Bispecific chimeric antigen receptor targeting BCMA and CD19 and application thereof

Abstract

The invention discloses a bispecific chimeric antigen receptor targeting BCMA and CD19 and an application of the bispecific chimeric antigen receptor. Specifically, the invention discloses a chimeric antigen receptor with an amino acid sequence of SEQ ID No.1, a CAR-T cell containing the chimeric antigen receptor and application of the chimeric antigen receptor. According to the invention, the CAR-T cell targeting CD19 and BCMA is constructed, and experiments prove that the CAR-T cell disclosed by the invention can well secrete T cell specific effector molecules IFN-gamma, has very high cytotoxicity and has very good killing ability on tumor cells. In-vivo tests prove that the CAR-T cell provided by the invention can significantly inhibit tumor growth, has good in-vivo tumor killing activity, and has a longer inhibition effect. The CAR-T cell is safe and efficient, can be widely applied to treatment of autoimmune diseases and tumors related to B cells, and has good clinical transformation value and wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of cellular immunotherapy, and specifically relates to a bispecific chimeric antigen receptor targeting BCMA and CD19 and applications thereof. Background Art

[0002] Chimeric antigen receptor T cells (CAR-T cells) are immune cells modified by genetic engineering technology for precise targeted treatment of tumors and autoimmune diseases. CAR-T cell technology is to modify T cells in vitro through biotechnology, so that they can recognize antigens on the surface of target cells and achieve the effect of targeted killing of target cells. CAR-T cell therapy has achieved encouraging results in blood tumors and solid tumors. In particular, for blood tumors such as B cell lymphoma, B cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL), CAR-T cell therapy has shown precise, rapid, efficient and sustained killing effects, which can significantly prolong the survival of patients and even achieve complete remission of the disease. In recent years, more and more clinical studies have confirmed that CAR-T cell therapy is also a promising new option for the treatment of autoimmune diseases. Autoimmune diseases (AIDs) are a class of diseases associated with excessive activation or proliferation of B cells. Under the action of certain factors (infection, trauma, drugs, etc.), the body's autoimmune response reaches a certain level or lasts too long, resulting in the destruction of the body's own immune tolerance, causing pathological damage or dysfunction of the body's own cell tissues, and the appearance of corresponding clinical symptoms. Common AIDs include systemic lupus erythematosus (SLE), multiple sclerosis (MS), pemphigus vulgaris (PV), rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic sclerosis (SS), inflammatory bowel disease (IBD) and myasthenia gravis (MG), etc. This type of disease is often caused by excessive activation of B cells. Studies have shown that the activation of B cells is involved in the pathogenesis of a variety of AIDs, and autoreactive B cells play an important role in the pathogenesis of AIDs. For example, in SLE patients, B cell reactivity increases, producing a variety of autoantibodies, which combine with corresponding antigens in the body to form immune complexes that are deposited in various tissues and cause tissue damage. In view of the abnormal activation of autoreactive B cells in the onset of various AIDs, CAR-T cells are expected to target and kill B cells, thereby effectively controlling the progression of the disease and alleviating disease symptoms.

[0003] In view of this, targeting B cell targets, exploring and designing new CAR-T cell drugs, and developing new CAR-T cell therapies have broad clinical application prospects in the fields of B cell malignancies and autoimmune diseases, and are expected to provide more effective and safe treatment strategies for these intractable diseases. Summary of the invention

[0004] One of the purposes of the present invention is to provide a bispecific chimeric antigen receptor targeting BCMA and CD19, or an immune effector cell containing the chimeric antigen receptor, or an application thereof. The technical problems to be solved are not limited to the described technical subjects, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0005] To achieve the above objectives, the present invention first provides a chimeric antigen receptor, which includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain includes a single domain antibody targeting BCMA (which may be named BCMA VHH) and a single chain antibody targeting CD19 (which may be named CD19-ScFV), the amino acid sequence of the single domain antibody may be as shown at positions 22-138 of SEQ ID No.1, and the amino acid sequence of the single chain antibody may be as shown at positions 154-401 of SEQ ID No.1.

[0006] Further, the single-domain antibody BCMA VHH may be located at the N-terminus or C-terminus of the single-chain antibody CD19-ScFV. Specifically, the single-domain antibody BCMA VHH may be located at the N-terminus of the single-chain antibody CD19-ScFV.

[0007] Furthermore, the single-domain antibody BCMA VHH and the single-chain antibody CD19-ScFV can be connected via a linker.

[0008] The linker is non-restrictive and its purpose is to prevent steric hindrance. Those skilled in the art are familiar with other replaceable linkers, as long as the linker has a certain flexibility to allow the proteins on both sides to perform their respective independent functions.

[0009] The linker may be a flexible peptide linker, such as a peptide linker comprising glycine, serine, proline and / or lysine residues. The peptide linker may consist of 1-40 amino acids.

[0010] Further, the linker includes, but is not limited to: (G)n, (S)n, (GxS)n, (SxG)n, (GSSGG)n (SEQ ID No.5), (GGSGG)n (SEQ ID No.6), (GSGGSG)n (SEQ ID No.7), (GSGSGS)n (SEQ ID No.8), (GGQGG)n (SEQID No.9), (EAAAK)n (SEQ ID No.10) and IEGRMD (SEQ ID No.11), 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 invention, the linker is (G4S)3.

[0011] The single-domain antibody BCMA VHH and the single-chain antibody CD19-ScFV can be connected through a linker to form a dual-epitope antibody targeting BCMA and CD19, and the dual-epitope antibody constitutes the antigen binding domain of the chimeric antigen receptor of the present invention.

[0012] Furthermore, the antigen binding domain may be the single domain antibody BCMA VHH, the linker, and the single chain antibody CD19-ScFV from the N-terminus to the C-terminus. Specifically, the amino acid sequence of the antigen binding domain may be as shown in positions 22-401 of SEQ ID No.1.

[0013] The intracellular signaling domain may include a co-stimulatory domain and an activation domain.

[0014] Further, the intracellular signaling domain may comprise one, two or more costimulatory domains. The costimulatory domain may comprise the entire intracellular portion of a costimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" may refer 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.

[0015] The co-stimulatory domain can be an intracellular signaling domain from a co-stimulatory molecule, for example, the co-stimulatory domain can be derived from the 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.

[0016] The activation domain may be derived from the intracellular signaling domain of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66d.

[0017] Furthermore, the co-stimulatory domain may be derived from 4-1BB, and the activation domain may be derived from CD3ζ.

[0018] Furthermore, the co-stimulatory domain may be the intracellular signaling domain of 4-1BB.

[0019] Furthermore, the activation domain may be the intracellular signaling domain of CD3ζ.

[0020] The transmembrane domain may be derived from the α, β or ζ chain of the T cell receptor, CD3ζ, CD8a (CD8α), CD4, CD9, CD45, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152 or the transmembrane domain of CD154.

[0021] Furthermore, the transmembrane domain is located between the antigen binding domain and the intracellular signaling domain, and is responsible for maintaining the stability of the CAR molecule on the T cell membrane.

[0022] Furthermore, the transmembrane domain may be a CD8a transmembrane region (TM).

[0023] The chimeric antigen receptor may further include a hinge domain located at the N-terminus of the transmembrane domain.

[0024] Further, the hinge domain may 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.

[0025] Furthermore, the hinge domain may be a CD8a hinge region (Hinge).

[0026] Furthermore, the chimeric antigen receptor includes, from N-terminus to C-terminus, the antigen binding domain, the CD8a hinge region and transmembrane region, the co-stimulatory domain 4-1BB and the activation domain CD3ζ.

[0027] Furthermore, the chimeric antigen receptor also includes a signal peptide at the N-terminus and a safety switch at the C-terminus, and the safety switch includes a protein encoded by a suicide gene.

[0028] Furthermore, the suicide genes include herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (iCasp9), truncated epidermal growth factor receptor (tEGFR / EGFRt), CD20, cytosine deaminase (CD), RQR8, cytochrome P450, purine nucleoside phosphorylase (PNP) and interleukin-1β converting enzyme (ICE) genes.

[0029] Furthermore, the suicide gene may be herpes simplex virus thymidine kinase (HSV-TK).

[0030] Furthermore, the protein encoded by the suicide gene is connected or fused to the C-terminus of the chimeric antigen receptor via a linker.

[0031] Furthermore, the protein encoded by the suicide gene is connected or fused to the C-terminus of the activation domain CD3ζ via a linker.

[0032] Furthermore, the linker may be a cleavable linker (also called a cleavable linker).

[0033] Further, the cleavable linker includes P2A, F2A, T2A and E2A, or any combination thereof, but is not limited thereto. The amino acid sequence of the P2A may be as shown in SEQ ID No. 12. The amino acid sequence of the F2A may be as shown in SEQ ID No. 13. The amino acid sequence of the T2A may be as shown in SEQ ID No. 14. The amino acid sequence of the E2A may be as shown in SEQ ID No. 15.

[0034] Furthermore, the cleavable linker may be P2A.

[0035] Although the chimeric antigen receptor designed and constructed in the present invention uses P2A as a linker to connect the safety switch, the present invention is not limited to this specific linker, and those skilled in the art can replace P2A in the chimeric antigen receptor of the present invention, for example, F2A, T2A and E2A, etc., which have the same "self-cleavage" function as P2A, can be used. As long as the constructed chimeric antigen receptor has the same function as the chimeric antigen receptor of the present invention, it is regarded as a chimeric antigen receptor equivalent to the chimeric antigen receptor of the present invention.

[0036] The signal peptide may be derived from the signal peptide of CD4, CD8, CD8a, CD28, CD137 and GM-CSF.

[0037] Furthermore, the signal peptide may be a human CD8 signal peptide.

[0038] The chimeric antigen receptor described in this article can be CD8 signal peptide (Signal), single-domain antibody BCMAVHH, linker, single-chain antibody CD19-ScFV, CD8a hinge region (Hinge) and transmembrane region, co-stimulatory domain 4-1BB, activation domain CD3ζ, cleavable linker and safety switch HSV-TK from N-terminus to C-terminus.

[0039] Furthermore, the chimeric antigen receptor may be any of the following:

[0040] A1) a protein with an amino acid sequence as shown in SEQ ID No. 1;

[0041] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.1;

[0042] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0043] The substitutions may be conservative substitutions.

[0044] The connection in A3) can be directly connected via a peptide bond, or connected via a linker.

[0045] In order to facilitate the separation, purification, detection and / or positioning of the chimeric antigen receptor described herein, a label protein can be connected to the amino terminus or carboxyl terminus of the chimeric antigen receptor. The label includes, but is not limited to: GST (glutathione sulfhydryl transferase) label protein, Trx (thioredoxin) label protein, nitrogen utilization substrate A (NusA) label protein, His label protein (His-tag), MBP (maltose binding protein) label protein, Flag label protein, SUMO label protein, HA (influenza hemagglutinin) label protein, Myc label protein, LacZ label protein, CBD (cellulose binding domain) label protein, bacteriophage T7 protein kinase (T7PK) label protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag label protein. Those skilled in the art know how to select a suitable label protein according to the desired purpose. The use of the label does not change the function of the target protein, and its purpose is to separate, purify, detect or trace, so the label protein suitable for the present application is not limited to a specific type. The tag can be separated from the target protein by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site to remove the tag by cleavage with TEV protease).

[0046] The present invention also provides a biomaterial, which may be any of the following:

[0047] B1) a nucleic acid molecule encoding any chimeric antigen receptor described herein;

[0048] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0049] B3) a recombinant vector containing the nucleic acid molecule described in B1);

[0050] B4) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0051] B5) A recombinant host cell containing the nucleic acid molecule described in B1).

[0052] The biological materials can all express any chimeric antigen receptor described herein.

[0053] The recombinant vector may be a recombinant vector obtained by cloning a nucleic acid molecule encoding any of the chimeric antigen receptors described herein (such as a DNA molecule shown in SEQ ID No. 2) into a backbone vector. Although the backbone vectors used in the embodiments provided by the present invention are pUC57 vectors and retroviral vectors MP71, the present invention may not be limited to this specific vector. Those skilled in the art may use other suitable vectors, such as lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, non-viral vector transposons, CRISPR-Cas systems, or other backbone vectors for cloning or expression. As long as the vector is capable of cloning or expressing a nucleic acid molecule encoding any of the chimeric antigen receptors described herein.

[0054] Furthermore, the recombinant vector may specifically be a recombinant retroviral vector MP71-BCMA CD19 CAR-TK. The recombinant retroviral vector MP71-BCMA CD19 CAR-TK 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 whose nucleotide sequence is SEQ ID No. 2 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0055] In the above biological material, the nucleic acid molecule may be a DNA molecule with a coding sequence or a nucleotide sequence such as that shown in SEQ ID No.2.

[0056] The DNA molecule shown in SEQ ID No.2 encodes a chimeric antigen receptor having an amino acid sequence of SEQ ID No.1.

[0057] Those skilled in the art can easily mutate the nucleotide sequence encoding any chimeric antigen receptor described herein by using known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis and cassette mutagenesis, etc.) or directed evolution (including error-prone PCR, DNA shuffling and in vitro random priming recombination, etc.). Those artificially modified nucleotide sequences that have 75% or more identity with the nucleotide sequence encoding any chimeric antigen receptor described herein (such as SEQ ID No. 2) are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode any chimeric antigen receptor described herein and have the same function as any chimeric antigen receptor described herein.

[0058] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No. 2. Considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression of specific species as needed.

[0059] Variants with improved affinity to any of the chimeric antigen receptor sequences described herein can be obtained by methods known in the art and are included within the scope of the present invention. For example, amino acid substitutions can be used to obtain chimeric antigen receptors with further improved affinity. Alternatively, codon optimization of nucleotide sequences can also be used to improve translation efficiency in expression systems used to produce chimeric antigen receptors.

[0060] In certain embodiments, substitutions, insertions or deletions may occur in one or more complementary determining regions or framework regions in a single domain antibody and / or single chain antibody in any of the chimeric antigen receptors of the present invention, as long as such changes do not substantially reduce the ability of the chimeric antigen receptor to bind antigen. For example, conservative changes (e.g., conservative substitutions, as is well known to those skilled in the art, 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, which do not substantially reduce binding affinity. For example, such changes may be outside the antigen contact residues in the complementary determining regions.

[0061] The present invention also provides a modified immune effector cell, wherein the modified immune effector cell comprises any chimeric antigen receptor or nucleic acid molecule described herein.

[0062] The modified immune effector cell may be any chimeric antigen receptor modified immune effector cell described herein (i.e., CAR-modified immune effector cell, CAR cell), which expresses any chimeric antigen receptor described herein.

[0063] Furthermore, the immune effector cells include T cells, NK cells, B cells, macrophages, dendritic cells (DC), peripheral blood mononuclear cells (PBMC) and monocytes (MC).

[0064] The immune effector cells may also include (or be derived from) 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).

[0065] The immune effector cells can be autologous or non-autologous (allogeneic, syngeneic or xenogeneic).

[0066] Furthermore, the immune effector cells are autologous or allogeneic.

[0067] 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), CAR-monocytes (CAR-MC cells), CAR-iPSC cells and CAR-PSC cells but are not limited thereto.

[0068] Furthermore, the immune effector cells may be T cells.

[0069] The T cells may include CD8 + T cells, CD4 + T cells, regulatory T cells (Treg), initial T cells, effector T cells, memory T cells (including stem memory T cells, central memory T cells and effector memory T cells), αβT cells, γδT cells, natural killer T cells (Natural killer T cells, NKT cells) and mucosal-associated invariant T cells (Mucosal-associated invariant T cells, MAIT cells), etc., but not limited thereto.

[0070] Furthermore, the T cells may be derived from tumor patients or healthy volunteers.

[0071] Furthermore, the T cells may be derived from peripheral blood, monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue and tumor but are not limited thereto.

[0072] The T cells can be separated and obtained using a variety of techniques known to those skilled in the art (eg, density gradient separation technique, adhesion separation method, magnetic bead separation technique, flow cytometry sorting technique, etc.).

[0073] Furthermore, the modified immune effector cells may be CAR-T cells.

[0074] Furthermore, the CAR-T cells may be BCMA CD19 CAR-T cells.

[0075] The BCMA CD19 CAR-T cells are CAR-T cells obtained by transferring the gene of the chimeric antigen receptor constructed by the present invention into T cells for stable expression through a genetic engineering in vitro recombination method.

[0076] Furthermore, the BCMA CD19 CAR-T cells contain and / or express the DNA molecule shown in SEQ ID No.2.

[0077] The BCMA CD19 CAR-T cells may have at least one of the following characteristics:

[0078] (1) Secretion of T cell-specific effector molecule IFN-γ;

[0079] (2) Specific killing of BCMA and / or CD19-positive tumor cells;

[0080] (3) inhibiting the proliferation of BCMA and / or CD19-positive tumor cells;

[0081] (4) Inhibit the growth of BCMA and / or CD19 positive tumors.

[0082] The BCMA CD19 CAR-T cells include all classes and subclasses of CAR-modified T lymphocytes, including CAR-modified CD8 + T cells, CD4 + T cells, regulatory T cells (Treg), initial T cells, effector T cells, memory T cells (including stem memory T cells, central memory T cells and effector memory T cells), αβT cells, γδT cells, natural killer T cells (Natural killer Tcell, NKT cells) and mucosal-associated invariant T cells (Mucosal-associated invariant T cells, MAIT cells), etc. The proportion of each category and subclass in the BCMA CD19 CAR-T cells can be random or fixed.

[0083] The present invention also provides the use of any chimeric antigen receptor, biomaterial or modified immune effector cell described herein in the preparation of a product having any of the following functions:

[0084] C1) for preventing or treating B cell related diseases;

[0085] C2) for preventing or treating B cell-related autoimmune diseases or B cell-related tumors;

[0086] C3) for preventing or treating BCMA and / or CD19 target related diseases;

[0087] C4) is used to kill BCMA and / or CD19 positive tumor cells;

[0088] C5) is used to inhibit the proliferation of BCMA and / or CD19 positive tumor cells;

[0089] C6) is used to inhibit the growth of BCMA and / or CD19 positive tumors.

[0090] The B cell-related disease may be caused by B cell abnormalities, such as B cell overactivation, activation or proliferation.

[0091] In the above-mentioned use, the B cell-related autoimmune diseases include systemic lupus erythematosus (SLE), multiple sclerosis (MS), pemphigus vulgaris (PV), rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic sclerosis (SS), inflammatory bowel disease (IBD) and myasthenia gravis (MG).

[0092] In the above-mentioned use, the B cell-related tumors include B cell lymphoma, B cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL), follicular lymphoma (FL), transformed follicular lymphoma (tFL), marginal zone lymphoma (MZL), diffuse large B cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), B cell non-Hodgkin lymphoma (B-NHL), small B cell lymphoma (SLL), primary mediastinal large B cell lymphoma (PMBCL), gray zone lymphoma (GZL), primary central nervous system lymphoma (PCNSL) and multiple myeloma (MM).

[0093] In the above uses, the B cell-related tumors include early, middle, late, late, recurrent, metastatic and refractory tumors.

[0094] The present invention also provides a pharmaceutical composition comprising any of the chimeric antigen receptors, biomaterials or modified immune effector cells described herein, and one or more pharmaceutically acceptable carriers.

[0095] The pharmaceutical composition may have at least one of the following uses:

[0096] (1) For preventing or treating B cell-related diseases;

[0097] (2) For preventing or treating B cell-related autoimmune diseases or B cell-related tumors;

[0098] (3) Used to prevent or treat BCMA and / or CD19 target-related diseases;

[0099] (4) Used to kill BCMA and / or CD19-positive tumor cells;

[0100] (5) Used to inhibit the proliferation of BCMA and / or CD19-positive tumor cells;

[0101] (6) Used to inhibit the growth of BCMA and / or CD19 positive tumors.

[0102] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, cosolvents, diluents (such as water, saline, PBS (phosphate buffer), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78), pH adjusters, stabilizers (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactants (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption accelerators (such as chitosan), thickeners (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidants (such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, etc.), plasticizers (such as glycerol, sorbitol, phthalates, etc.), propellants (such as hydrofluoroalkanes, dimethyl ether, etc.), aerosolizers, suspending agents, dispersants, colorants (such as TiO2, pigments, etc.) and flavoring agents.

[0103] Excipients are usually used in medicines to shape the medicines, change the physical state of the medicines, and play a supporting role. Excipients include, but are not limited to: (1) Excipients for injections: such as solvent water, alcohols, ethers, amides, sulfoxides, esters, etc.; (2) Excipients for injection powders: such as sucrose, lactose, mannitol, etc.; (3) Excipients for sprays: such as soy lecithin, propylene glycol, borneol, ethanol, phenol, etc.; (4) Excipients for tablets: such as starch, sucrose, dextrin, methyl cellulose, gelatin, polyethylene glycol, tartaric acid, boric acid, etc.; (5) Excipients for eye drops: such as sodium hyaluronate, disodium ethylenediaminetetraacetic acid (EDTA-Na2), etc.; (6) Excipients for suppositories: such as cocoa butter, semi-synthetic or fully synthetic fatty acid glycerides, glycerol gelatin, polyethylene glycol, etc.; (7) Excipients for granules: such as corn flour, bentonite, zeolite powder, etc.; (8) Excipients for capsules: such as gelatin, etc.; (9) Excipients for gels: such as gelatin, pectin, gum arabic, etc.; (10) Excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soaps, higher fatty alcohols, fatty alcohol sulfates, polyols, polyethylene glycol, FAPG, etc.; (11) Excipients for patches: such as ethylene-vinyl acetate copolymer (EVA), pressure-sensitive adhesive (PSA), etc.; (12) Excipients for films: such as gelatin, shellac, gum arabic, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0104] Biological drug preparations are easily contaminated by exogenous microorganisms during production, transportation and storage, which may affect the quality of the drug. Therefore, preservatives are usually added to inhibit the growth and reproduction of microorganisms. The preservatives include but are not limited to thimerosal, formaldehyde, phenol and m-cresol.

[0105] Protective agents generally refer to any substance that can protect the activity of drugs, including freeze-drying protective agents. Freeze-drying protective agents can be used to change the physical and chemical environment of biological samples during freeze-drying, reduce cell damage, maintain original biological activity, improve the stability of proteins and cells, etc. The freeze-drying protective agents include but are not limited to sugars / polyols (such as sucrose, trehalose, lactose, glucose, maltose, maltodextrin, mannitol, sorbitol, etc.), polymers (such as HES, PVP, PEG, dextran, albumin, etc.), anhydrous solvents (such as polyethylene glycol, ethylene glycol, glycerol, DMSO, DMF, etc.), surfactants (such as Tween-80, etc.), amino acids (such as L-serine, sodium glutamate, alanine, glycine, sarcosine, acetyltryptophan, etc.), and salts and amines (such as phosphates, acetates, citrates, etc.).

[0106] The dosage form of the pharmaceutical composition includes, but is not limited to, injections (including injection solutions and powder injections for injection), gels, eye drops (including eye drops and intraocular injection solutions), oral solutions, suppositories, effervescent tablets, capsules, ointments, creams, sprays, aerosols, external solutions, tablets, powders, pills, granules, drops, paints, patches and long-acting sustained-release preparations.

[0107] Injections generally refer to solutions and emulsions for injection into the body made from extracted and purified medicinal materials, as well as sterile powders prepared into solutions before use, including injection solutions and powder injections for injection. The preparation method of injections is well known to those skilled in the art, for example, powder injections for injection can be prepared by vacuum freeze drying technology, spray drying technology, spray freeze drying technology. Injections can be prepared by preparing a solution with a drug and a solvent by a concentrated or diluted method, and then by filtering (such as surface filtration and / or deep filtration), encapsulation, sterilization and other steps.

[0108] In order to prepare the pharmaceutical composition into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, a commonly used diluent in the art, such as water, physiological saline, PBS (phosphate buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc., can be used as a solvent to prepare an injection. In addition, in order to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, a conventional cosolvent, buffer, pH adjuster and other carriers can also be added.

[0109] The administration methods of the pharmaceutical composition include, but are not limited to, injection (such as administration in the form of injection), mucosal administration (such as administration in the form of sprays, aerosols, tablets, eye drops, suppositories, granules, capsules, etc.) and transdermal administration (such as administration in the form of gels, ointments, patches, films, etc.).

[0110] The pharmaceutical composition may also include a cell freezing solution. The cell freezing solution is used to freeze cells to prevent ice crystals from forming during freezing and thawing to damage the cells. Its main components may include: dimethyl sulfoxide, sodium chloride, human albumin, dextran glucose, dextrose and / or plasma electrolyte-A.

[0111] The active ingredient of the pharmaceutical composition may be any modified immune effector cell described herein (such as BCM ACD19 CAR-T cell).

[0112] The present invention also provides a method for preparing any modified immune effector cell described herein, the method comprising: introducing a nucleic acid molecule encoding any chimeric antigen receptor described herein into an immune effector cell for expression to obtain the modified immune effector cell.

[0113] In the above method, the immune effector cells may be T cells.

[0114] Furthermore, the introduction method may include calcium phosphate co-precipitation, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e., electrotransfection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc., but is not limited thereto.

[0115] Furthermore, the modified immune effector cells may be CAR-T cells.

[0116] Furthermore, the method for preparing the CAR-T cells may include the following steps:

[0117] (1) cloning a nucleic acid molecule encoding any chimeric antigen receptor described herein into a viral vector to obtain a recombinant viral vector;

[0118] (2) introducing the recombinant viral vector into packaging cells for packaging to obtain a recombinant virus;

[0119] (3) Infecting (transfecting) human T cells with the recombinant virus to obtain the CAR-T cells.

[0120] Furthermore, the nucleic acid molecule may be a BCMA CD19 CAR-TK gene having a nucleotide sequence as shown in SEQ ID No.2.

[0121] Further, 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). Specifically, it may be a retrovirus, adenovirus, adeno-associated virus or lentivirus.

[0122] Furthermore, the viral vector may be a retroviral vector (such as retroviral vector MP71).

[0123] Further, the packaging cell is used to package a viral vector plasmid lacking at least one gene encoding a protein required for viral packaging, which can provide comprehensive viral proteins for viral packaging into recombinant viruses, and itself cannot produce any form of viral particles. Any cell that provides a protein or polypeptide 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 Palt E cells, 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.

[0124] In one or more embodiments of the present invention, the packaging cells are PG13 cells and Phoenix-ECO cells.

[0125] In the above preparation method, before step (1) or (2), a step of separating and extracting peripheral blood mononuclear cells (PBMC) and a step of separating and activating T cells may be included.

[0126] The mononuclear cells (PBMC) can be separated by using a density gradient centrifugation method known in the art to separate PBMC. This method utilizes the difference in cell specific gravity and separates cells by using a sucrose polymer (Ficoll) reagent.

[0127] The separation and activation of T cells can achieve effective enrichment of T cells and their subpopulations through CD3 / CD28 monoclonal antibodies coupled to magnetic beads. At the same time, CD3 is a surface marker of T cells, CD28 is a second signal for T cell activation, and CD3 / CD28 magnetic beads can also effectively activate T cells. The technology of separating and activating T cells by CD3 / CD28 magnetic beads is known in the art.

[0128] In the above preparation method, after step (3), the steps of expanding the culture and detecting the CAR-T cells may also be included.

[0129] The present invention also provides a kit for preparing CAR-T cells, the kit comprising any chimeric antigen receptor or biological material described herein.

[0130] Furthermore, the kit may also include one or more of the following reagents: lymphocyte separation fluid, CD3 / CD28 monoclonal antibody-coated magnetic beads (CD3 / CD28 magnetic beads), phosphate buffer (such as Dulbecco's phosphate buffer), cell culture medium, IL-2, IL-7, IL-15, IL-21, antibiotics, cell freezing solution and viral vector.

[0131] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0132] The components of the kit may be provided in the form of a solution, such as an aqueous solution. In the case of being in the form of an aqueous solution, the concentration or content of these components can be easily determined by those skilled in the art according to different needs. For example, for storage purposes, the concentration of the components may be present in a higher form, and when in working state or use, the concentration may be reduced to the working concentration by diluting the above-mentioned higher concentration solution.

[0133] The present invention also provides a method for preventing or treating a B cell-related disease, comprising administering any of the modified immune effector cells or the pharmaceutical composition described herein to a subject suffering from the B cell-related disease.

[0134] In the above method, the modified immune effector cell can be any T cell modified by the chimeric antigen receptor described herein.

[0135] Furthermore, the modified immune effector cells may be the BCMA CD19 CAR-T cells described in the present invention.

[0136] Furthermore, the BCMA CD19 CAR-T cells can be autologous or allogeneic.

[0137] Furthermore, the method may include the following steps:

[0138] (1) Isolating and extracting mononuclear cells (PBMC) from samples (such as peripheral blood) of subjects (including patients or healthy volunteers);

[0139] (2) isolating activated T cells from the PBMCs;

[0140] (3) Genetically modifying the T cells using any chimeric antigen receptor gene described in the present invention to obtain CAR-T cells;

[0141] (4) expanding the culture and quality testing of the CAR-T cells to obtain CAR-T cells of qualified quality;

[0142] (5) administering the qualified CAR-T cells to subjects suffering from B cell-related diseases.

[0143] Furthermore, the sample may be derived from peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue and tumor but is not limited thereto.

[0144] In the above method, the B cell-related disease may be a B cell-related autoimmune disease or a B cell-related tumor.

[0145] In the above method, the B cell-related tumors include B cell lymphoma, B cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL), follicular lymphoma (FL), transformed follicular lymphoma (tFL), marginal zone lymphoma (MZL), diffuse large B cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), B cell non-Hodgkin lymphoma (B-NHL), small B cell lymphoma (SLL), primary mediastinal large B cell lymphoma (PMBCL), gray zone lymphoma (GZL), primary central nervous system lymphoma (PCNSL) and multiple myeloma (MM).

[0146] In the above method, the B cell-related tumors include early, middle, late, late, recurrent, metastatic and refractory tumors.

[0147] In the above method, the B cell-related autoimmune diseases include systemic lupus erythematosus (SLE), multiple sclerosis (MS), pemphigus vulgaris (PV), rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic sclerosis (SS), inflammatory bowel disease (IBD) and myasthenia gravis (MG).

[0148] In the above method, the amount of administration may be a therapeutically effective amount. The therapeutically effective amount may refer to the amount of the drug used to (i) treat or prevent a specific disease, condition or disorder; (ii) alleviate, improve or eliminate one or more symptoms of a specific disease, condition or disorder; or (iii) prevent or delay 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.

[0149] Furthermore, the therapeutically effective amount described herein may be about (0.05-35)×10 6 pcs, (0.05~30)×10 6 pcs, (0.05~25)×10 6 pcs, (0.05~20)×10 6 pcs, (0.05~15)×10 6 pcs, (0.05~10)×10 6 pcs, (0.05~9)×10 6 pcs, (0.05~8)×10 6 pcs, (0.05~7)×10 6 pcs, (0.05~6)×10 6 pcs, (0.05~5)×10 6 pcs, (0.05~4)×106 pcs, (0.05~3)×10 6 pcs, (0.05~2)×10 6 pcs, (0.05~1)×10 6 pcs, (0.1~35)×10 6 pcs, (0.1~30)×10 6 pcs, (0.1~25)×10 6 pcs, (0.1~20)×10 6 pcs, (0.1~15)×10 6 pcs, (0.1~10)×10 6 pcs, (0.1~9)×10 6 pcs, (0.1~8)×10 6 pcs, (0.1~7)×10 6 pcs, (0.1~6)×10 6 pcs, (0.1~5)×10 6 pcs, (0.1~4)×10 6 pcs, (0.1~3)×10 6 pcs, (0.1~2)×10 6 pcs, (0.1~1)×10 6 pcs, (0.3~35)×10 6 pcs, (0.3~30)×10 6 pcs, (0.3~25)×10 6 pcs, (0.3~20)×10 6 pcs, (0.3~15)×10 6 pcs, (0.3~10)×10 6 pcs, (0.3~9)×10 6 pcs, (0.3~8)×10 6 pcs, (0.3~7)×10 6 pcs, (0.3~6)×10 6 pcs, (0.3~5)×10 6 pcs, (0.3~4)×10 6 pcs, (0.3~3)×10 6 pcs, (0.3~2)×10 6 pcs, (0.3~1)×10 6 pcs, (1~35)×10 6 pcs, (1~30)×10 6 pcs, (1~25)×10 6 pcs, (1~20)×10 6 pcs, (1~15)×10 6 pcs, (1~10)×10 6pcs, (1~9)×10 6 pcs, (1~8)×10 6 pcs, (1~7)×10 6 pcs, (1~6)×10 6 pcs, (1~5)×10 6 pcs, (1~4)×10 6 pcs, (1~3)×10 6 pcs, (1~2)×10 6 pcs, (2~35)×10 6 pcs, (2~30)×10 6 pcs, (2~25)×10 6 pcs, (2~20)×10 6 pcs, (2~15)×10 6 pcs, (2~10)×10 6 pcs, (2~9)×10 6 pcs, (2~8)×10 6 pcs, (2~7)×10 6 pcs, (2~6)×10 6 pcs, (2~5)×10 6 pcs, (2~4)×10 6 pcs, (2~3)×10 6 pcs, (4~35)×10 6 pcs, (4~30)×10 6 pcs, (4~25)×10 6 pcs, (4~20)×10 6 pcs, (4~15)×10 6 pcs, (4~10)×10 6 pcs, (4~9)×10 6 pcs, (4~8)×10 6 pcs, (4-7)×10 6 pcs, (4~6)×10 6 or (4-5)×10 6 The number of modified immune effector cells (such as the CAR-T cells of the present invention) is preferably (0.05-25)×10 per kg body weight. 6 or (1~10)×10 6 More preferably, (1 to 9) × 10 per kg body weight 6 pcs, (2~9)×10 6 pcs, (1~5)×10 6 or (2~5)×10 6 A modified immune effector cell (such as the CAR-T cell of the present invention).

[0150] Furthermore, the administration includes but is not limited to intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumor injection, mucosal administration, oral administration, oral and nasal spraying, aerosol inhalation, in vivo implantation and in vitro device administration.

[0151] The preferred mode of administration (route of administration) is parenteral (such as intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular) administration. It should be understood by the technician that the route of administration will change according to the intended purpose. In certain embodiments, the modified immune effector cells (such as CAR-T cells) or pharmaceutical compositions of the present invention can be administered by injection or continuous infusion (including but not limited to intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular and portal vein).

[0152] In some embodiments, the prevention or treatment methods provided by the present invention can also be used in combination with other therapies (including hematopoietic stem cell transplantation, surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, etc.), for example, a second therapeutic agent can be used for treatment, and the second therapeutic agent can be an immune checkpoint inhibitor (such as PD-1 inhibitor, CTLA-4 inhibitor), an immunomodulator (Immunomodulatory drugs, IMiDs) (such as lenalidomide and pomalidomide), immunosuppressants (such as methotrexate, cyclosporine, DMF and azathioprine), cytokines (such as recombinant human IL-12), anti-tumor drugs, anti-inflammatory drugs or other therapeutically active drugs, for example, may include but are not limited to decitabine, ibrutinib, gefitinib, imatinib, icotinib, sorafenib, pembrolizumab, infliximab, adalimumab, rituximab, etanercept, filgrastim, pegfilgrastim, epirubicin, capecitabine, cisplatin, carboplatin, gemcitabine, pemetrexed, paclitaxel, oxaliplatin and fluorouracil.

[0153] The modified immune effector cells (such as CAR-T cells) or pharmaceutical compositions of the present invention can be administered to the subject separately (administered simultaneously or substantially simultaneously via different routes), sequentially (administered at different times, the administration routes may be the same or different), or simultaneously (administered simultaneously or substantially simultaneously via the same route) with the second therapeutic agent.

[0154] The present invention also provides any of the following methods:

[0155] D1) Methods for inhibiting the growth of BCMA-positive tumors or killing BCMA-positive tumor cells;

[0156] D2) Methods for inhibiting CD19 positive tumor growth or killing CD19 positive tumor cells;

[0157] D3) Methods for inhibiting the growth of BCMA and CD19 positive tumors or killing BCMA and CD19 positive tumor cells;

[0158] The method comprises contacting a BCMA and / or CD19 positive tumor or tumor cell with any of the modified immune effector cells or the pharmaceutical composition described herein.

[0159] Furthermore, the method may be an in vitro method or an in vivo method.

[0160] The purposes of the uses and methods described in the present invention may be disease diagnosis, disease prognosis and / or disease treatment, or non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or non-disease diagnosis, non-disease prognosis and / or non-disease treatment.

[0161] The products described herein may include reagents, kits, preparations, drugs, and pharmaceutical compositions, among others.

[0162] The subject described herein may be a human or a non-human animal. In certain embodiments, the subject suffers from a B cell-related disease.

[0163] The non-human animal described herein may be a non-human mammal.

[0164] The non-human mammals described herein may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but are not limited thereto.

[0165] The CD19 and / or BCMA target-related diseases described herein include systemic lupus erythematosus (SLE), multiple sclerosis (MS), pemphigus vulgaris (PV), rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic sclerosis (SS), inflammatory bowel disease (IBD), myasthenia gravis (MG), B-cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell The main types of leukemia include primary mediastinal large B-cell lymphoma (MCL), follicular lymphoma (FL), transformed follicular lymphoma (tFL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), B-cell non-Hodgkin lymphoma (B-NHL), small B-cell lymphoma (SLL), primary mediastinal large B-cell lymphoma (PMBCL), gray zone lymphoma (GZL), primary central nervous system lymphoma (PCNSL), and multiple myeloma (MM).

[0166] CD19 is a transmembrane protein that regulates B cell activation in an antigen-receptor dependent manner. CD19 begins to be expressed in bone marrow B progenitor cells and continues throughout the B cell maturation period until it differentiates into plasma cells. Studies have shown that more than 95% of B cell tumors express CD19, and CD19 is an effective and widely used target for the treatment of B cell tumors.

[0167] B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily, also known as CD269 or TNFRSF17, and is a type III transmembrane protein. BCMA is highly expressed in mature B cells and plasma cells, but not in hematopoietic stem cells or non-blood cells, making it an ideal therapeutic target. In patients with multiple myeloma, BCMA is expressed at high levels on abnormal plasma cells.

[0168] After extensive and in-depth research, the inventors of this application have designed a bispecific chimeric antigen receptor (CAR) targeting CD19 and BCMA. The bispecific CAR adopts a dual epitope design, and in order to improve the safety of treatment, so that the genetically modified CAR-T cells can be quickly eliminated when adverse reactions occur, a safety switch (suicide gene) is also fused to the CAR molecule. Its structure is as follows Figure 1 As shown in B. The antigen binding domain of the bispecific CAR of the present invention is formed by connecting a single domain antibody (BCMA VHH) targeting BCMA and a single chain antibody (ScFV) (CD19-ScFV) targeting CD19 through a linker, which is mainly responsible for recognizing and specifically binding to tumor surface antigens CD19 and / or BCMA. The N-terminus of CAR carries a signal peptide for guiding the peptide chain of the antigen binding domain to transfer to the outside of the cell, thereby recognizing tumor cell surface antigens. The hinge region (Hinge) connects the extracellular antigen binding domain and the transmembrane region, which can promote the stable expression of CAR molecules on the surface of the T cell membrane, provide certain flexibility for the antigen binding domain, and avoid steric hindrance. The transmembrane region (TM) is used to anchor the antigen binding domain to the cell membrane. The intracellular signaling domain includes the costimulatory domain 4-1BB and the activation domain CD3ζ. CD3ζ and 4-1BB provide the first signal and the second signal for the activation of CAR-T cells, respectively, so that CAR-T cells can be fully activated after being stimulated by antigens to kill tumor cells.

[0169] The inventors infected T cells with the designed bispecific CAR through retrovirus and constructed CAR-T cells (BCMACD19 CAR-T cells) targeting CD19 and BCMA. Experiments have shown that the BCMA CD19 CAR-T cells of the present invention can well secrete the T cell-specific effector molecule IFN-γ, have high cytotoxicity, and have good killing ability against tumor cells. In addition, in vivo experiments have confirmed that BCMA CD19 CAR-T cells can significantly inhibit tumor growth, have good in vivo tumoricidal activity, and have a longer-lasting inhibitory effect. The BCMCD19 CAR-T cells of the present invention are safe and efficient, and can be widely used in the treatment of B cell-related autoimmune diseases and B cell-related tumors, with good clinical transformation value and broad application prospects.

[0170] Definition of terms

[0171] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0172] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a target gene. A typical expression cassette comprises a promoter, an MCS (multiple cloning site) and a terminator. The expression cassette may also include a target gene, a marker gene (such as a TK gene, a DHFR gene, a CAT gene and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette may be directly connected or indirectly connected via a linker.

[0173] The term "vector" generally refers to a vector that can carry foreign DNA or target gene into host cells for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into host cells by transformation, transduction or transfection, so that the genetic material elements it carries are amplified and / or expressed in the host cells. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the properties of the recipient cells. The vector includes, but is not limited to, plasmid, phage (e.g., lambda phage or M13 phage), cosmid, phagemid, shuttle vector (e.g., yeast expression vector), Ti plasmid, artificial chromosome (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC), or Ti plasmid artificial chromosome (TAC)), viral vector (e.g., baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (e.g., SV40), herpesvirus (e.g., herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including, but not limited to, promoter sequence, transcription initiation sequence, enhancer sequence, selection element, and reporter gene. In addition, the vector may also contain a replication initiation site.

[0174] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria may be from Escherichia sp. (such as Escherichia coli), Erwinia sp., Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium sp., Alcaligenes sp., Pseudomonas sp. and Bacillus sp. (such as Bacillus), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from Saccharomyces sp. (such as Saccharomyces cerevisiae, methanol yeast, Pichia pastoris), Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., and Cephalosporium sp., etc. The actinomycetes may be from Streptomyces sp. (such as Streptomyces). The algae may be from Cyanophyta (such as cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphoras sp., Ankistrodesmus sp., Asteromonas sp., and Boekelovia sp., etc.

[0175] The term "host cell" is also referred to as a recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as a plant cell and an animal cell. The host cell can be understood to refer not only to a specific recipient 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. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like plant cells but are not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), Chinese hamster ovary cell substrain (CHO-K1 cell), African green monkey kidney cell (Vero cell), SV40-transformed African green monkey kidney cell (COS cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc., but are not limited thereto.

[0176] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0177] The term "recombinant microorganism" generally refers to a recombinant microorganism with changed functions obtained by manipulating and modifying the genes of a target microorganism, such as introducing an exogenous target gene or a recombinant vector into the target microorganism, or directly editing the endogenous genes of the target microorganism.

[0178] The term "recombinant host cell" generally refers to a recombinant host cell whose function has been changed by manipulating and modifying the genes of the host cell, such as introducing an exogenous target gene or a recombinant vector into the host cell, or directly editing the endogenous genes of the host cell.

[0179] The term "link" generally 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 (joint).

[0180] The term "linker" may also be referred to as a connecting peptide, a peptide linker or a connector, and is used to fuse, couple, link or join two proteins or polypeptides to prevent steric hindrance. A linker is an amino acid chain that acts as a link between two fusion proteins, and has a certain flexibility to allow the proteins on both sides to perform their respective independent functions. It should be understood that the presence of a linker is optional, and the length of the flexible linker can also be adjusted to allow the fusion protein to fold correctly or achieve optimal biological activity. The characteristics of the linker and its suitability for a specific purpose are known in the art, and those skilled in the art can independently select and optimize each peptide linker.

[0181] The term "signal peptide" generally refers to a short peptide chain that guides the newly synthesized protein to the secretory pathway. The antigen binding region of the chimeric antigen receptor of the present invention may be preceded by a signal peptide (N-terminal). The signal peptide of the CAR structure can guide the antigen binding region peptide chain to transfer outside the cell, thereby recognizing tumor cell surface antigens.

[0182] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is generally expressed as a percentage. Identity as described herein may refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies with exactly the same sequence have 100% identity. It is known to those skilled in the art that the identity of an amino acid sequence or a nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST webpage of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existencecost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, then the value (%) of identity can be obtained. In addition, sequence analysis software (such as CLC MainWorkbench and MegAlignTM) can be used for determination, such as computer programs BLAST using default parameters, especially BLASTP or TBLASTN. The 75% or more identity described herein can be at least 75%, 80%, 85%, 90% or 95% or more identity. The 80% or more identity described herein can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity.

[0183] The term "conservative substitution" generally refers to replacing an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions generally do not cause significant changes in the conformational structure of the protein and do not substantially change the biological activity of the protein. Conservative substitutions in a protein sequence that are expected to have little or no effect on the protein structure or function can be easily designed by a person of ordinary skill in the art.

[0184] The term "single domain antibody (sdAb)" is also called nanobody, which usually refers to an antibody consisting of only one H chain V region, also known as VHH antibody. The ability of single domain antibodies to bind to antigens is extremely stable, which is basically the same as that of complete antibodies.

[0185] The term "single-chain antibody (ScFv)" generally refers to a light chain and a heavy chain variable region gene connected with an appropriate oligonucleotide linker to express a single polypeptide chain, which is called a single-chain antibody (ScFv). The polypeptide chain can spontaneously fold into a natural conformation, maintaining the specificity and affinity of Fv.

[0186] The term "introduction" generally refers to the transfer of foreign genes into recipient cells such as eukaryotic recipient cells or prokaryotic recipient cells. The method of introduction is not particularly limited, and any known transformation method can be used as long as it can transfer the target gene (such as the DNA molecule of the present invention) into the recipient cell. The method of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) directly transferring the target gene into plant receptor cells by physical or chemical methods, such as chemical stimulation, electric shock, liposome-mediated method, microinjection, gene gun method, laser microbeam method, pollen tube channel method, ultrasonic method, air gun method, and vortex method. (4) Using vectors as media to transfer target genes into plant recipient cells, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method. (5) Introducing target genes into ex vivo animal cells (transfection) by calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc. (6) Introducing target genes into in vivo animal cells by microinjection method, retrovirus vector method, somatic cell nuclear transfer method, sperm vector method or embryonic stem cell method, etc.

[0187] The term "CD19 positive tumor or tumor cell" generally refers to a tumor or tumor cell that expresses CD19 protein. Methods for detecting CD19 protein expression in tumors or tumor cells are well known to those skilled in the art. For example, the expression of CD19 protein can be determined by immunological detection methods based on antigen-antibody specific reactions (such as immunohistochemistry, FACS, etc.), or the expression of CD19 mRNA can be determined by real-time fluorescence quantitative PCR, Northern blotting or RNA in situ hybridization.

[0188] The term "tumor" generally refers to any new and abnormal growth, especially a new growth of tissue, in which cell growth is uncontrolled and progressive.

[0189] The term "B cell-related tumor" generally refers to a tumor caused by abnormal B cells (such as B cell overactivation, activation or proliferation, etc.).

[0190] The term "pharmaceutically acceptable carrier" generally refers to a substantially chemically inert and non-toxic component of a pharmaceutical composition or drug that does not inhibit the effectiveness and / or safety of the drug. Pharmaceutically acceptable carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0191] The term "prevention" generally refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom in a subject.

[0192] The term "treatment" generally refers to a method implemented to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, reduction in the extent of the disease, stabilization of the disease (i.e., no longer worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. In addition, treatment may also refer to prolonging the survival of the subject compared to the expected survival if the subject had not received treatment.

[0193] The term "cleavable linker (also referred to as a cleavable linker)" generally refers to a linker comprising a cleavage site so that it can be selectively cleaved when expressed to produce two or more products. The cleavable linker includes P2A, F2A, T2A and E2A, or any combination thereof, but is not limited thereto.

[0194] The term "chimeric antigen receptor (CAR)" generally refers to a transmembrane molecule encoded by an artificially constructed fusion gene, which can guide immune cells to specifically track, identify and remove tumor cells expressing relevant target ligands, and has a high degree of targeting. The basic structure of CAR can be divided into four parts, from the extracellular to the intracellular direction, which are the extracellular tumor antigen binding region (antigen binding domain), hinge region (hinge domain), transmembrane region (transmembrane domain) and intracellular signal region (intracellular signal domain) that can provide T cell activation signals. The method for preparing a chimeric antigen receptor and an immune effector cell (such as a T cell) comprising the chimeric antigen receptor is known in the art, and may include transfecting cells with at least one nucleic acid molecule (CAR gene) encoding CAR, and expressing the nucleic acid molecule in the cell. For example, the nucleic acid molecule encoding the CAR of the present invention can be constructed into an expression vector (such as a retroviral vector), and the expression vector can be expressed in a host cell such as a T cell to prepare the CAR.

[0195] The term "CAR cell" is also referred to herein as a modified immune effector cell, and generally refers to the introduction of a chimeric antigen receptor (CAR) gene (CAR gene) into an immune effector cell by molecular biology and genetic engineering techniques, and the expression of CAR on the surface of the immune effector cell, so as to obtain a class of genetically modified immune effector cells that specifically recognize and kill target cells. Relative to CAR-T cells, CAR cells are not limited to specific receptor cell types, and CAR 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), CAR-monocytes (CAR-MC cells), CAR-iPSC cells and CAR-PSC cells, but not limited thereto.

[0196] The term "CAR-T cells (chimeric antigen receptor T cells)" generally refers to T cells that express chimeric antigen receptors (CARs), which can specifically recognize and kill cells that express target antigens recognized by chimeric antigen receptors. CAR-T cells can be prepared by transferring the CAR gene into T cells in vitro using various vectors. CAR-T cells are activated after specifically recognizing tumor antigens through CAR molecules, and achieve specific killing of tumor cells by releasing perforin, granzyme B, cytokine IFN-γ, etc. The definition of CAR-T cells covers all categories and subclasses of T lymphocytes, including CD8 + T cells, CD4+ T cells, regulatory T cells (Treg), initial T cells, effector T cells, memory T cells (including stem memory T cells, central memory T cells and effector memory T cells), αβT cells, γδT cells, natural killer T cells (Natural killer T cell, NKT cells) and mucosal-associated invariant T cells (Mucosal-associated invariant Tcells, MAIT cells), etc.

[0197] The term "antigen binding region" is also called antigen binding domain, which usually refers to the part of the chimeric antigen receptor that specifically binds to the target antigen and is responsible for the recognition of tumor antigens. It can include 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. The antigen binding region may be preceded (N-terminal) by a signal peptide.

[0198] The term "hinge region (Hinge)" is also called the hinge domain, which generally refers to any oligopeptide or polypeptide used to connect the extracellular antigen binding region and the transmembrane region, and is a peptide chain of CAR located outside the cell. Selecting a suitable hinge region can increase the flexibility of the antigen recognition region and reduce the spatial constraints between the antigen and the CAR, thereby promoting the synaptic formation between CAR-T cells and target cells. 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.

[0199] The term "transmembrane domain (TD)" is also called a transmembrane domain, which is used to connect the antigen binding region and intracellular signaling region of the chimeric antigen receptor, and is mainly responsible for maintaining the stability of the CAR molecule on the T cell membrane. It can be natural or synthetic, or it can be derived from any membrane-bound protein or transmembrane protein, usually composed of homologous or heterodimeric membrane proteins. The transmembrane region of the chimeric antigen receptor of the present invention can be any protein structure known in the art, as long as it can be thermodynamically stable in the cell membrane (particularly eukaryotic cell membrane). The transmembrane region can be derived from the transmembrane domain of α, β or ζ chain, CD3ζ, CD8a (CD8α), CD4, CD9, CD45, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152 or CD154 of the T cell receptor.

[0200] The term "intracellular signaling region (also referred to as an intracellular signaling domain)" generally refers to the part of a chimeric antigen receptor that converts antigen stimulation into a T cell activation signal. 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, such as an intracellular signaling domain that may be derived from 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. 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.

[0201] The term "immune effector cell" generally refers to an immune cell that participates in an immune response and performs immune effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). The immune effector cells include T cells (e.g., CD8 + T cells, CD4 +T cells, regulatory T cells (Treg), initial T cells, effector T cells, memory T cells (including stem memory T cells, central memory T cells and effector memory T cells), αβT cells, γδT cells, natural killer T cells (Natural killer T cell, NKT cells) and mucosal-associated invariant T cells (Mucosal-associated invariant T cells, MAIT cells) etc.), NK cells, B cells, macrophages, dendritic cells (DC), peripheral blood mononuclear cells (PBMC) and monocytes (MC). Immune effector cells described herein 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, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSC), totipotent stem cells or hematopoietic stem cells (HSC). 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). The immune effector cells can be autologous or non-autologous (allogeneic, isogenic or xenogeneic), preferably autologous or allogeneic. In one embodiment of the invention, the immune effector cells are T cells. 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 isolated using a variety of techniques known to those skilled in the art (e.g., density gradient separation technology, adhesion separation method, magnetic bead separation technology, flow cytometry sorting technology, etc.).

[0202] The term "safety switch" generally refers to a "switch molecule" that can be "turned off" and / or "turned on" introduced into the CAR molecule in order to avoid the toxicity or side effects of cell therapy (such as cytokine release syndrome, etc.), so that the activation of CAR cells is controlled by the switch molecule, increasing the safety of CAR cell therapy. For example, CAR cells can be targeted to tumor cells by linker molecules (such as FITC or folic acid), that is, CAR cells do not directly target tumor antigens, but target linker molecules that can bind to tumor antigens. Only in the presence of linker molecules can CAR cells be activated by tumor cells expressing surface antigens; CAR cell activation can also be inhibited by introducing suicide genes. For example, in one or more embodiments of the present invention, a herpes simplex virus thymidine kinase (HSV-TK) suicide gene is introduced into CAR-T cells, and the expression of HSV-TK can be activated by the administration of ganciclovir, thereby inducing rapid apoptosis of CAR-T cells.

[0203] The term "suicide gene" usually refers to a gene that can cause the death of cells expressing suicide genes. These genes can be activated in the presence of specific drugs to induce apoptosis of CAR cells. For example, HSV-TK can convert non-toxic nucleotide analogs (such as ganciclovir) into metabolites that are toxic to cells; iCasp9 can bind to specific small molecule drugs to activate the caspase cascade in cells, leading to cell self-destruction; EGFRt polypeptides can eliminate T cells by administering anti-EGFR monoclonal antibodies (such as cetuximab); CD20 antigen, as a suicide gene, can be triggered by rituximab (anti-CD20 monoclonal antibody) to perform its function.

[0204] The term "autologous" generally refers to cells derived from the same subject.

[0205] The term "allogeneic" generally refers to cells of the same species that are genetically different from the cells being compared.

[0206] The term "isogenic" generally refers to cells from a different subject that are genetically identical to the cells being compared.

[0207] The term "xenogeneic" generally refers to cells that are of a different species than the cells being compared.

[0208] The term "comprising" is not intended to be limiting, but is intended to be inclusive and means that there may be other elements besides the listed elements, which can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figure 1 Schematic diagram of the BCMA CD19 CAR-TK molecular structure design. Figure 1 A in the middle is a schematic diagram of the structure of the CD19 CAR molecule used as a control; Figure 1 Figure B is a schematic diagram of the BCMA CD19 CAR-TK molecular structure design.

[0210] Figure 2 Detection of CAR expression in BCMA CD19 CAR-T cells.

[0211] Figure 3 Detection of IFN-γ molecule expression in BCMA CD19 CAR-T cells.

[0212] Figure 4 Detection of CD107a molecule expression on the membrane of BCMA CD19 CAR-T cells.

[0213] Figure 5To detect the killing effect of BCMA CD19 CAR-T cells in vivo. Figure 5 A in the middle is the experimental timeline of the B-NDG mouse model; Figure 5 Middle B is the bioluminescence imaging of B-NDG mice after CAR-T administration; Figure 5 C in the middle is the tumor quantitative analysis corresponding to Figure B. DETAILED DESCRIPTION

[0214] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0215] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0216] The retroviral vector MP71 in the following examples is described in the following document: Engels B, Cam H, et al. Retroviral Vectors for High-Level Transgene Expression in T Lymphocytes [J]. Human Gene Therapy, 2003, 14 (12): 1155-1168. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0217] The Raji cells in the following examples were derived from Kyowa Cell Bank, resource number: 4201HUM-CCTCC00191.

[0218] The human multiple myeloma cells RPMI-8226 in the following examples are derived from the Kyowa Cell Bank, resource number: 1101HUM-PUMC000083.

[0219] The Phoenix-ECO cells in the following examples are ATCC products with the number CRL-3214.

[0220] The PG13 cells in the following examples are ATCC products with the number CRL-3597.

[0221] The human peripheral blood mononuclear cells (PBMC) in the following examples were derived from the peripheral blood of healthy volunteers.

[0222] The RPMI-1640 culture medium in the following examples was purchased from Gibco, catalog number 11875093.

[0223] Example 1. Structure and sequence of chimeric antigen receptor

[0224] The chimeric antigen receptors (CARs) constructed in this example are named CD19CAR and BCMA CD19CAR-TK.

[0225] From the N-terminus to the C-terminus, CD19 CAR consists of the CD8 signal peptide, CD19-ScFV antibody, CD8a hinge region and transmembrane region, co-stimulatory domain 4-1BB and activation domain CD3ζ. The structural diagram is shown in Figure 1 As shown in A.

[0226] From N-terminus to C-terminus, BCMA CD19 CAR-TK consists of CD8 signal peptide (Signal), dual-epitope antibody (BCMA-VHH G4SlinkerCD19-ScFV), CD8a hinge region (Hinge) and transmembrane region, co-stimulatory domain 4-1BB, activation domain CD3ζ, cleavable linker P2A and safety switch HSV-TK. The structural diagram is shown in the figure. Figure 1 As shown in B.

[0227] The amino acid sequence of BCMA CD19 CAR-TK is shown in SEQ ID No.1. Among them: positions 1-21 of SEQ ID No.1 are CD8 signal peptide (Signal); positions 22-138 of SEQ ID No.1 are single domain antibodies targeting BCMA (BCMAVHH); positions 139-153 of SEQ ID No.1 are linkers (linker); positions 154-401 of SEQ ID No.1 are single chain antibodies (ScFV) targeting CD19 (CD19-ScFV); positions 402-470 of SEQ ID No.1 are CD8a hinge region (Hinge) and transmembrane region; positions 471-517 of SEQ ID No.1 are co-stimulatory domains 4-1BB; positions 518-629 of SEQ ID No.1 are activation domains CD3ζ; positions 630-651 of SEQ ID No.1 are cleavable linkers P2A; positions 652-1026 of SEQ ID No.1 are safety switches HSV-TK.

[0228] Positions 22-401 of SEQ ID No. 1 are dual-epitope antibodies (BCMA-VHH G4Slinker CD19-ScFV).

[0229] The nucleotide sequence of the gene encoding BCMA CD19 CAR-TK (BCMA CD19 CAR-TK gene) is shown in SEQ ID No. 2.

[0230] The amino acid sequence of CD19 CAR is shown in SEQ ID No.3, and the nucleotide sequence of the gene encoding CD19 CAR (CD19 CAR gene) is shown in SEQ ID No.4.

[0231] Example 2. Preparation of BCMA CD19 CAR-T cells and detection of CAR expression

[0232] This example prepares bispecific chimeric antigen receptor T cells that specifically target BCMA and CD19, named BCMA CD19 CAR-T cells. BCMA CD19 CAR-T cells are recombinant cells obtained by transferring the BCMA CD19 CAR-TK gene (SEQ ID No.2) into T cells for stable expression. At the same time, chimeric antigen receptor T cells targeting only CD19 (as a control) were also prepared, named CD19 CAR-T cells. CD19CAR-T cells are recombinant cells obtained by transferring the CD19 CAR gene (SEQ ID No.4) into T cells for stable expression. The specific steps are as follows:

[0233] 1. Construction of recombinant retroviral vector

[0234] (1) The recombinant vector pUC57-BCMA CD19 CAR-TK was double-digested with NotI (NEB) and EcoRI (NEB), and the target gene fragment was recovered by gel excision. The recombinant vector pUC57-BCMA CD19 CAR-TK is a recombinant vector obtained by cloning the BCMA CD19 CAR-TK gene (SEQ ID No. 2) into the pUC57 vector, and was synthesized and provided by Qingke Biotechnology Co., Ltd.

[0235] (2) The retroviral vector MP71 was double-digested with NotI and EcoRI, and the large vector fragment was recovered by gel excision.

[0236] (3) The target gene fragment and the large vector fragment were connected using T4 ligase (NEB) to obtain a recombinant retroviral vector carrying the BCM ACD19 CAR-TK gene, which was named MP71-BCMA CD19 CAR-TK.

[0237] (4) The recombinant retroviral vector MP71-BCMA CD19 CAR-TK was transformed into competent Escherichia coli DH5α, and the plasmid was extracted and purified using Qiagen's plasmid purification kit (purchased from Qiagen, catalog number 12362) to obtain MP71-BCMA CD19 CAR-TK plasmid.

[0238] The recombinant retroviral vector MP71-BCMA CD19 CAR-TK 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 whose nucleotide sequence is SEQ ID No. 2 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0239] The construction method of the recombinant retroviral vector MP71-CD19 CAR refers to the construction method of MP71-BCMA CD19 CAR-TK. The recombinant retroviral vector MP71-CD19 CAR 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 having a nucleotide sequence of SEQ ID No. 4 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0240] 2. Retroviral packaging

[0241] The MP71-BCMA CD19 CAR-TK plasmid and MP71-CD19 CAR 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:

[0242] a) Day 1: Phoenix-ECO cells should be less than 20 generations and not overgrown. 6 / mL cell density, add 10mL DMEM medium (10% Fetal Bovine Serum, 2mM L-glutamine, 1% Penicillin / Streptomycin) to a 10cm cell culture dish, mix the cells thoroughly, and culture at 37°C overnight;

[0243] b) Day 2: When the confluency of Phoenix-ECO cells reaches about 90%, transfection is performed (usually about 14-18 hours after plating). Chloroquine is added half an hour before transfection. Prepare 15μg of plasmid, 250μL of 1.5M CaCl2, and 1mL of H2O to a total volume of 1.25mL. Add 2×HBS in an equal volume to the plasmid complex, mix well, and leave at room temperature for 15 minutes. Then add the mixture to the Phoenix-ECO cell dish. Culture at 37℃ for 6 hours, remove the culture medium, wash once with PBS, and add 10mL of preheated fresh culture medium again.

[0244] c) Day 4: 48 hours after transfection, collect the supernatant, filter with a 0.45 μm filter to obtain the retroviral solution, and store it in aliquots at -80°C;

[0245] d) Add 1.2 mL of 15 ug / mL Retronectin coating solution to each well of a 6-well NTC plate and incubate at 4°C overnight;

[0246] e) Carefully remove the blocking solution, add 2 mL / well PBS for washing, add 5 mL of the above virus solution to each well, centrifuge at 32°C, 2000×g for 2 h, and discard the unbound virus supernatant;

[0247] f) Take logarithmic phase PG13 cells, wash them once with 10 mL PBS, add 1 mL 0.25% recombinant trypsin, and let stand at room temperature for 2-3 minutes;

[0248] g) Add 5 mL of complete medium containing 10% FBS to terminate digestion, and centrifuge at 1500 rpm for 5 min;

[0249] h) Discard the supernatant and adjust the cell density to 0.5×10 5 cell / mL, and add 3 mL / well to the NTC 6-well plate coated with the above virus to make the final cell number 1.5×10 5 cell / well;

[0250] i) culturing at 37°C, 5% CO2 for 48 h;

[0251] j) After 1-2 generations of subculture, the cells were transferred to T175 culture flasks and cultured in DEME medium containing 12% FBS for 2 days;

[0252] k) Replace with new DEME medium containing 12% FBS and continue culturing for 48 hours, then collect the supernatant, filter with a 0.45 μm filter to obtain a retrovirus solution, and store it in aliquots at -80°C.

[0253] 3. Retrovirus infection of human T cells

[0254] a) Thaw frozen healthy human peripheral blood PBMCs and adjust the cell density to 1×10 in RPMI-1640 medium containing 10% fetal bovine serum (FBS). 6 -2×10 6 cells / mL.

[0255] b) PBMCs were separated using Ficoll separation solution (Tianjin Haoyang, LTS1077-1) to obtain lymphocytes, and then CD3+T cells were separated using a magnetic bead method (Miltenyi, catalog number 130-097-043). Clinical-grade Dynabeads Human T ExpanderCD3 / CD28 magnetic beads (Invitrogen, catalog number: 11141D) were added to activate T cells at a ratio of magnetic beads:CD3+ cells = 3:1. T cells were cultured in a 24-well cell culture plate to obtain an activated T cell suspension.

[0256] c) RetroNectin (TAKARA, catalog number T100A) diluted with PBS to a final concentration of 15 μg / mL was coated on non-tissue treated culture plates, 1.2 mL per well of a 6-well plate. Protect from light and store at 4°C overnight for later use.

[0257] d) After two days of T cell activation culture, the coated 6-well plate was taken out, the coating solution was discarded, and PBS was added to wash the plate once.

[0258] e) Add 5-6 mL of the retrovirus 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.

[0259] f) After cell infection, the cell density was observed every day, and T cell culture medium containing 500 U / mL IL-2 was added in time to maintain the T cell density at 5×10 5 / mL, which is convenient for cell expansion.

[0260] g) CAR-T cells infected with the retrovirus prepared in step 2 were obtained and named BCMCD19 CAR-T cells (containing the recombinant vector MP71-BCMA CD19 CAR-TK) and CD19 CAR-T cells (containing the recombinant vector MP71-CD19 CAR).

[0261] The constructed CD19 CAR-T cells, BCMA CD19 CAR-T cells and No CAR-T cells (i.e., T cells without virus transfection, used as negative control) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and recorded as D0 day. Various functional tests were performed until D8 day.

[0262] 4. Detection of CAR expression in CD19 CAR-T cells and BCMA CD19 CAR-T cells

[0263] a) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1×PBS containing 2% FBS) to each well of a 96-well round bottom plate to resuspend the cells, and centrifuge at 1500 rpm for 5 min;

[0264] b) Add 60 μL of prepared fluorescently labeled anti-human CD8-PerCP-Cy5 (BD, 341049) and Anti-CD19 (FMC63)-FITC antibody (ACROBiosystems, FM3-FY45G0) to each well, resuspend and mix, and incubate at 4°C for 30 min;

[0265] c) Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min;

[0266] d) Discard the supernatant, resuspend the cells in 400 μL FACS buffer, transfer to a flow cytometer, and read the cells using a flow cytometer (BD Canto-II).

[0267] Test results such as Figure 2 As shown in the figure, CD19 CAR-T cells and BCMA CD19 CAR-T cells can express CD19 molecules well, while T cells without virus transduction (No CAR) do not express it, indicating that the expression of CAR molecules has reached the expected design.

[0268] Example 3: Functional detection of secreted effector molecule IFN-γ

[0269] 1. Adjust the cell density of the test cells CD19 CAR-T cells, BCMA CD19 CAR-T cells and No CAR T cells (i.e., T cells without virus transfection, as negative control) to 2×10 6 / mL, take 100 μL of each and add it to a 96-well U-bottom plate, add double-positive target cell line Raji cells (CD19+BCMA+) or single-positive cell line RPMI-8226 cells (CD19-BCMA+) at a ratio of test cells: target cells = 1:1, add Brefeldin A (Med ChemExpress, HY-16592) with a final concentration of 5 μg / mL to each well, and incubate in a 37°C incubator for 6 hours.

[0270] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0271] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1× PBS containing 2% FBS) to each well to resuspend the cells, centrifuge at 1500 rpm for 5 min, and repeat this step twice;

[0272] (2) Add 60 μL of prepared fluorescently labeled CD8-PerCP-Cy5 (BD, 341049) to each well, resuspend and mix, and incubate at room temperature for 10 min in the dark;

[0273] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0274] (4) Add 150 μL of Cytofix / Cytoperm (BD Biosciences, Catalog No. 55472) to each well, resuspend and mix, and incubate at room temperature for 15 min in a dark place.

[0275] (5) Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Catalog No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0276] (6) Add 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, Catalog No. 506510) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark;

[0277] (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 the cells.

[0278] Test results such as Figure 3 As shown in the figure, BCMA CD19 CAR-T cells co-cultured with Raji and RPMI-8226 target cells (targeting CD19 and / or BCMA antigens) can specifically and highly express the T cell effector molecule IFN-γ; while CD19 CAR-T cells only highly express IFN-γ when co-cultured with CD19-positive Raji cells, and do not produce IFN-γ when co-cultured with CD19-negative RPMI-8226 target cells, which is similar to the No CAR-T control.

[0279] Example 4. Detection of CD107a expression in BCMA CD19 CAR-T cells

[0280] CD107a is used as a marker for degranulation and effector cell lytic function. Based on CD107a labeling, T cell toxicity was detected by flow cytometry. The specific operation is as follows:

[0281] 1. The cell density of the test cells CD19 CAR-T cells, BCMA CD19 CAR-T cells and No CAR T cells (i.e., T cells without virus transfection, as negative control) was adjusted to 2×10 6 / mL, take 100μL and add it to 96-well U-bottom plate, add double positive target cell line Raji cells (CD19+BCMA+) or single positive cell line RPMI-8226 cells (CD19-BCMA+) at a ratio of test cells: target cells = 1: 1. Add 1μL APC anti-human CD107a antibody to each well and incubate in the incubator for 1h.

[0282] 2. Add 1 μL of Monensin solution (1 mg / mL) to each well and continue incubation for 3 hours. After incubation, perform flow cytometry staining. The steps are as follows:

[0283] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) to each well, resuspend, centrifuge at 1500 rpm for 5 min, and repeat this step twice;

[0284] (2) Add 60 μL of the prepared fluorescently labeled CD8 percp-cy5.5 to each well, resuspend and mix, and incubate at room temperature for 10 min in the dark;

[0285] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0286] (4) Resuspend the cells in 400 μL 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 CD107a in the cells.

[0287] Test results such as Figure 4As shown, BCMA CD19 CAR-T cells can specifically upregulate CD107a molecules (targeting CD19 and / or BCMA antigens) when co-cultured with Raji and RPMI-8226 target cells (both are BCMA-positive); while CD19 CAR-T cells can only specifically upregulate CD107a molecules (targeting CD19 antigen) when co-cultured with CD19-positive Raji cells, and express CD107a molecules at a low level when co-cultured with CD19-negative RPMI-8226 target cells, which is similar to the No CAR-T control.

[0288] Example 5. Detection of in vivo tumoricidal activity of BCMA CD19 CAR-T cells

[0289] 1. Cells and experimental animals

[0290] Cell line: Raji-Luc cells carrying the luciferase gene.

[0291] The construction method of Raji-Luc cells is as follows: luciferase gene (GenBank accession number: 116160065) was cloned into the vector pcDNA TM 3.1 (purchased from Invitrogen, catalog number V79020), a recombinant vector was obtained, and the recombinant vector was transfected into Raji cells to obtain Raji-Luc cells carrying the luciferase gene.

[0292] Experimental animals: NOD.CB17-PrkdcscidIl2rgtm1 / Bcgen mice (B-NDG, Biocytogen).

[0293] Animal information: SPF level, 7 to 8 weeks old when inoculated with cells, all female.

[0294] 2. Modeling and grouping

[0295] Take the Raji-Luc cells in the logarithmic growth phase and resuspend them in 1× PBS precooled at 4°C to 5.0×10 5 All mice were inoculated with 0.3 mL of cell suspension in the tail vein under sterile conditions. Five days after the mice were inoculated with Raji-Luc cells, 13 animals were randomly divided into three groups according to cell signal screening: Raji (No T, CTR) group (4 mice), CD19 CAR-T group (4 mice), and BCMA CD19 CAR-T group (5 mice). All animals were given a single injection through the tail vein, 3.0×10 6CAR-T cells / mouse, the day of administration was defined as D0. The cell bioluminescence signal was detected by IVIS Spectrum small animal imager on D7, D14, D21, D28, D35, and D43 after administration. The animals were euthanized on D43, and the survival of the mice was observed during this period.

[0296] The results are as follows Figure 5 As shown in the figure, during the experiment, compared with the CTR group, the BCMA CD19 CAR-T group and the CD19 CAR-T group had a significant inhibitory effect on tumor cell proliferation. At D28, there was no Raji cell growth in the BCMA CD19 CAR-T group, while some mice in the CD19CAR-T group began to relapse. At D28, all mice in the CTR group died, and at D43, all mice in the BCMA CD19 CAR-T group and the CD19CAR-T group survived.

[0297] In summary, both BCMA CD19 CAR-T and CD19 CAR-T cells can specifically recognize and eliminate the growth of CAR antigen-positive cells in the body, and dual-target BCMA CD19 CAR-T cells can kill BCMA and CD19-positive tumors at the same time, which can enhance the ability to resist heterogeneous tumor activity. For example, in the treatment of B cell-related autoimmune diseases, dual-target CAR-T cells can eliminate B cells and plasma cells that produce antibodies at different developmental stages. Therefore, they can more significantly inhibit tumor growth, kill tumors better and more completely, and have a longer-lasting inhibitory effect than CD19 CAR-T cells.

[0298] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A chimeric antigen receptor, characterized in that The chimeric antigen receptor includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain, the antigen binding domain includes a single domain antibody targeting BCMA and a single chain antibody targeting CD19, the amino acid sequence of the single domain antibody is shown in positions 22-138 of SEQ ID No.1, and the amino acid sequence of the single chain antibody is shown in positions 154-401 of SEQ ID No.

1.

2. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor comprises, from N-terminus to C-terminus, the antigen binding domain described in claim 1, the CD8a hinge region and the transmembrane region, the co-stimulatory domain 4-1BB and the activation domain CD3ζ.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that The chimeric antigen receptor further comprises a signal peptide at the N-terminus and a safety switch at the C-terminus, wherein the safety switch comprises a protein encoded by a suicide gene.

4. The chimeric antigen receptor according to claim 3, characterized in that The suicide genes include herpes simplex virus thymidine kinase, inducible caspase 9, truncated epidermal growth factor receptor, CD20, cytosine deaminase, RQR8, cytochrome P450, purine nucleoside phosphorylase and interleukin-1β converting enzyme genes.

5. The chimeric antigen receptor according to any one of claims 1 to 4, characterized in that The chimeric antigen receptor is any of the following: A1) a protein with an amino acid sequence as shown in SEQ ID No. 1; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.1; A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

6. Biomaterial, characterized in that The biological material is any of the following: B1) a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 5; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1); B4) a recombinant microorganism containing the nucleic acid molecule described in B1); B5) A recombinant host cell containing the nucleic acid molecule described in B1).

7. The biomaterial according to claim 6, characterized in that The nucleic acid molecule is a DNA molecule whose coding sequence or nucleotide sequence is shown as SEQ ID No.

2.

8. A modified immune effector cell, characterized in that The modified immune effector cell comprises the chimeric antigen receptor according to any one of claims 1 to 5 or the nucleic acid molecule according to claim 6 or 7.

9. The modified immune effector cell according to claim 8, characterized in that The immune effector cells include T cells, NK cells, B cells, macrophages, dendritic cells, peripheral blood mononuclear cells and monocytes.

10. The modified immune effector cell according to claim 8 or 9, characterized in that The immune effector cells are T cells.

11. Use of the chimeric antigen receptor according to any one of claims 1 to 5, the biomaterial according to claim 6 or 7, or the modified immune effector cell according to any one of claims 8 to 10 in the preparation of a product having any of the following functions: C1) for preventing or treating B cell related diseases; C2) for preventing or treating B cell-related autoimmune diseases or B cell-related tumors; C3) for preventing or treating BCMA and / or CD19 target related diseases; C4) is used to kill BCMA and / or CD19 positive tumor cells; C5) is used to inhibit the proliferation of BCMA and / or CD19 positive tumor cells; C6) is used to inhibit the growth of BCMA and / or CD19 positive tumors.

12. The use according to claim 11, characterized in that The B cell-related autoimmune diseases include systemic lupus erythematosus, multiple sclerosis, pemphigus vulgaris, rheumatoid arthritis, ankylosing spondylitis, systemic sclerosis, inflammatory bowel disease and myasthenia gravis.

13. The use according to claim 11, characterized in that The B cell-related tumors include B cell lymphoma, B cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, mantle cell lymphoma, follicular lymphoma, transformed follicular lymphoma, marginal zone lymphoma, diffuse large B cell lymphoma, non-Hodgkin lymphoma, B cell non-Hodgkin lymphoma, small B cell lymphoma, primary mediastinal large B cell lymphoma, gray zone lymphoma, primary central nervous system lymphoma and multiple myeloma.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimeric antigen receptor according to any one of claims 1 to 5, the biological material according to claim 6 or 7 or the modified immune effector cell according to any one of claims 8 to 10, and one or more pharmaceutically acceptable carriers.

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