the retroviral solution, and store in aliquots at -80°C.

[0279] d) Add 1.2 mL of 15 μg / mL Retronectin coating solution to each well of a 6-well NTC plate and incubate at 4°C overnight.

[0280] e) Carefully aspirate the blocking solution, add 2 mL / well PBS for washing, add 5 mL of the above virus solution to each well, centrifuge at 2000 × g at 32°C for 2 h, and discard the unbound virus supernatant;

[0281] f) Wash logarithmic phase PG13 cells once with 10 mL of PBS, add 1 mL of 0.25% recombinant trypsin, and incubate at room temperature for 2-3 minutes;

[0282] g) Add 5 ml of complete medium containing 10% FBS to terminate the digestion, and centrifuge at 1500 rpm for 5 minutes;

[0283] h) Discard the supernatant and adjust the cell density to 0.5×10 5 cell / mL, and add 3 mL / well to the virus-coated NTC 6-well plate to make the final cell count 1.5×10 5 cell / hole;

[0284] i) incubation at 37°C, 5% CO2 for 48 h;

[0285] j) After passage 1-2, the cells were transferred to T175 culture flasks and cultured in DEME medium containing 12% FBS for 2 days;

[0286] k) Replace with fresh DEME medium containing 12% FBS and continue culturing for 48 hours. Collect the supernatant, filter with a 0.45 μm filter to obtain a retroviral solution, and store in aliquots at -80°C.

[0287] 3. Retrovirus infection of human T cells

[0288] a) Thaw frozen healthy human peripheral blood PBMCs and adjust the cell density to 1×10 6 -2×10 6 cells / mL.

[0289] b) PBMCs were separated using Ficoll separation buffer (Tianjin Haoyang, LTS1077-1) to obtain lymphocytes, and then CD3+ T cells were isolated using a Miltenyi magnetic bead method. Clinical-grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen) were added at a ratio of 3:1 to activate T cells. The T cells were cultured in a 24-well cell culture plate to obtain an activated T cell suspension.

[0290] c) Coat non-tissue-treated culture plates with RetroNectin (TAKARA) diluted with PBS to a final concentration of 15 μg / mL, using 1.2 mL per well of a 6-well plate. Protect from light and incubate at 4°C overnight.

[0291] d) After two days of T cell activation culture, the coated 6-well plate was removed, the coating solution was discarded, and the plate was washed once with PBS.

[0292] e) Add 5-6 mL of the retroviral solution prepared in step 2 to each well. Centrifuge at 32°C, 2000 × g for 2 h, and discard the supernatant. Add 3 mL of activated T cell suspension containing hIL-2 (500 U / mL) to each well and continue culturing for 1 day.

[0293] f) After cell infection, the cell density was observed daily and T cell culture medium containing 500 U / mL IL-2 was added as appropriate to maintain the T cell density at 5×10 5 / mL, which is convenient for cell expansion.

[0294] g) CAR-T cells (BCMA CAR-T cells) infected with the retrovirus prepared in step 2 are obtained and named BCMA CAR1-T cells and BCMA CAR2-T cells, respectively. BCMA CAR1-T cells are T cells expressing the BCMA CAR1 gene with a nucleotide sequence of SEQ ID NO: 2, and BCMA CAR2-T cells are T cells expressing the BCMA CAR2 gene with a nucleotide sequence of SEQ ID NO: 4.

[0295] The constructed BCMA CAR-T cells (BCMA CAR1-T cells and BCMA CAR2-T cells) and CTR T cells (i.e., T cells without virus transfection, serving as negative control) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and recorded as day 0. Various functional tests were performed until day 10.

[0296] 4. Detection of CAR expression in BCMA CAR-T cells

[0297] a) After centrifugation (1500 rpm for 5 min), discard the supernatant, add 200 μL of FACS buffer (1× PBS containing 2% FBS) to each well of a 96-well round-bottom plate, resuspend the cells, and centrifuge at 1500 rpm for 5 min.

[0298] b) Add 60 μL of prepared fluorescently labeled anti-human CD27-PE (Biolegend, 356406) / BCMA(rp)-APC (APC anti-human IgG Fc Antibody, Biolegend, 410712) to each well, resuspend and mix, and incubate at 4°C for 30 min.

[0299] c) Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 minutes;

[0300] d) Discard the supernatant, resuspend the cells in 400 μL FACS buffer, transfer the cells to a flow cytometer, read the cells using a flow cytometer (BD Canto-II), and analyze the percentage of BCMA antibodies in BCMA CAR-T cells.

[0301] The test results are shown in Figure 3. BCMA CAR1-T cells and BCMA CAR2-T cells can well express BCMA antibodies and CD27 proteins, indicating that the expression of CAR molecules in BCMA CAR-T cells has met the expected design.

[0302] Example 4: Functional detection of secreted effector molecule IFN-γ

[0303] IFN-γ has anti-tumor, antiviral, and immunomodulatory properties. High levels of IFN-γ are an important indicator of well-functioning CAR-T cells. Its expression is consistent with T cell cytotoxicity and can indicate the activation level of CAR-T cells. Detection is performed by flow cytometry after intracellular staining. The steps are as follows:

[0304] 1. Test cells BCMA CAR-T cells (BCMA CAR1-T cells and BCMA CAR2-T cells) and CTR T cells (i.e., T cells without virus transfection, as negative control) were adjusted to a cell density of 2×10 6 100 μL of the solution was added to a 96-well U-bottom plate, and MM1.S cells were added at a ratio of test cells: target cells = 1:1. Brefeldin A (Med Chem Express, HY-16592) was added to each well at a final concentration of 5 μg / mL, and the cells were incubated at 37°C in a culture medium for 6 hours.

[0305] CTR+MM1.S: 1 mL per well, total 1×10 6 1×10 T cells without virus transfection (CTR T cells) were added with 1 mL of 6 MM1.S cells;

[0306] BCMA CAR-T cells + MM1.S: 1 mL per well, 1 × 106 1×10 BCMA CAR-T cells were added to 1 mL. 6 MM1.S cells;

[0307] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0308] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μL FACS buffer (1× PBS containing 2% FBS) to each well for resuspending. Centrifuge at 1500 rpm for 5 min. Repeat this step twice.

[0309] (2) Add 60 μL of the prepared fluorescently labeled BCMA(rp)-PE to each well, resuspend and mix thoroughly, and incubate at room temperature for 10 min in the dark.

[0310] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0311] (4) Add 150 μL of Cytofix / Cytoperm (BD Biosciences, Cat. No. 55472) to each well, resuspend and mix, and incubate at room temperature for 15 min in the dark.

[0312] (5) Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Cat. No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0313] (6) Add 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, Cat. No. 506510) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark.

[0314] (7) Add 200 μL of Perm buffer to each well and centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells in 400 μL of FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) to analyze the percentage of the functional effector molecule IFN-γ in BCMA CAR1-T cells, BCMA CAR2-T cells, and control cells.

[0315] The test results are shown in Figure 4. After co-culture with MM1.S target cells, both BCMA CAR1-T cells and BCMA CAR2-T cells were able to secrete the T cell-specific effector molecule IFN-γ well.

[0316] Example 5. In vitro tumorigenicity assay of BCMA CAR-T cells

[0317] In vitro killing experiments were performed to detect the killing effect of BCMA CAR-T cells on tumor cells. The steps are as follows:

[0318] 1. Adjust the cell density of effector cells BCMA CAR1-T cells, BCMA CAR2-T cells, and CTR T cells (i.e., T cells without virus transfection, as negative control) to 1×10 6 100uL of each solution was added to a 96-well V-bottom plate. 100uL of target cells MM1.S-LUC at different densities was added according to different effector-target ratios (9:1, 3:1, 1:1, 1:3). The cells were mixed and cultured in a 96-well V-bottom plate at 37°C for 4 hours.

[0319] 2. Prepare and add fluorescein sodium salt diluent (1 μL / mL) in a dark place, add 100 μL to each well in a dark place.

[0320] 3. Measure the fluorescence value using a TECAN spark microplate reader and take the average value of three replicate wells to calculate the specific cytotoxicity of the effector cells.

[0321] Cytotoxicity%=100-100×(Eexp-Emin) / (Tmax-Tmin)

[0322] Eexp: RLU value when effector cells and target cells are co-cultured;

[0323] Emin: RLU value of spontaneous death of effector cells in the absence of target cells;

[0324] Tmax: RLU value of spontaneous death of target cells in the absence of effector cells;

[0325] Tmin: RLU value under conditions of maximum killing rate.

[0326] The test results are shown in Figure 5. Both BCMA CAR1-T cells and BCMA CAR2-T cells can effectively and specifically kill BCMA. + Target cells (MM1.S-LUC cells), with the increase of the effector-target ratio, the killing effect of BCMA CAR1-T cells and BCMA CAR2-T cells on tumor cells was significantly increased (P values ​​< 0.01). Under the same effector-target ratio, the killing effect of BCMA CAR2-T cells was also significantly increased compared with BCMA CAR1-T cells (P < 0.01). BCMA CAR2-T cells have more efficient killing activity, with the highest killing activity close to 100%. + No killing effect on target cells.

[0327] Example 6. Detection of in vivo tumoricidal activity of BCMA CAR-T cells

[0328] 1. Tumor cells and experimental animals

[0329] Tumor cell line: human multiple myeloma cells MM1.S-Luc carrying luciferase and GFP genes.

[0330] Culture medium conditions: RPMI1640 medium containing 10% fetal bovine serum, 1× penicillin-streptomycin, and 1 μg / mL puromycin.

[0331] Experimental animals: NOD.CB17-PrkdcscidIl2rgtm1 / Bcgen mice (B-NDG, Biocytogen).

[0332] Animal information: SPF level, 7-8 weeks old when inoculated with tumor cells, all female.

[0333] 2. Modeling and grouping

[0334] MM1.S-Luc cells in the logarithmic growth phase were taken and resuspended to 4.0×10 6 All mice were inoculated with 0.3 mL of cell suspension in the tail vein under sterile conditions.

[0335] Five days after the mice were inoculated with MM1.S-Luc cells, the tumor signal intensity of the tumor-bearing mice was measured by a small animal in vivo imaging instrument (IVIS Spectrum, PerkinElmer). At the same time, animals were enrolled according to the tumor signal intensity, with 3 to 4 animals per group. Then, on the sixth day of tumor bearing, CTR-T was used as a control (T cells not infected with the virus) and BCMA CAR1-T cells and BCMA CAR2-T cells cultured for 8 days after virus-infected T cells were collected and the cell density was adjusted to 3.0×10 6 CAR-T cells / 0.3mL. All animals were dosed via tail vein with a single injection of 0.3mL. The day of administration was designated as D0.

[0336] 3. Tumor Signals

[0337] All animals received an intraperitoneal injection of 150 mg / kg (15 mg / mL, 10 mL / kg) of D-luciferin potassium (Yishen Biotech) on Days 1, 6, 14, 20, 27, 35, 42, 49, 56, and 63 before grouping. Tumor signal intensity was measured using a small animal in vivo imaging device under isoflurane anesthesia 10 to 15 minutes after injection, with imaging lasting 60 seconds. The imaging results from Days 1 to 63 are summarized in Figures 6A and 6B.

[0338] In the CTR group, the tumor size of animals was positively correlated with time, and the average tumor signal intensities on D-1, D06, D14, D20, D27, D35, and D42 were 2.62×10 6 , 1.12×10 7 , 5.89×10 7 , 4.28×10 8 , 6.68×10 9 , 5.41×10 10 and 2.28×10 11 P / S.

[0339] In the BCMA CAR1-T group, the average tumor signal intensities on D-1, D06, D14, D20, D27, D35, D42, D49, and D56 were 4.99×10 6 , 8.50×10 5 9.99×10 5 , 1.80×10 6 , 3.25×10 7 , 5.33×10 8 , 2.19×10 9 , 1.61×10 10 and 1.38×10 11 P / S.

[0340] In the BCMA CAR2-T group, the average tumor signal intensity on D-1, D06, D14, D20, D27, D35, D42, D49, and D56 was 4.01×10 6 , 1.01×10 6 , 1.24×10 6 , 7.52×10 5 , 8.44×10 5 , 5.78×10 6 , 3.51×10 7 , 7.04×10 7 and 7.59×10 8 P / S.

[0341] Compared with the CTR group, mice in the BCMA CAR1-T and BCMA CAR2-T groups showed significant tumor suppression on D06 (P<0.0001), and tumor cell clearance was maintained from D06 to D35. Compared with BCMA CAR1-T cells, BCMA CAR2-T cells showed a better tumor suppression effect (P<0.01). In addition, as the observation time prolonged, 3 / 4 of the mice in the BCMA CAR1-T group relapsed on D35; 1 / 4 of the mice in the BCMA CAR2-T group relapsed on D35, and the remaining mice did not relapse until D63. The above results suggest that both BCMA CAR1-T cells and BCMA CAR2-T cells can significantly inhibit tumor growth, and BCMA CAR2-T cells have a better tumor suppression effect.

[0342] 4. Mouse survival

[0343] The experimental observation endpoint was D63, and the survival curves of tumor-bearing mice in each group during this period are shown in Figure 6C.

[0344] All animals in the CTR group (n=3) died on D51; 3 / 4 mice in the BCMA CAR1-T group (n=4) died on D56 / D56 / D58, respectively; no animals in the BCMA CAR2-T group (n=4) died during the experiment.

[0345] The results suggest that administration of BCMA CAR1-T and BCMA CAR2-T groups can significantly prolong the survival time of mice (P<0.01), among which the mice in the BCMA CAR2-T group had a longer survival time (P<0.01).

[0346] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art. Industrial Applicability

[0347] The present application utilizes BCMA recombinant protein to immunize alpacas to screen and obtain high-affinity anti-BCMA single-domain antibodies. This single-domain antibody can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system. It can be further developed into an anti-tumor drug as an anti-tumor antibody with clinical application prospects by targeting and binding to the tumor antigen BCMA with high affinity. At the same time, it can be used as an antibody to detect BCMA protein in applications such as ELISA, Western blot, and immunohistochemistry and developed into a detection product, which can greatly save scientific research costs and time. The single-domain antibody drug of the present application has a stable structure, small molecules, easy recombinant expression, and low production cost. It can be used alone or as a drug delivery system to carry related drugs. It has very broad prospects and important significance in the fields of drug application and clinical diagnosis.

[0348] The BCMA-targeting CAR-T cells (BCMA CAR-T cells) with a novel structure further designed and developed based on the above-mentioned single-domain antibody can effectively secrete the T cell-specific effector molecule IFN-γ, effectively and specifically kill BCMA+ target cells, and have good in vivo tumoricidal activity. They can not only significantly inhibit the proliferation of tumor cells in mice, but also significantly prolong the survival of mice. The BCMA CAR-T cells of this application have good anti-tumor ability and can be used for immunotherapy of BCMA target-related diseases, with broad clinical application prospects.

Claims

1. A single-domain antibody targeting BCMA, characterized in that: The single-domain antibody comprises a complementarity determining region CDR1 having amino acid sequences of positions 179-186 of SEQ ID NO: 1, a complementarity determining region CDR2 having amino acid sequences of positions 204-211 of SEQ ID NO: 1, and a complementarity determining region CDR3 having amino acid sequences of positions 250-260 of SEQ ID NO:

1.

2. The single domain antibody according to claim 1, characterized in that The single domain antibody is any of the following: A1) a protein whose amino acid sequence is positions 154-271 of SEQ ID NO: 1; A2) a protein having at least 80% identity with the protein of A1) and having the same function as the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1; A3) A fusion protein having the same function obtained by connecting a tag or signal peptide to the N-terminus and / or C-terminus of A1) or A2).

3. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the single domain antibody of claim 1 or 2.

4. The chimeric antigen receptor according to claim 3, characterized in that The chimeric antigen receptor further comprises a second single domain antibody, wherein the second single domain antibody is any one of the following: B1) a protein whose amino acid sequence is positions 22-138 of SEQ ID NO: 1; B2) a protein having at least 80% identity with the protein of B1) and having the same function as the protein of B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1 from positions 22 to 138; B3) A fusion protein having the same function obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of B1) or B2).

5. The chimeric antigen receptor according to claim 3 or 4, characterized in that The chimeric antigen receptor further comprises a hinge region, a transmembrane region, an intracellular signaling region and CD27.

6. The chimeric antigen receptor according to any one of claims 3 to 5, characterized in that The hinge region and transmembrane region are derived from CD8a or CD28.

7. The chimeric antigen receptor according to any one of claims 3 to 6, characterized in that The chimeric antigen receptor is any one of the following: C1) a protein whose amino acid sequence is positions 22-781 of SEQ ID NO: 1; C2) a protein whose amino acid sequence is positions 22-778 of SEQ ID NO: 3; C3) a protein having at least 80% identity with the protein of C1) and having the same function as the protein of C1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID NO: 1; C4) a protein having at least 80% identity with the protein of C2) and having the same function as the protein of C2) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of positions 22-778 of SEQ ID NO: 3; C5) A fusion protein having the same function obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of C1), C2), C3) or C4).

8. Biomaterial, characterized in that The biological material is any one of the following: D1) a nucleic acid molecule encoding the single domain antibody of claim 1 or 2; D2) a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 3 to 7; D3) an expression cassette containing the nucleic acid molecule described in D1) or D2); D4) a recombinant vector containing the nucleic acid molecule described in D1) or D2); D5) a recombinant microorganism containing the nucleic acid molecule described in D1) or D2); D6) A recombinant host cell containing the nucleic acid molecule described in D1) or D2).

9. The biomaterial according to claim 8, characterized in that The nucleic acid molecule is any of the following: E1) the coding sequence or nucleotide sequence is the DNA molecule shown in positions 460-813 of SEQ ID NO: 2; E2) the coding sequence or nucleotide sequence is positions 64-2343 of SEQ ID NO: 2 or the DNA molecule set forth in SEQ ID NO: 2; E3) The coding sequence or nucleotide sequence is positions 64-2334 of SEQ ID NO: 4 or the DNA molecule shown in SEQ ID NO:

4.

10. A modified immune effector cell, characterized in that The modified immune effector cell comprises the single domain antibody of claim 1 or 2, the chimeric antigen receptor of any one of claims 3 to 7, or the nucleic acid molecule of claim 8 or 9.

11. The modified immune effector cell according to claim 10, characterized in that The immune effector cells include T cells, NK cells, NKT cells, γδT cells, αβT cells, regulatory T cells, MAIT cells, B cells, macrophages, dendritic cells, peripheral blood mononuclear cells and monocytes.

12. Use of the single domain antibody of claim 1 or 2, the chimeric antigen receptor of any one of claims 3 to 7, the biomaterial of claim 8 or 9, or the modified immune effector cell of claim 10 or 11 in the preparation of a product having any of the following functions: F1) Prevention or treatment of tumors; F2) Prevent or treat BCMA target-related diseases; F3) Killing BCMA-positive tumor cells; F4) inhibit the proliferation of BCMA-positive tumor cells; F5) inhibiting the growth of BCMA-positive tumors; F6) Detection of BCMA-expressing tumor cells or BCMA protein.

13. Any of the following uses of the single domain antibody according to claim 1 or 2 or the nucleic acid molecule encoding the single domain antibody according to claim 1 or 2: G1) Use in the preparation of products for screening, diagnosis or auxiliary diagnosis of BCMA target-related diseases; G2) Use in the preparation of a product for binding BCMA protein; G3) Use in the preparation of a product that mediates drug-specific recognition of tumors expressing BCMA antigen; G4) Use in the preparation of a product for in vivo imaging of BCMA protein; G5) in the preparation of CAR cells targeting BCMA.

14. A pharmaceutical composition for preventing or treating BCMA target-related diseases, characterized in that: The pharmaceutical composition comprises the single domain antibody of claim 1 or 2 or the modified immune effector cell of claim 10 or 11, and one or more pharmaceutically acceptable carriers.

15. A kit, characterized in that The kit contains the single domain antibody of claim 1 or 2, the chimeric antigen receptor of any one of claims 3 to 7, or the modified immune effector cell of claim 10 or 11.

16. An antibody-drug conjugate comprising an antibody portion and a conjugate portion, characterized in that: The antibody portion comprises the single domain antibody of claim 1 or 2.

17. A method for preventing or treating a BCMA target-related disease, characterized in that: The method comprises administering the single domain antibody of claim 1 or 2, the modified immune effector cell of claim 10 or 11, or the pharmaceutical composition of claim 14 to a subject suffering from a BCMA target-related disease.

18. A method for screening, diagnosing or assisting in the diagnosis of BCMA target-related diseases, characterized in that: The method comprises isolating and obtaining a sample from a subject, then using the single-domain antibody according to claim 1 or 2 to detect the content of BCMA protein in the sample, and performing screening, diagnosis, or auxiliary diagnosis of BCMA target-related diseases based on the content of the BCMA protein.

19. A method for preparing the modified immune effector cells according to claim 10 or 11, characterized in that: The method comprises: introducing a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 3 to 7 into an immune effector cell for expression, thereby obtaining the modified immune effector cell.

20. A method for preparing the single domain antibody according to claim 1 or 2, characterized in that: The method comprises: constructing a recombinant expression vector containing a nucleic acid molecule encoding the single-domain antibody according to claim 1 or 2; introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the single-domain antibody; culturing the recombinant cell, and obtaining the single-domain antibody through separation and purification.

Images