CN108018299B

CN108018299B - Chimeric antigen receptor targeting BCMA and uses thereof

Info

Publication number: CN108018299B
Application number: CN201610932365.7A
Authority: CN
Inventors: 黄飞; 金涛; 王海鹰; 何凤; 史子啸
Original assignee: Shanghai Hrain Biotechnology Co Ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co.,Ltd.
Priority date: 2016-11-01
Filing date: 2016-11-01
Publication date: 2020-03-17
Anticipated expiration: 2036-11-01
Also published as: CN108018299A

Abstract

The invention relates to a chimeric antigen receptor targeting BCMA and application thereof, in particular to a polynucleotide sequence selected from (1) a polynucleotide sequence containing a coding sequence of an anti-BCMA single-chain antibody, a coding sequence of a human CD8 α hinge region, a coding sequence of a human CD8 transmembrane region, a coding sequence of a human 41BB intracellular region, a coding sequence of a human CD3 zeta intracellular region and an optional coding sequence of a fragment of EGFR containing an extracellular domain III and an extracellular domain IV, which are connected in sequence, and (2) (1) a complementary sequence of the polynucleotide sequence.

Description

Chimeric antigen receptor targeting BCMA and uses thereof

Technical Field

The invention belongs to the field of chimeric antigen receptors, and particularly relates to a BCMA (brain cell activating antigen) targeted chimeric antigen receptor and application thereof.

Background

Multiple myeloma is a malignant plasma cell disease, which is characterized by malignant clonal proliferation of bone marrow plasma cells, secretion of monoclonal immunoglobulin or a fragment thereof (M protein), and damage to relevant target organs or tissues such as bones and kidneys, and is commonly and clinically manifested by bone pain, anemia, renal insufficiency, infection and the like [ Multiple myelotoma.N Engl J Med,2011.364(11): p.1046-60 ]. Multiple myeloma is the second most serious malignancy in the blood system, accounting for 10% of the malignancy in the blood system, and is frequently developed in men, the incidence rate of which increases year by year with the increase of age, and the multiple myeloma is more likely to become younger in recent years [ Siegel, R., et al, Cancer statistics,2014.CA Cancer J Clin,2014.64(1): p.9-29 ].

The B Cell Maturation Antigen (BCMA), also called CD269, consists of 184 amino acid residues, the intracellular region of which contains 80 amino acid residues and has a very short sequence, and only one carbohydrate recognition domain is a B cell surface molecule. BCMA belongs to the type I transmembrane signal protein lacking a signal peptide, and is a member of the Tumor necrosis factor receptor family (TNFR), which binds to the B cell activator factor BAFF or proliferation-induced ligand (APRIL), respectively [ Tumor necrosis factor receptor ligand-receptor binding. current Opin Struct Biol,2004.14(2): p.154-60 ]. In normal tissues, BCMA is expressed on the surface of mature B cells and plasma cells, BCMA knockout mice have normal immune system, normal spleen structure, normal development of B lymphocytes but obviously reduced plasma cell number, and the BCMA plays an important role in maintaining the survival of plasma cells, and the mechanism mainly comprises the combination of BCMA and BAFF protein, and up-regulation of anti-apoptosis genes Bcl-2, Mcl-1, Bclw and the like, and the maintenance of cell growth [ BCMA is the important for the survival of the low-live bone marrow cells plasma. J Exp Med,2004.199(1): p.91-8 ]. Similarly, this mechanism also functions in myeloma cells and plays an important role in promoting malignant proliferation of myeloma cells [ BAFF and APRIL protected myelomas cells from apoptosis induced by interferon 6 depletion and demethasone. blood,2004.103(8): p.3148-57 ]. It has been shown that BCMA is ubiquitously expressed in multiple myeloma cell lines and that detection in multiple myeloma patients also gives consistent results [ Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival. blood,2004.103(2): p.689-94 ]. Based on the reports, Kochenderfer et al further applied Q-PCR, Flow Cytometry and immunohistochemistry to study the expression characteristics of BCMA, and confirmed that BCMA is not expressed in normal human tissues except mature B cells and plasma cells, and also not expressed in CD34+ hematopoietic cells [ B-cell growth inhibitor is a developmental target for additive T-cell therapy of multiple myocyte. Clin Cancer Res,2013.19(8): p.2048-60 ]. Combined with the high similarity of BCMA expression profiles to CD19, and the successful progress of anti-CD 19CAR T cell therapy, suggests that our BCMA can be used as one of the CAR-T cell targets for cellular immunotherapy of multiple myeloma.

Chimeric Antigen Receptor-T cell (CAR-T) T cell refers to a T cell that is genetically modified to recognize a specific Antigen of interest in an MHC non-limiting manner and to continuously activate expanded T cells. The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. CAR-T cell back-infusion therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of studies show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and remarkably improve the survival condition of patients.

Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a tumor-associated antigen (TAA) binding region (usually the scFV fragment from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.

With the continuous development of Chimeric Antigen Receptor T cell (CAR-T) technology, CAR-T can be divided into four generations.

The first generation CAR-T cells consist of an extracellular binding domain-single chain antibody (scFV), a transmembrane domain (TM), and an intracellular signaling domain-Immunoreceptor Tyrosine Activation Motif (ITAM), wherein the chimeric antigen receptor portions are linked as follows: scFv-TM-CD3 ζ. Although some specific cytotoxicity could be seen in the first generation CARs, it was found to be less effective when summarized in 2006 in clinical trials. The reason for this is because the first generation of CAR-T cells are rapidly depleted in the patient and have so poor persistence that CAR-T cells already apoptotic when they have not yet come into contact with a large number of tumor cells can elicit an anti-tumor cytotoxic effect, but rather less cytokine secretion, but their short survival time in vivo fails to elicit a persistent anti-tumor effect [ simple g2D-modified T cells inhibition system T-cell lymphoma growth in a mannenrinating multiple cytokines and cytotoxic pathways, Cancer 2007, 67 (22): 11029 vs 11036).

Optimization of T cell activation signaling regions in CAR design of second generation CAR-T cells remains a hotspot of research. Complete activation of T cells relies on dual signaling and cytokine action. Wherein the first signal is a specific signal initiated by the recognition of an antigen peptide-MHC complex on the surface of an antigen presenting cell by the TCR; the second signal is a co-stimulatory signal. Second generation CAR appeared as early as 1998 (J Immunol.1998; 161 (6): 2791-7). The 2 nd generation CAR adds a costimulatory molecule in the intracellular signal peptide region, namely the costimulatory signal is assembled into the CAR, and can better provide an activation signal for CAR-T cells, so that the CAR can simultaneously activate the costimulatory molecule and the intracellular signal after identifying tumor cells, double activation is realized, and the proliferation and secretion capacity of the T cells and the anti-tumor effect can be obviously improved. The first well-studied T cell costimulatory signal receptor was CD28, which was capable of binding to a B7 family member on the surface of target cells. Co-stimulation of CD28 promotes T cell proliferation, IL-2 synthesis and expression, and enhances T cell resistance to apoptosis. Costimulatory molecules such as CD134(OX40) and 41BB (4-1BB) are subsequently presented to increase cytotoxicity and proliferative activity of T cells, maintain T cell responses, prolong T cell survival, and the like. Such second generation CARs produced unexpected results in subsequent clinical trials, with shaking frequently triggered since 2010 based on clinical reports of second generation CARs, with complete remission rates of up to 90% and above, especially for relapsed, refractory ALL patients.

The third generation CAR signal peptide region integrates more than 2 costimulatory molecules, which can lead the T cells to continuously activate and proliferate, lead the cytokines to be continuously secreted, and lead the capability of the T cells to kill tumor cells to be more remarkable, namely, the new generation CAR can obtain stronger anti-tumor response (Mol ther., 2005, 12 (5): 933-941). Most typically, U Pen Carl June is added with a 41BB stimulating factor under the action of CD28 stimulating factor.

The fourth generation CAR-T cells are supplemented with cytokines or co-stimulatory ligands, for example the fourth generation CAR can produce IL-12, which can modulate the immune microenvironment-increase the activation of T cells, and simultaneously activate innate immune cells to act to eliminate target antigen negative cancer cells, thus achieving a bi-directional regulatory effect [ TRUCKs: the four generation of cars, Expert Opin Biol ther, 2015; 15(8): 1145-54 ].

One advantage of CAR-T cells is that they are active drugs, and once infused, physiological mechanisms regulate T cell balance, memory formation, and antigen-driven expansion. However, this treatment is not complete and T cells can miss the target and attack other tissues or expand too much beyond what is needed for treatment. Given that CAR-T cells have been included in the standard therapeutic range, it is very useful to design patient or drug-controlled turn-on or turn-off mechanisms to regulate the presence of CAR-T cells. For technical reasons, the shutdown mechanism is more easily applied to T cells. As one of them, the iCas9 system is under clinical study. When the cell expresses the iCas9, the small molecule compound can induce the iCas9 precursor molecule to form a dimer and activate an apoptosis pathway, thereby achieving the purpose of removing the cell. Small molecule AP1903 has been used to induce iCas9 dimers and clear T cells in graft versus host disease, demonstrating the feasibility of this approach (Making cutter clinical antibiotic candidates for adaptive T-cell therapy, Clin Cancer Res; 22(8), 2016, 15/4).

In addition, the clinical clearing antibody can be used to make CAR-T cells simultaneously express the protein against which these antibodies are directed, such as tEGFR, and after the treatment-related toxic reaction occurs or the treatment is completed, the corresponding CAR-T cells can be cleared by administration of antibody drugs (Rational resolution and characterization of humanized-EGFR variant III clinical anti-cancer receptors T cells for cytolethama, SciTransl Med 2015; 7: 275ra 22).

A successful treatment of 1 relapsing refractory Multiple Myeloma (MM) patient with CD19 molecular targeted CAR-T cell therapy was published by Carl June research group in New England journal, top-level journal of the medical community at 9.2015 [ Chimeric Antigen Receptor T Cells against CD19for Multiple Myeloma.N Engl JMed,2015.373(11): p.1040-7 ]. While multiple myeloma does not typically express CD19 as a B cell line tumor, CD19 is not a target for multiple myeloma immunotherapy. A trace of multiple myeloma clones with drug resistance and disease recurrence properties have been reported to have a B cell phenotype (i.e., CD19 positive). After it was clear that BCMA could be a target for CAR T cells, the american national cancer institute Kochenderfer et al successfully constructed anti-BCMA CAR T cells, and preclinical studies showed that the CAR T cells specifically recognized BCMA, and after activation by BCMA, amplified in large amounts, secreted cytokines and exerted killing function, and also had anti-tumor effect in mouse tumorigenesis models [ B-cell growth inhibition is infecting target for adaptive T-cell therapy of multiple myeloloma. clin cancer res,2013.19(8): p.2048-60 ]. Phase I clinical studies of anti-BCMA CAR T cell therapy for multiple myeloma were performed by the national cancer institute in 2014, and clinical safety and efficacy of anti-BCMA CAR T cells was validated against multiple myeloma patients that did not respond to current standard treatment protocols (clinical trials. gov Identifier: NCT 02215967). At 57 th American blood year date, beginning at 12 months of 2015, the professor team of Syed Abbas Ali, national cancer institute medical oncology, reported phase I clinical trial results for CAR-T cell therapy in multiple myeloma patients. The study included 12 patients with refractory relapsed multiple myeloma who failed over-line 3 chemotherapy, with some patients having more than 50% myeloma cells in their bone marrow. After infusion of BCMA CAR-T cells into these patients, 1 patient was completely relieved, 3 patients were partially relieved, and the rest were stable, thus demonstrating for the first time that anti-BCMA CAR-T cell therapy was effective in multiple myeloma without major side effects and was evaluated as one of the most influential clinical studies in the year of ASH (Late-Breaking extracts, Abstract number of university: LAB-1). Currently, the Abelmoscon cancer center, university of Abelmoschus, has also registered a phase I clinical trial of anti-BCMA CART cells for the treatment of multiple myeloma and was in the development of a Ting Rong Drum study (clinical trials. gov Identifier: NCT 02546167).

Disclosure of Invention

In a first aspect, the present invention provides a polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence comprising the coding sequence of an anti-BCMA single-chain antibody, the coding sequence of a human CD8 α hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3 zeta intracellular region, and optionally the coding sequence of a fragment of EGFR comprising extracellular domain III and extracellular domain IV, which are linked in sequence, and

(2) (1) the complement of the polynucleotide sequence.

In one or more embodiments, the polynucleotide sequence further comprises a coding sequence for a signal peptide before the coding sequence of the anti-BCMA single-chain antibody, in one or more embodiments, the amino acid sequence of the signal peptide is represented by amino acids 1-21 of SEQ ID NO. 2 in one or more embodiments, the amino acid sequence of the light chain variable region of the anti-BCMA single-chain antibody is represented by amino acids 22-132 of SEQ ID NO. 2 in one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-BCMA single-chain antibody is represented by amino acids 148-264 of SEQ ID NO. 2 in one or more embodiments, the amino acid sequence of the hinge region of human CD8 α is represented by amino acids 265-311 of SEQ ID NO. 2 in one or more embodiments, the amino acid sequence of the CSF hinge region is represented by amino acids 312-333 of SEQ ID NO. 2 in one or more embodiments, the amino acid sequence of the CSF polypeptide sequence of the CSF 8 is represented by amino acids 312-333 of the amino acids of SEQ ID NO. 2, the extracellular domain of the polypeptide coding sequence of the EGFR polypeptide, or the signal peptide of the extracellular domain of the polypeptide coding sequence of the EGFR NO. 25, the polypeptide sequence of the EGFR polypeptide GM NO. 35, the amino acids of the polypeptide sequence of SEQ ID NO. 10, the polypeptide sequence of the amino acids of the extracellular domain of the polypeptide coding sequence of the EGFR NO. 35, the polypeptide sequence of the polypeptide GM NO. 10, the polypeptide sequence of the polypeptide, the polypeptide GM NO. 10, the polypeptide sequence of the EGFR polypeptide, the polypeptide sequence of the EGFR polypeptide sequence of the polypeptide, the polypeptide sequence of the EGFR NO. 25, the polypeptide sequence of the polypeptide GM NO. 25, the polypeptide GM NO. 10, the polypeptide sequence of the polypeptide sequence of the polypeptide, the polypeptide sequence of the polypeptide III.

In one or more embodiments, the coding sequence of the signal peptide preceding the coding sequence of the anti-BCMA single-chain antibody is represented by the nucleotide sequence from position 1 to 63 of SEQ ID NO. 1, in one or more embodiments, the coding sequence of the light chain variable region of the anti-BCMA single-chain antibody is represented by the nucleotide sequence from position 64 to 396 of SEQ ID NO. 1, in one or more embodiments, the coding sequence of the heavy chain variable region of the anti-BCMA single-chain antibody is represented by the nucleotide sequence from position 442 to position 792 of SEQ ID NO. 1. in one or more embodiments, the coding sequence of the hinge region of human CD8 α is represented by the nucleotide sequence from position 793 ζ of SEQ ID NO. 1. in one or more embodiments, the coding sequence of the transmembrane region of human CD8 is represented by the nucleotide sequence from position 934-999 of SEQ ID NO. 1, the nucleotide sequence from position 999 of SEQ ID NO. 1 or the coding sequence of the intracellular domain of SEQ ID NO. 1 or the polynucleotide is represented by the nucleotide sequence from position 1144 to position 2624 of SEQ ID NO. 1, the amino acid sequence from position 2621 to position 15512 of SEQ ID NO. 1, or the polynucleotide is represented by the polynucleotide from position 1144 of SEQ ID NO. 1, the coding sequence of SEQ ID NO. 1, the polynucleotide as SEQ ID NO. 1, the coding sequence from position 1144, the coding sequence of SEQ ID NO. 1, the polynucleotide or the polynucleotide as SEQ ID NO. 1, the polynucleotide is represented by the polynucleotide as SEQ ID NO. 1, the amino acid sequence of SEQ ID NO. 1, in one or the polynucleotide as SEQ ID NO. 1, in one or the polynucleotide as SEQ ID NO. 1, in one or the polynucleotide 15512 or the polynucleotide as the amino acid sequence of SEQ ID NO. 1, in one or the polynucleotide as the amino acid sequence of SEQ ID NO. 1, in one or the polynucleotide as the amino acid sequence of SEQ ID NO. 1, the polynucleotide as the amino acid sequence of SEQ ID NO. 1, the polynucleotide of SEQ ID NO. 1, in one or the polynucleotide of SEQ.

In a second aspect, the invention provides a fusion protein selected from the group consisting of:

(1) a sequence encoding a fusion protein comprising an anti-BCMA single-chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human 41BB intracellular region and a human CD3 zeta intracellular region, which are linked in this order, and optionally a fragment of EGFR comprising ectodomain III and ectodomain IV, and

(2) a fusion protein derived from (1) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (1) and retaining the activity of activated T cells;

preferably, the anti-BCMA single chain antibody is the anti-BCMA monoclonal antibody c11d5.3.

In one or more embodiments, the fusion protein further comprises a signal peptide at the N-terminus of the anti-BCMA single chain antibody, in one or more embodiments, the amino acid sequence of the signal peptide is represented by amino acids 1-21 of SEQ ID NO. 2 in one or more embodiments, the amino acid sequence of the light chain variable region of the anti-BCMA single chain antibody is represented by amino acids 22-132 of SEQ ID NO. 1 in one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-BCMA single chain antibody is represented by amino acids 148-264 of SEQ ID NO. 1 in one or more embodiments, the amino acid sequence of the hinge region of human CD8 α is represented by amino acids 265-311 of SEQ ID NO. 1 in one or more embodiments, the amino acid sequence of the transmembrane domain of human CD8 is represented by amino acids 312-333 of SEQ ID NO. 1 in one or more embodiments, the extracellular domain of the polypeptide CSF 121, the extracellular domain of the polypeptide GM NO. 1, the polypeptide amino acids NO. 35, the amino acids of the extracellular domain of the polypeptide GM NO. 35, the polypeptide amino acids of the polypeptide GM NO. 35, the polypeptide amino acids of the amino acids 20, the polypeptide amino acids of the polypeptide GM NO. 11, the polypeptide amino acids 20, the polypeptide amino acids of the polypeptide GM NO. 11, the polypeptide amino acid sequence of the polypeptide GM NO. 11, the polypeptide GM NO. 1, the polypeptide amino acids 20, the polypeptide amino acid sequence of the polypeptide GM NO. 11, the polypeptide amino acid sequence of the polypeptide GM NO. 1, the polypeptide amino acid sequence of the polypeptide amino acids NO. 11, the polypeptide amino acid sequence of the polypeptide GM NO. 1, the polypeptide amino acid sequence of the polypeptide GM NO. 1, the polypeptide NO. 11, the polypeptide amino acid sequence of the polypeptide NO. 11, the polypeptide GM NO. 11, the polypeptide amino acid sequence of the polypeptide NO. 1, the polypeptide SEQ ID NO. 1, the polypeptide NO. 11.

In a third aspect, the invention provides a nucleic acid construct comprising a polynucleotide sequence as described herein.

In one or more embodiments, the nucleic acid construct is a vector. In one or more embodiments, the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR, a polynucleotide sequence described herein, and optionally a selectable marker.

In a fourth aspect, the invention provides a retrovirus containing a nucleic acid construct as described herein, preferably containing the vector, more preferably containing the retroviral vector.

In a fifth aspect, the invention provides a genetically modified T cell comprising a polynucleotide sequence as described herein, or comprising a nucleic acid construct as described herein, or infected with a retrovirus as described herein, or stably expressing a fusion protein as described herein and optionally a fragment of EGFR comprising extracellular domain III, extracellular domain IV and optionally a transmembrane region.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a genetically modified T cell as described herein.

In a seventh aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct or retrovirus as described herein in the preparation of an activated T cell.

In an eighth aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct, retrovirus, or genetically modified T cell as described herein, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a BCMA mediated disease.

In one or more embodiments, the BCMA-mediated disease is multiple myeloma.

Drawings

FIG. 1 is a schematic representation of a BCMA-CAR retroviral expression vector (BCMA-41 BBz). SP: a signal peptide; VL: a light chain variable region; and Lk: joint (G)₄S)₃VH is the heavy chain variable region, H is the CD8 α hinge region, TM is the CD8 transmembrane region.

FIG. 2 is a peak plot of the partial sequencing results of the BCMA-CAR retroviral expression vector (BCMA-41 BBz).

FIG. 3 is a schematic representation of the BCMA-tEGFR-CAR retroviral expression vector (BCMACAR-tEGFR). SP: a signal peptide; VL: a light chain variable region; and Lk: joint (G)₄S)₃VH is the heavy chain variable region, H is the CD8 α hinge region, TM is the CD8 transmembrane region, 2A is the P2A peptide.

FIG. 4 is a partial sequencing peak plot of pRetro-BCMA-tEGFR-CAR retroviral expression vector (BCMACAR-tEGFR).

FIG. 5 shows the BCMA-tEGFR-CAR + expression efficiency of retroviral infected T cells for 72 hours by flow cytometry.

Figure 6 shows target cell BCMA expression by flow cytometry.

FIG. 7 is a preparation of 5-day BCMA-tEGFR-CART cells co-cultured with target cells for 5 hours of CD107a expression.

FIG. 8 shows INF-gamma secretion from 5-day-old BCMA-tEGFR-CART cells co-cultured with target cells for 5 hours.

FIG. 9 shows the killing effect on tumor cells after 5 days of preparation of BCMA-tEGFR-CART cells and 20 hours of co-culture with target cells.

Detailed Description

The invention provides a Chimeric Antigen Receptor (CAR) targeting BCMA, the CAR comprising, in sequence, an anti-BCMA single chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human 41BB intracellular region, a human CD3 ζ intracellular region, and optionally, an extracellular domain III and extracellular domain IV-containing fragment of EGFR.

anti-BCMA single chain antibodies suitable for use in the present invention can be derived from a variety of anti-BCMA monoclonal antibodies known in the art.

Optionally, the light chain variable region and the heavy chain variable region may be linked together by a linker sequence. Such single chain antibodies that may be exemplified include, but are not limited to, C11D5.3, J22.9. In certain embodiments, the monoclonal antibody is a monoclonal antibody having clone number C11D5.3. In certain embodiments, the amino acid sequence of the variable region of the light chain of the anti-BCMA single chain antibody is represented by amino acid residues 22 to 132 of SEQ ID No. 2. In other embodiments, the amino acid sequence of the heavy chain variable region of the anti-BCMA single chain antibody is as shown in amino acid residues 148-264 of SEQ ID NO: 2.

The amino acid sequence of the human CD8 α hinge region suitable for use in the present invention can be shown as amino acids 265 and 311 of SEQ ID NO 2.

The human CD8 transmembrane region suitable for use in the present invention can be the various human CD8 transmembrane region sequences commonly used in the art for CARs. In certain embodiments, the amino acid sequence of the transmembrane region of human CD8 is depicted as amino acids 312-333 of SEQ ID NO 2.

The 41BB suitable for use in the present invention can be any of the various 41 BBs known in the art for use in CARs. As an illustrative example, the present invention uses the 41BB shown in the amino acid sequence at position 334-381 of SEQ ID NO. 2.

The intracellular domain of human CD3 ζ suitable for use in the present invention may be various intracellular domains of human CD3 ζ conventionally used in CARs in the art. In certain embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 382-492 of SEQ ID NO 2.

The above-mentioned portions forming the fusion protein of the present invention, such as the light chain variable region and the heavy chain variable region of an anti-BCMA single chain antibody, the human CD8 α hinge region, the human CD8 transmembrane region, 41BB, and the human CD3 zeta intracellular region, etc., may be directly linked to each other, or may be linked by a linker sequence, which may be a linker sequence known in the art, such as a linker sequence comprising G and S, typically, a linker comprises one or more motifs repeating back and forth, e.g., GGGS, GGGGS, SSSGS, GSGSA, and GGSGG, preferably, the motifs are adjacent in the linker sequence without intervening amino acid residues between the repeats, the linker sequence may comprise 1, 2,3, 4, or 5 repeating motifs, the length of the linker may be 3 to 25 amino acid residues, e.g., 3 to 15, 5 to 15, 10 to 20 amino acid residues, in some embodiments, the linker sequence is a polyglycine linker sequence, the number of glycine residues in the linker sequence is not particularly limited, typically 2, 2 to 2 amino acid residues, e.g., 15 to 20 amino acid residues in the linker sequence, e.g., 2 to 15 amino acid residues in the linker sequence, e.g., 15 amino acid residues in the amino acid sequence (amino acid sequence), and the amino acid sequence of the heavy chain variable region of the linker sequence of the present invention, e.g., preferably, L, the amino acid sequence of the present invention, and the heavy chain variable region of the present invention may comprise amino acid sequence of the present invention (e.g., preferably, E_nAnd (b) connecting, wherein n is an integer of 1-5.

In certain embodiments, the amino acid sequence of a CAR of the invention is as set forth in amino acids 22-492 of SEQ ID NO 2 or as set forth in amino acids 1-492 of SEQ ID NO 2. In certain embodiments, the CAR of the invention further comprises within its amino acid sequence extracellular domain III and extracellular domain IV-containing fragments of EGFR, as described below, signal peptides thereof, and linker sequences.

It will be appreciated that in gene cloning procedures it is often necessary to design appropriate cleavage sites which will introduce one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, facilitate expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of a host cell, or facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-terminus or the carboxy-terminus of the fusion protein of the invention (i.e., the CAR) may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty 1. These tags can be used to purify proteins.

The invention also includes a CAR represented by the amino acid sequence at positions 24-495 of SEQ ID NO. 2, a CAR represented by the amino acid sequence at positions 24-878 of SEQ ID NO. 2, a CAR represented by the amino acid sequence at positions 1-495 of SEQ ID NO. 2, or a mutant of the CAR represented by SEQ ID NO. 2. These mutants include: an amino acid sequence that has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retains the biological activity (e.g., activating T cells) of the CAR. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI.

Mutants also include amino acid sequences as shown at positions 22-492 of SEQ ID NO. 2, amino acid sequences as shown at positions 22-874 of SEQ ID NO. 2, amino acid sequences as shown at positions 1-492 of SEQ ID NO. 2, or amino acid sequences as shown in SEQ ID NO. 2 that have one or several mutations (insertions, deletions, or substitutions) in the amino acid sequences as shown at positions 22-874 of the CAR, while still retaining the biological activity of the CAR.

The present invention includes polynucleotide sequences encoding the fusion proteins of the present invention. The polynucleotide sequences of the invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e., nucleotide sequences which encode the same amino acid sequence but differ in nucleotide sequence.

The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding the fusion proteins described herein is as set forth in nucleotides 64 to 1476 of SEQ ID NO. 1, or as set forth in nucleotides 1 to 1476 of SEQ ID NO. 1.

In certain embodiments, the polynucleotide sequences of the invention further comprise nucleotide sequences encoding fragments of EGFR.

The EGFR suitable for use in the present invention may be an EGFR known in the art, e.g., from human. EGFR contains N-terminal extracellular domains I and II, extracellular domain III, extracellular domain IV, transmembrane, juxtamembrane domain and tyrosine kinase domain. The present invention preferably uses a truncated EGFR ("tfegfr", i.e., a fragment of EGFR as described herein), particularly a truncated EGFR that does not include its intracellular regions (membrane proximal domain and tyrosine kinase domain). In certain embodiments, EGFR that does not include an intracellular region may be further truncated to include no extracellular domains I and II. Thus, in certain embodiments, the EGFR used in the present invention contains or consists of the extracellular domain III, the extracellular domain IV and the transmembrane region of EGFR. In certain embodiments, the tEGFR comprises or consists of the amino acid sequence at positions 310 and 646 of the human EGFR, wherein the amino acid sequence at positions 310 and 480 is the extracellular domain III of the human EGFR, the amino acid sequence at positions 481 and 620 is the extracellular domain IV of the human EGFR, and the amino acid sequence at positions 621 and 646 is the transmembrane region of the human EGFR. In certain embodiments, the extracellular domains III and IV of the amino acid sequence of tEGFR have the amino acid sequences as shown in amino acids 518-539 of SEQ ID NO 2

In certain embodiments, the invention uses a signal peptide from the GM-CSF receptor ("GMCSFR") α chain, in certain embodiments, the amino acid sequence of the signal peptide is as set forth in SEQ ID NO:2, amino acids 522-543.

In addition, the signal peptide and the coding sequence for tEGFR can be linked to the coding sequence for the intracellular domain of human CD3 ζ in the CAR of the invention by the coding sequence for the P2A polypeptide. In one or more embodiments, the amino acid sequence of the P2A peptide is depicted as amino acids 493 521 of SEQ ID NO 2.

Thus, in certain embodiments, the polynucleotide sequence of the invention comprises a coding sequence for a CAR of the invention, a coding sequence for a P2A polypeptide, a coding sequence for a signal peptide from the chain of GM-CSF receptor α, and a coding sequence for tEGFR.

The invention also relates to nucleic acid constructs comprising the polynucleotide sequences described herein, and one or more control sequences operably linked to these sequences. The polynucleotide sequences of the invention can be manipulated in a variety of ways to ensure expression of the fusion proteins (CAR and/or tfegfr). The nucleic acid construct may be manipulated prior to insertion into the vector, depending on the type of expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

In certain embodiments, the nucleic acid construct is a vector. Expression of a polynucleotide sequence of the invention is typically achieved by operably linking the polynucleotide sequence to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The polynucleotide sequences of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

For example, in certain embodiments, the invention uses a retroviral vector that contains a replication initiation site, a 3 'LTR, a 5' LTR, polynucleotide sequences described herein, and optionally a selectable marker.

Another example of a suitable promoter is the extended growth factor-1 α (EF-1 α). however, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40(SV40) early promoter, mouse breast cancer virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoLV promoter, the avian leukemia virus promoter, the EB virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein.

Methods for introducing and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell by any method known in the art, for example, mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly retroviral vectors, which have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Thus, in certain embodiments, the invention also provides a retrovirus for activating T cells, the virus comprising a retroviral vector as described herein and corresponding packaging genes, such as gag, pol and vsvg.

T cells suitable for use in the present invention may be of various types from various sources. For example, T cells may be derived from PBMCs of B cell malignancy patients.

In certain embodiments, after T cells are obtained, activation may be stimulated with an appropriate amount (e.g., 30-80 ng/ml, such as 50ng/ml) of CD3 antibody prior to culturing in an appropriate amount (e.g., 30-80 IU/ml, such as 50IU/ml) of IL2 medium for use.

Thus, in certain embodiments, the invention provides a genetically modified T cell comprising a polynucleotide sequence as described herein, or comprising a retroviral vector as described herein, or infected with a retrovirus as described herein, or prepared by a method as described herein, or stably expressing a fusion protein as described herein and optionally a tfegfr.

The CAR-T cells of the invention can undergo robust in vivo T cell expansion and sustained at high levels in the blood and bone marrow for extended amounts of time, and form specific memory T cells. Without wishing to be bound by any particular theory, the CAR-T cells of the invention can differentiate into a central memory-like state in vivo upon encountering and subsequently depleting target cells expressing a surrogate antigen.

The invention also includes a class of cell therapies in which T cells are genetically modified to express a CAR and optionally a tfegfr as described herein, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing tumor cells of the recipient. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

The anti-tumor immune response elicited by the CAR-T cells can be an active or passive immune response. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells induce an immune response specific for the antigen-binding portion in the CAR.

Thus, the diseases that can be treated with the CARs, their coding sequences, nucleic acid constructs, expression vectors, viruses, and CAR-T cells of the invention are preferably BCMA-mediated diseases.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as relevant cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise CAR-T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Dosage of individual cells/kg body weight. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressive agents. For example, treatment may be in conjunction with radiation or chemotherapeutic agents known in the art for the treatment of BCMA mediated diseases.

Herein, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

"patient," "subject," "individual," and the like are used interchangeably herein and refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

The invention uses the gene sequence of anti-BCMA antibody (specifically scFV derived from clone number C11D5.3) and searches the gene sequence information of human CD8 α hinge region, human CD8 transmembrane region, human 41BB intracellular region and human CD3 zeta intracellular region from NCBI GenBank database, the whole gene synthesizes the gene fragment of chimeric antigen receptor anti-BCMA scFv-CD8 hinge region-CD 8TM-41BB-CD3 zeta and anti-BCMA-CD 8 hinge region-CD 8TM-41BB-CD3 zeta-GMCSFR leader sequence-tEGFR, which is inserted into a retroviral vector, recombinant plasmid packages virus in 293T cells, infects T cells, making T cells express the chimeric antigen receptor, the invention realizes the transformation of chimeric antigen receptor gene modified T lymphocytes, the invention realizes the transformation method of chimeric antigen receptor gene modified T lymphocytes, which is based on the retrovirus transformation method, which has high transformation efficiency, can stably express foreign gene, and can reach the amount of T cells cultured in vitro, the T cells, the invention can be used as a safe transfer marker of anti-BCMA antibody, the invention can be used as a safe transfer marker, the anti-T lymphocyte, the invention can be used for the anti-T lymphocyte, the anti-EGFR-lymphocyte, the invention can be used for the detection of the anti-BCMA antibody, the anti-BCMA antibody-CD receptor gene-T cell can be used for the invention, the invention can be used for the detection of the invention, the invention can be used for the invention, the invention can be used for the detection of the invention, the invention of the.

The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Example 1 determination of the sequence of the BCMA-scFv-CD8 α -CD28-41BB-CD3 zeta Gene

The sequence information of human CD8 α hinge region, human CD8 α transmembrane region, 41BB intracellular region and human CD3 zeta intracellular region gene is searched from NCBI website database, the cloning number of the anti-BCMA single-chain antibody is C11D5.3, and the sequences are subjected to codon optimization on website http:// sg.

Connecting the sequences by adopting overlapping PCR according to the sequences of anti-BCMA scFv, a human CD8 α hinge region gene, a human CD8 α transmembrane region gene, a 41BB intracellular region gene and a human CD3 zeta intracellular region gene in sequence, and introducing different enzyme cutting sites at the connection positions of the sequences to form complete BCMA-CAR gene sequence information.

The nucleotide sequence of the CAR molecule was double-digested with NotI (NEB) and EcoRI (NEB), inserted into the NotI-EcoRI site of the retrovirus MSCV (Addgene) by T4 ligase (NEB) and transformed into competent E.coli (DH5 α).

The recombinant plasmid is sent to Shanghai Biotechnology Limited company for sequencing, and the sequencing result is compared with the fitted BCMACAR sequence to verify whether the sequence is correct. The sequencing primer is as follows:

and (3) sense: AGCATCGTTCTGTGTTGTCTC (SEQ ID NO: 3);

antisense: TGTTTGTCTTGTGGCAATACAC (SEQ ID NO: 4).

After the sequencing is correct, plasmids are extracted and purified by using a plasmid purification kit of Qiagen company, and 293T cells are transfected by a plasmid calcium phosphate method for purifying the plasmids to carry out a retrovirus packaging experiment.

The plasmid map constructed in this example is shown in FIG. 1. FIG. 2 shows a partial sequencing peak plot of the retroviral expression plasmid.

Example 2: determination of BCMACAR-GMCSFR leader sequence-tEGFR Gene sequence

The gene sequence information of the human EGFR extracellular region is searched from an NCBI website database, and the sequence is subjected to codon optimization on a website http:// sg. idtdna. com/site, so that the coding sequence is more suitable for human cell expression under the condition of unchanging an encoding amino acid sequence.

The sequences are connected in sequence by adopting overlapping PCR according to the BCMACAR, GMCSFR leader sequence and tEGFR of the embodiment 1, and different enzyme cutting sites are introduced at the connection positions of the sequences to form complete BCMACAR-GMCSFR leader sequence-tEGFR gene sequence information.

The recombinant plasmid is sent to Shanghai Biotechnology Limited company for sequencing, and the sequencing result is compared with the sequence of the synthesized BCMACAR-GMCSFR leader sequence-tEGFR to verify whether the sequence is correct. The sequencing primer is as follows:

and (3) sense: AGCATCGTTCTGTGTTGTCTC (SEQ ID NO: 5);

antisense: TGTTTGTCTTGTGGCAATACAC (SEQ ID NO: 6).

The plasmid map constructed in this example is shown in FIG. 3. FIG. 4 shows a partial sequencing peak plot of the retroviral expression plasmid.

Example 3: retroviral packaging

1. Day 1: 293T cells should be less than 20 passages, but not overgrown.At 0.6X 10⁶Plating cells/ml, adding 10ml of DMEM medium into a 10cm dish, fully and uniformly mixing the cells, and culturing at 37 ℃ overnight;

2. day 2: the 293T cell fusion degree reaches about 90%, and transfection is carried out (generally, the plate laying time is about 14-18 h); preparing plasmid complex, wherein the amount of each plasmid is 12.5ug of MSCV skeleton, 10ug of Gag-pol, 6.25ug of VSVg, and CaCl₂250ul，H₂O1 ml, the total volume is 1.25 ml; in another tube, an equal volume of HBS to plasmid complex was added, and the plasmid complex was vortexed for 20 seconds. The mixture was gently added to 293T dishes, incubated at 37 ℃ for 4h, medium removed, washed once with PBS, and re-added to the pre-warmed fresh medium.

3. Day 4: after transfection for 48h, the supernatant was collected, filtered through a 0.45um filter, dispensed and stored at-80 ℃, and preheated fresh DMEM medium was added continuously.

Example 4: retroviral infection of human T cells

1. Separating with Ficcol separation solution (tertiary sea of Tianjin) to obtain relatively pure CD3+ T cells, and adjusting cell density to 1 × 10 with medium containing 5% AB serum X-VIVO (LONZA)⁶and/mL. The cells were inoculated at 1 ml/well with anti-human 50ng/ml CD3 antibody (Beijing Hokkimeiyuan) and 50ng/ml CD28 antibody (Beijing Hokkimeiyuan), and 100IU/ml interleukin 2 (Beijing Erlu) was added to stimulate and culture for 48 hours, and then infected with the virus prepared in example 3;

2. every other day after T cell activation culture, the plates were plated in 24-well plates with 250. mu.l/well in Retronectin (Takara) coated non-tissue-treated plates diluted in PBS to a final concentration of 15. mu.g/ml. Protected from light and kept at 4 ℃ overnight for use.

3. After two days of T cell activation culture, 2 coated 24-well plates were removed, the coating solution was aspirated away, and HBSS containing 2% BSA was added and blocked at room temperature for 30 min. The volume of blocking solution was 500. mu.l per well, and the blocking solution was aspirated and the plate washed twice with HBSS containing 2.5% HEPES.

4. The virus solution prepared in example 3 was added to wells 2ml of virus solution per well, centrifuged at 32 ℃ and 2000g for 2 h.

5. The supernatant was discarded, and activated T cells were added to each well of a 24-well plate at 1X 10⁶The volume is 1ml, and the culture medium is T cell culture medium added with IL-2200 IU/ml. Centrifuge at 30 ℃ for 10min at 1000 g.

6. After centrifugation, the plates were placed at 37 ℃ in 5% CO₂Culturing in an incubator.

7. 24h after infection, the cell suspension was aspirated and centrifuged at 1200rpm, 4 ℃ for 7 min.

8. After the cells are infected, the density of the cells is observed every day, and a T cell culture solution containing IL-2100 IU/ml is supplemented at a proper time to maintain the density of the T cells at 5 x 10⁵Cells were expanded at around/ml.

Thereby, CART cells infected with the retroviruses shown in example 3, respectively, were obtained, and named BCMA CART cells (expressing BCMACAR of example 1) and BCMA-tfegfr CART cells (expressing BCMACAR and tfgfr of example 2), respectively.

Example 5: flow cytometry for detecting proportion of infected T lymphocytes and expression of surface CAR protein

The CAR-T cells and NT cells (control) prepared in example 4 were collected by centrifugation 72 hours after infection, washed 1 time with PBS, the supernatant was discarded, the corresponding antibody was added and washed with PBS 30min in the dark, resuspended, and finally detected by flow cytometry. CAR + was detected by anti-mouse IgG F (ab') antibody (Jackson Immunoresearch).

FIG. 5 shows that the expression efficiency of BCMA-tEGFRCRAR + was 24.51% 72 hours after T cells were infected with the retrovirus prepared in example 3.

Fig. 6 shows that the percentage of BCMA in the target cells, which was detected by BCMA antibody, was 95.5% in U266 cells, demonstrating that the target cells are highly expressing BCMA.

Example 6: detection of CD107a expression following coculture of CAR-T cells with target cells

1. A V-bottom 96-well plate was prepared, 2X 105 CART/NT cells and 2X 105 target (U266)/control (K562) cells were added to each well, resuspended in 100ul of IL-2-free X-VIVO complete medium, BD GolgiStop (containing monesin, 1. mu.l BD GolgiStop per 1ml of medium) was added, 2ul CD107a antibody (1:50) was added to each well, and the cells were incubated at 37 ℃ for 4 hours to harvest.

2. The samples were centrifuged to remove the medium, washed once with PBS, 400g, and centrifuged at 4 ℃ for 5 minutes. The supernatant was discarded, and appropriate amounts of specific surface antibodies CD3, CD4, and CD8 were added to each tube, and the volume of the suspension was 100ul, followed by incubation for 30 minutes on ice in the absence of light.

3. Cells were washed 1 time with 3mL PBS per tube and centrifuged at 400g for 5 min. The supernatant was carefully aspirated.

4. The appropriate amount of PBS was resuspended and CD107a was detected by flow cytometry.

Shown in fig. 7. Fig. 7 shows that the percentage of CD107a secretion by BCMA-tfegfr CART cells in CD8 positive U266 cells was 35.9%, respectively, and the percentage of CD107a secretion by BCMA-tffr cells in CD4 positive U266 cells was 27.8%, respectively.

Example 7: INF-gamma secretion assay after co-culture of CAR-T cells with target cells

1. Prepared CAR-T cells were taken, resuspended in Lonza medium, and the cell concentration was adjusted to 1X 106/mL.

2. The experimental groups contained 2X 105 target cells (U266) or negative control cells (K562) per well, 2X 105 CAR-T cells, 200. mu.l Lonza medium without IL-2. Mix well and add to 96-well plate. BD GolgiPlug (containing BFA, 1. mu.l BD GolgiPlug per 1ml cell culture medium) was added at the same time, mixed well and incubated at 37 ℃ for 5-6 hours. Cells were collected as experimental groups.

3. Cells were washed 1 time with 1mL of PBS per tube and centrifuged at 300g for 5 minutes. The supernatant was carefully aspirated or decanted.

4. After washing the cells with PBS, 250. mu.l/EP tube Fixation/Permeabilization solution was added and incubated at 4 ℃ for 20 minutes to fix the cells and rupture the membranes. Using 1 XBD Perm/Wash^TMbuffer washes cells 2 times, 1 mL/time.

5. Staining with intracellular factor, taking appropriate amount of IFN-gamma cytokine fluorescent antibody or negative control, and performing BD Perm/Wash^TMbuffer diluted to 50. mu.l. Resuspending the fixed and disrupted cells thoroughly with the antibody dilution, incubating at 4 ℃ in the dark for 30min, 1 XBD Perm/Wash^TMbuffer 1 mL/wash cells 2 times, then use PBS heavy suspension.

6. And (4) detecting by using a flow cytometer.

Shown in fig. 8. FIG. 8 shows that the percentage of INF- γ secretion by BCMA-tEGFR CART cells in U266 cells positive for CD8 was 16.2%, respectively, and the percentage of INF- γ secretion by BCMA-tEGFR cells in U266 cells positive for CD4 was 13.4%, respectively.

Example 8: detection of tumor-specific cell killing after Co-culture of CAR-T cells with target cells

1. K562 cells (negative control cells of target cells without BCMA target protein) were resuspended in serum-free medium (1640), adjusted to a cell concentration of 1X 106/ml, and the fluorescent dye BMQC (2,3,6,7-tetrahydro-9-bromomethyl-1H,5 Hquinozino (9,1-gh) coumarins) was added to a final concentration of 5. mu.M.

2. Mixing, and incubating at 37 deg.C for 30 min.

3. Centrifugation was carried out at 1500rpm for 5min at room temperature, the supernatant was discarded and the cells resuspended in cytotoxic medium (phenol red-free 1640+ 5% AB serum) and incubated for 60min at 37 ℃.

4. Fresh cytotoxic medium washed the cells twice and resuspended at a density of 1X 106/ml in fresh cytotoxic medium.

5. U266 cells (containing BCMA target protein, target cells) were suspended in PBS containing 0.1% BSA at a concentration of 1X 106/ml.

6. The fluorescent dye CFSE (fluorescent dye) (CFSE) was added to a final concentration of 1. mu.M.

7. Mixing, and incubating at 37 deg.C for 10 min.

8. After the incubation was completed, FBS in an equal volume to the cell suspension was added and incubated at room temperature for 2min to terminate the labeling reaction.

9. Cells were washed and resuspended in fresh cytotoxic medium at a density of 1X 106/ml.

9. Effector T cells were washed and suspended in cytotoxic medium at a concentration of 5X 106/ml.

10. In all experiments, the cytotoxicity of CAR-T cells was compared to that of uninfected negative control effector T cells (ntcells), and these effector T cells were from the same patient.

11. CAR-T and NT, according to effector cell: target cells were cultured in 5ml sterile test tubes (BDBiosciences) at a ratio of 10:1, 2:1, with two duplicate wells per group. In each co-culture group, 50,000 (50. mu.l) U266 cells were targeted, and 50,000K 562 cells (50. mu.l) were negative control cells. A panel was set up containing only U266 target cells and K562 negative control cells.

12. The co-cultured cells were incubated at 37 ℃ for 5 h.

13. After incubation was complete, cells were washed with PBS and immediately followed by rapid addition of 7-AAD (7-aminoactomycin D) at the concentrations recommended by the instructions and incubation on ice for 30 min.

14. The Flow-type detection is directly carried out without cleaning, and the data is analyzed by Flow Jo.

15. Assay the proportion of viable U266 target cells and viable K562 negative control cells after co-culture of T cells and target cells was determined using 7AAD negative viable cell gating.

16. For each group of co-cultured T cells and target cells

17. The% cytotoxic killer cells is 100-the% calibrated target cell survival, i.e., (number of U266 viable cells without effector cells-number of U266 viable cells with effector cells)/number of K562 viable cells.

The results are shown in fig. 9. Figure 9 shows that at a 10:1 effective target ratio, the killing rate of BCMA-tfegfr CART cells on U266 cells was 70%.

Sequence listing

<110> Shanghai Hengrunheng Dasheng Biotech Co., Ltd

<120> BCMA-targeting chimeric antigen receptor and use thereof

<170>PatentIn version 3.3

<210>1

<211>2628

<212>DNA

<213> Artificial sequence

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aggggtcctg ataactgtat ccaatgtgca cactacatag atggtcctca ctgtgtaaag 2400

acgtgccccg ccggagtaat gggtgaaaac aacaccctcg tgtggaagta cgccgatgcc 2460

gggcatgtct gtcatttgtg tcatcccaac tgcacatatg gctgtaccgg tcctggattg 2520

gagggctgtc caacaaacgg gccgaaaata ccgagtatcg caacaggcat ggtgggagca 2580

cttttgcttc tcctcgttgt cgccctgggc atcggcttgt tcatgtga 2628

<210>2

<211>874

<212>PRT

<213> Artificial sequence

<400>2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu

20 25 30

Ala Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu

35 40 45

Ser Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu

65 70 75 80

Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val Ala Ile Tyr Ser

100 105 110

Cys Leu Gln Ser Arg Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

145 150 155 160

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

165 170 175

Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu

180 185 190

Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala

195 200 205

Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

210 215 220

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr

225 230 235 240

Tyr Phe Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln

245 250 255

Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

305 310 315 320

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser

325 330 335

Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ala Lys Arg Gly

485 490 495

Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu

500 505 510

Glu Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys

515 520 525

Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn

530 535 540

Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr

545 550 555 560

Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His

565 570 575

Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro

580 585 590

Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr

595 600 605

Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His

610 615 620

Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly

625 630 635 640

Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu

645 650 655

Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn

660 665 670

Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly

675 680 685

Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser

690 695 700

Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly

705 710 715 720

Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser

725 730 735

Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro

740 745 750

Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys

755 760 765

Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn

770 775 780

Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr

785 790 795 800

Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr

805 810 815

Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr

820 825 830

Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys

835 840 845

Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu

850 855 860

Val Val Ala Leu Gly Ile Gly Leu Phe Met

865 870

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>3

agcatcgttc tgtgttgtct c 21

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>4

tgtttgtctt gtggcaatac ac 22

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>5

agcatcgttc tgtgttgtct c 21

<210>6

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>6

tgtttgtctt gtggcaatac ac 22

<210>7

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>7

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Leu

1 5 10 15

Gly Ser Thr Glu Phe

20

Claims

1. A polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence comprising the coding sequence of an anti-BCMA single-chain antibody, the coding sequence of a human CD8 α hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3 zeta intracellular region and the coding sequence of a fragment of EGFR containing extracellular domain III and extracellular domain IV, which are connected in sequence, wherein the amino acid sequence of the light chain variable region of the anti-BCMA single-chain antibody is shown as amino acids 22-132 of SEQ ID NO:2, the amino acid sequence of the heavy chain variable region of the anti-BCMA single-chain antibody is shown as amino acid 148-264 of SEQ ID NO:2, and

(2) (1) the complement of the polynucleotide sequence,

wherein the amino acid sequence of the hinge region of the human CD8 α is shown as the amino acid at the 265 rd and 311 th positions of SEQ ID NO. 2, the amino acid sequence of the transmembrane region of the human CD8 is shown as the amino acid at the 312 rd and 333 th positions of SEQ ID NO. 2, the amino acid sequence of the intracellular region of the human 41BB is shown as the amino acid at the 334 rd and 381 th positions of SEQ ID NO. 2, the amino acid sequence of the intracellular region of the human CD3 zeta is shown as the amino acid at the 382 nd and 492 nd positions of SEQ ID NO. 2, and the amino acid sequence of the EGFR fragment is shown as the amino acid at the 540 nd and 874 th positions of SEQ ID NO. 1.

2. The polynucleotide sequence of claim 1, further comprising a coding sequence for a signal peptide prior to the coding sequence for the anti-BCMA single chain antibody.

3. The polynucleotide sequence of claim 2, wherein the amino acid sequence of the signal peptide is as set forth in amino acids 1-21 of SEQ id No. 2.

4. The polynucleotide sequence of claim 2, further comprising a coding sequence for a GM-CSF receptor α chain signal peptide, wherein the GM-CSF receptor α chain signal peptide is disposed N-terminal to the EGFR fragment.

5. The polynucleotide sequence of claim 4, wherein the amino acid sequence of the signal peptide of the strand of GM-CSF receptor α is represented by amino acids 518-539 in SEQ ID NO 2.

6. The polynucleotide sequence of claim 4, further comprising a coding sequence for a linker sequence linking the GM-CSF receptor α chain signal peptide to the intracellular domain of human CD3 ζ.

7. The polynucleotide sequence of claim 6, wherein the amino acid sequence of the linker sequence is as shown in amino acids 493 517 of SEQ ID NO 2.

8. The polynucleotide sequence of claim 2, wherein the coding sequence for said signal peptide preceding the coding sequence for said anti-BCMA single-chain antibody is as set forth in the nucleotide sequence of SEQ ID No. 1 to 63.

9. The polynucleotide sequence of claim 1, wherein the coding sequence of the light chain variable region of the anti-BCMA single-chain antibody is represented by the nucleotide sequence of SEQ ID NO. 1 from 64 th to 396 th positions, the coding sequence of the heavy chain variable region of the anti-BCMA single-chain antibody is represented by the nucleotide sequence of SEQ ID NO. 1 from 442-

The polynucleotide sequence codes an amino acid sequence shown as 24 th to 495 th positions of SEQ ID NO. 2, or codes an amino acid sequence shown as 24 th to 874 th positions of SEQ ID NO. 2, or codes an amino acid sequence shown as SEQ ID NO. 2; or

The polynucleotide sequence comprises a nucleotide sequence shown in SEQ ID NO. 1 and 1 st to 1476 th sites of SEQ ID NO. 1, a nucleotide sequence shown in 64 th to 1476 th sites of SEQ ID NO. 1 or a nucleotide sequence shown in 64 th to 2628 th sites of SEQ ID NO. 1, or consists of a nucleotide sequence shown in 1 st to 1476 th sites of SEQ ID NO. 1 and SEQ ID NO. 1, a nucleotide sequence shown in 64 th to 1476 th sites of SEQ ID NO. 1 or a nucleotide sequence shown in 64 th to 2628 th sites of SEQ ID NO. 1.

10. The polynucleotide sequence of claim 4, wherein the coding sequence of the signal peptide of GM-CSF receptor α chain is as shown in nucleotide sequence 1555-1634 of SEQ ID NO. 1.

11. The polynucleotide sequence of claim 6, wherein the coding sequence for the linker sequence linking the signal peptide of GM-CSF receptor α chain to the intracellular domain of human CD3 ζ is as shown in SEQ ID NO 1, nucleotide sequence 1477-1554.

12. A fusion protein comprises a fusion protein of an anti-BCMA single-chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human 41BB intracellular region and a human CD3 zeta intracellular region and a coding sequence of a fragment containing an extracellular domain III and an extracellular domain IV of an EGFR, which are connected in sequence, wherein the amino acid sequence of a light chain variable region of the anti-BCMA single-chain antibody is shown as amino acids 22-132 of SEQ ID NO:2, the amino acid sequence of a heavy chain variable region of the anti-BCMA single-chain antibody is shown as amino acid 148-264 of SEQ ID NO:2,

wherein the amino acid sequence of the hinge region of the human CD8 α is shown as the amino acid at the 265 rd and 311 st positions of SEQ ID NO. 1, the amino acid sequence of the transmembrane region of the human CD8 is shown as the amino acid at the 312 rd and 333 rd positions of SEQ ID NO. 1, the amino acid sequence of the intracellular region of the human 41BB is shown as the amino acid at the 334 rd and 381 st positions of SEQ ID NO. 1, the amino acid sequence of the intracellular region of the human CD3 zeta is shown as the amino acid at the 382 nd and 492 nd positions of SEQ ID NO. 1, and the amino acid sequence of the fragment of the EGFR is shown as the amino acid at the 540 nd and 874 th positions of SEQ ID NO. 1.

13. The fusion protein of claim 12, further comprising a signal peptide at the N-terminus of the anti-BCMA single chain antibody.

14. The fusion protein of claim 13, wherein the signal peptide has an amino acid sequence as set forth in amino acids 1-21 of SEQ id No. 2.

15. The fusion protein of claim 13, further comprising a GM-CSF receptor α chain signal peptide, wherein the GM-CSF receptor α chain signal peptide is disposed N-terminal to the EGFR fragment.

16. The fusion protein of claim 15, wherein the amino acid sequence of the signal peptide of the chain of GM-CSF receptor α is represented by amino acids 518-539 in SEQ ID NO 2.

17. The fusion protein of claim 15, further comprising a linker sequence linking the GM-CSF receptor α chain signal peptide to the intracellular domain of human CD3 ζ.

18. The fusion protein of claim 17, wherein the amino acid sequence of the linker sequence is represented by amino acids 493 517 of SEQ ID NO 2.

19. The fusion protein of claim 12, wherein the amino acid sequence of the fusion protein is represented by amino acids 22-492 of SEQ ID No. 2, or by amino acids 22-874 of SEQ ID No. 2, or by amino acids 1-492 of SEQ ID No. 2, or by amino acid SEQ ID No. 2.

20. A nucleic acid construct comprising the polynucleotide sequence of any one of claims 1-11.

21. The nucleic acid construct of claim 20, wherein said nucleic acid construct is a vector.

22. The nucleic acid construct of claim 20, wherein the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR, and the polynucleotide sequence of any one of claims 1 to 11.

23. A retrovirus comprising the nucleic acid construct of any one of claims 20-22.

24. A genetically modified T-cell or a pharmaceutical composition comprising a genetically modified T-cell, wherein the cell comprises a polynucleotide sequence according to any one of claims 1 to 11, or comprises a nucleic acid construct according to any one of claims 20 to 22, or is infected with a retrovirus according to claim 23, or stably expresses a fusion protein according to any one of claims 12 to 19.

25. Use of a polynucleotide sequence of any one of claims 1-11, a fusion protein of any one of claims 12-19, a nucleic acid construct of any one of claims 20-22, or a retrovirus of claim 23, in the preparation of a reagent comprising an activated T cell.

26. Use of the polynucleotide sequence of any one of claims 1-11, the fusion protein of any one of claims 12-19, the nucleic acid construct of any one of claims 20-22, the retrovirus of claim 23, or the genetically modified T-cell of claim 24, or a pharmaceutical composition thereof, in the preparation of a medicament for treating a BCMA-mediated disease.

27. The use of claim 26, wherein the BCMA-mediated disease is multiple myeloma.