WO2020061796A1

WO2020061796A1 - Bcma-and-cd19-targeting chimeric antigen receptor and uses thereof

Info

Publication number: WO2020061796A1
Application number: PCT/CN2018/107520
Authority: WO
Inventors: Yarong Liu; Feng He; Haiying Wang; Zixiao SHI
Original assignee: Hrain Biotechnology Co Ltd
Current assignee: Hrain Biotechnology Co Ltd
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2020-04-02
Anticipated expiration: 2021-03-26
Also published as: JP2022501067A

Abstract

A BCMA-and-CD19 dual-target chimeric antigen receptor and uses thereof are provided. Specifically, provided is a polynucleotide sequence selected from the group consisting of: (1) a polynucleotide sequence comprising, as linked in sequence, sequences encoding an anti-BCMA and an anti-CD19 single chain antibodies, a sequence encoding the hinge region of human IgG4, a sequence encoding the transmembrane region of human CD28, a sequence encoding the intracellular domain of human 41BB, a sequence encoding the intracellular domain of human CD3ζ and optionally a sequence encoding a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV; and (2) a complementary sequence of the polynucleotide sequence of (1). Further provided are corresponding fusion proteins and vectors comprising the coding sequence, as well as uses of the fusion proteins and vectors.

Description

BCMA-and-CD19-targeting Chimeric Antigen Receptor and Uses Thereof

Technical Field

The present disclosure relates to the field of cell therapy. Specifically, it relates to a BCMA-and-CD19 dual-target chimeric antigen receptor and uses thereof.

Background

Chimeric Antigen Receptor-T-cells (CAR-T-cells) are the T-cells that are genetically modified to be capable of non-MHC-restricted antigen recognition and sustained activation to proliferation. At the 2012 Annual Meeting of the International Society for Cellular Therapy, it was reported that in addition to surgery, radiation and chemotherapy, biological immune cell therapy has been recognized as the fourth anti-tumor therapy, which will become an essential measure of anti-tumor therapy in future. CAR-T-cell autologous infusion is presently the anti-tumor immune therapy with the most definite efficacy. Extensive studies have shown that CAR-T-cells can effectively recognize tumor antigens, elicit specific anti-tumor immune response and significantly improve patients'well-being.

Chimeric antigen receptor (CAR) is core to CAR-T, which render T-cells the HLA-independent recognition of tumor antigens. This expands the spectrum of targets for CAR-modified T-cells, compared to native T-cell surface receptors (TCR) . A design of CAR may basically include a tumor-associated antigen (TAA) binding domain, which is usually derived from a scFV fragment of the antigen-binding domains of a monoclonal antibody, an extracellular hinge domain, a transmembrane region and an intracellular signaling domain. Selection of the target antigen is crux for specificity and efficacy of the CAR, as well as safety of the genetically modified T-cells per se.

CD19 is a 95kDa glycoprotein on the surface of B-cells, which is expressed by B-cells from the early stage of development until differentiation into plasma cells. CD19 is a member of the superfamily of immunoglobulins (Igs) and a component of the B-cell surface signaling complex, which participates in regulation of B-cell receptor signaling. In a CD19-deficient model in mouse, there can be observed reduced B-cells in peripheral lymphoid tissues, decreased response to vaccine and mitogen, accompanied by decreased serum Ig. Normally, it is believed that CD19 expression is limited to B-cell lineage, which is not seen on surface of multipotent hematopoietic stem cells. CD19 expression has also been observed on cells in most B cell lymphoma, mantle cell lymphoma, ALLs, CLLs, hairy cell leukemia and part of acute myeloid leukemia. Accordingly, for leukemia/lymphoma, CD19 is a valuable target of immunotherapy. Importantly, CD19 is not expressed on most normal cells (including multipotent hematopoietic stem cells) other than B-cells, and this makes it a safer therapy target, which minimizes the risk of autoimmune diseases and irreversible toxic injury to bone marrow in patients. Up till now, anti-CD19 antibodies and scFv fragments have been developed with promising future in applications as tested in mouse and human beings/primates.

The field of CD19 CAR T-cells has been quite competitive, while many large pharmaceutical companies have partnership ties with research institutes. In recurrent and refractory acute B cell lymphoma in children and adults, CD19 CAR T-cells expressing CD28 or 4-1BB provide a complete response rate of around 90%. Recently, CD19 CAR T-cell therapy is reported with an overall response rate of 50%-100%in diffuse large B cell lymphoma, follicular lymphoma or chronic lymphoma. CD19 CAR T-cell therapy has an clinical edge in multiple myeloma because terminally differentiated plasma cells do not express CD19, and malignant B cell precursors continuously produce malignant plasma cells.

Though CD19 is expressed most of the lifecycle of B-cells, the surface expression is low at the terminal stage, i.e., plasma cells. Accordingly, it is not the first pick target for MM therapy. B-cell maturation antigen (BCMA) , also known as CD269, consists of 184 amino acid residues, which comprises an intracellular domain comprising 80 amino acid residues and a very short extracellular domain comprising a single carbohydrate-recognition domain as a B-cell surface molecule. BCMA is a type I transmembrane signaling protein lacking signal peptide and is a member of the family of tumor necrosis factor receptors (TNFRs) . It is capable of binding to the B cell activation factor (BAFF) and the proliferation-induced ligand (APRIL) . In normal tissues, BCMA is surface expressed on mature B cells and plasmacytes. BCMA gene-knocked out mice exhibit normal immune system functionality, normal spleen structure, normal B lymphocyte development, except for decreased level of plasmacytes. This demonstrates that BCMA plays an important role in survival of plasmacytes, wherein the mechanism of action mainly involves the BCMA's binding to BAFF, up-regulation of anti-apoptosis genes like Bcl-2, Mcl-1, Bclw, etc., maintenance of cell growth. Similarly, the action also plays an important role in promoting malignant proliferation in myeloma cells. It has been shown that BCMA is ubiquitously expressed in multiple myeloma cell lines, which has been confirmed by the detection in multiple myeloma patients. Kochenderfer et al., on basis of the previously reported results, further explored the expression profile of BCMA using multiple techniques including Q-PCR, Flow Cytometry and immunohistochemistry, and concluded that BCMA is not expressed on normal human tissues except for mature B cells and plasmacytes, and is not expressed on CD34+ hematopoietic cells. Besides, BCMA-deficiency has no impact on the number of B-cells in mouse and is not fatal. In view of these, BCMA is a potential target of CAR-T-cells for use in cellular immunotherapy of multiple myeloma.

There have already been clinical practices of combining anti-CD19 CAR and anti-BCMA CAR to, treat MM. At the annual meeting of the American Society of Hematology (ASH) December 9-12, 2017, Professor FU, Cheng Cheng from the Department of Hematology of the First Affiliated Hospital of Soochow University presented "an initial report of safety and efficacy of combined CD19-and BCMA-specific CAR-T-cells infusion for treating r/r MM" by her team. Among ten (10) patients enrolled and receiving combined anti-CD19 and anti-BCMA CAR T-cells infusion, it reported an objective response rate (ORR) of 100%, a partial response (PR) of 90%, VGPR and above 30%and controllable CRS, wherein eight (8) patients underwent CRS of grades 1～2 and two (2) grade-3, while all the CRSs were put under control within 10 days without relapse. All these patients survive through the median follow-up time being 23 weeks (4～32 weeks) , wherein one achieved a stringent complete response (sCR) of 7 months, though not yet the median PFS time.

Clinical studies show, multiple myeloma patients having been treated with BCMA-CART are found actually positive for CD19 expression, while a trace of CD19 expression can lead to relapse. Anti-BCMA-CD19 bi-specific CAR T-cells can effectively treat recurrent or refractory MM attributable to residual CD19 expression.

The present invention utilizes a CAR component dually targeting CD19 and BCMA. The anti-CD19-BCMA bi-specific CAR T-cells exhibit a strong killing of target cells, which sets a sound basis for future clinical trials and therapy.

Summary of invention

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

(1) a polynucleotide sequence comprising the followings linked in sequence: sequences encoding an anti-BCMA and an anti-CD19 single chain antibodies, a sequence encoding the hinge region of human IgG4, a sequence encoding the transmembrane region of human CD28, a sequence encoding the intracellular domain of human 41BB, a sequence encoding the intracellular domain of human CD3ζ and optionally a sequence encoding a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV; and

(2) a complementary sequence of the polynucleotide sequence of (1) .

In one or more embodiments, there is a sequence encoding a signal peptide lying ahead of the sequence encoding the anti-BCMA single chain antibody and having the sequence of nucleotides 1-63 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the light chain variable region of said anti-BCMA single chain antibody has the sequence of nucleotides 64-396 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the heavy chain variable region of said anti-BCMA single chain antibody has the sequence of nucleotides 442-792 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the heavy chain variable region of said anti-CD19 single chain antibody has the sequence of nucleotides 853-1212 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the light chain variable region of said anti-CD19 single chain antibody has the sequence of nucleotides 1267-1587 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding said hinge region of human IgG4 has the sequence of nucleotides 1588-1623 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the transmembrane region of human CD28 has the sequence of nucleotides 1627-1707 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the intracellular domain of human 41BB has the sequence of nucleotides 1708-1833 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the intracellular domain of human CD3ζ has the sequence of nucleotides 1834-2169 as set forth in SEQ ID NO: 1.

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

(1) a fusion protein comprising the followings linked in sequence: an anti-BCMA single chain antibody and an anti-CD19 single chain antibody, the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB and the intracellular domain of human CD3ζ; and

(2) a fusion protein derived from (1) , comprising one or more substitution (s) , deletion (s) or addition (s) in the amino acid sequence of (1) while retaining the activity of T-cell activation;

preferably, the anti-CD19 single chain antibody is of the anti-CD19 monoclonal antibody FMC63;

preferably, the anti-BCMA single chain antibody is of the anti-BCMA monoclonal antibody C11D5.3.

In the third aspect, the present disclosure provides a nucleic acid construct comprising the polynucleotide sequence according to the present disclosure.

In one or more embodiments, the nucleic acid construct is a vector. In one or more embodiments, the nucleic acid construct is a retrovirus vector comprising an origin of replication, a 3’LTR, a 5’LTR and the polynucleotide sequence of the present disclosure, as well as an optional selection marker.

In the fourth aspect, the present disclosure provides a retrovirus which comprises the nucleic acid construct, preferably the vector, more preferably the retrovirus vector according to the present disclosure.

In the fifth aspect, the present disclosure provides a pharmaceutical composition comprising the genetically modified T-cell according to the present disclosure.

In the sixth aspect, the present disclosure provides use of the polynucleotide sequence, the fusion protein, the nucleic acid construct or the retrovirus according to the present disclosure for producing activated T-cells.

In the seventh aspect, the present disclosure provides use of the polynucleotide sequence, the fusion protein, the nucleic acid construct, the retrovirus, the genetically modified T-cell or the pharmaceutical composition according to the present disclosure for manufacturing medicaments for treating diseases mediated by BCMA.

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

Description of Drawings

FIG. 1 schematically depicts the retrovirus expression vector RV-BCMA-CD19 -BBz.

FIG. 2 shows the flow cytometry of BCMA-CD19 -BBz CART expression by T-cells infected with the retrovirus for 72 hours.

FIG. 3 shows the flow cytometry of CD19 expression on surface of the target cell MM. 1S-CD19.

FIG. 4 shows the CD107a expression by a 5-day preparation of BCMA-CD19 CART-cells incubated with different target cells for 5 hours.

FIG. 5 shows the INF-γ secretion by a 5-day preparation of BCMA-CD19 CART-cells incubated with different target cells for 5 hours.

FIG. 6 shows the killing of tumor cells by a 5-day preparation of BCMA-CD19 CART-cells after incubation with different target cells for 5 hours.

Detailed Description of the invention

The present disclosure provides a chimeric antigen receptor (CAR) dually targeting BCMA and CD19. The CAR comprises the followings linked in sequence: an anti-BCMA single chain antibody and an anti-CD19 single chain antibody, the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB, the intracellular domain of human CD3ζ and optionally a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV of the receptor.

A suitable anti-BCMA single chain antibody may be one derived from any of the anti-BCMA monoclonal antibodies known to a person in the art.

A suitable anti-CD19 single chain antibody may be one derived from any of the anti-CD19 monoclonal antibodies known to a person in the art.

Optionally, the light chain variable region and the heavy chain variable region may be joined by a linker. In some embodiments, the anti-BCMA single chain antibody has a light chain variable region having the sequence of amino acids 22-132 as set forth in SEQ ID NO: 2. In some other embodiments, the anti-BCMA single chain antibody has a heavy chain variable region having the sequence of amino acids 148-264 as set forth in SEQ ID NO: 2. In some embodiments, the anti-CD19 single chain antibody has a heavy chain variable region having the sequence of amino acids 285-404 as set forth in SEQ ID NO: 2. In some other embodiments, the anti-CD19 single chain antibody has a light chain variable region having the sequence of amino acids 423-529 as set forth in SEQ ID NO: 2.

The hinge region of human IgG4 useful in the present disclosure may have the sequence of amino acids 530-541 as set forth in SEQ ID NO: 2.

The transmembrane region of human CD28 useful in the present disclosure may be any one of the human CD28 transmembrane sequences useful in CARs. In some embodiments, the transmembrane region of human CD28 has the sequence of amino acids 532-569 as set forth in SEQ ID NO: 2.

The 41BB useful in the present disclosure may be any one of the 41BB molecules useful in CARs. In an illustrative example, the 41BB used in the present disclosure has the sequence of amino acids 570-611 as set forth in SEQ ID NO: 2.

The intracellular domain of human CD3ζ useful in the present disclosure may any one of the intracellular domains of human CD3ζ useful in CARs. In some embodiments, the intracellular domain of human CD3ζ has the sequence of amino acids 612-723 as set forth in SEQ ID NO: 2.

These components of the fusion protein of the present disclosure, say the light chain variable region and the heavy chain variable region of the anti-BCMA and anti-CD19 single chain antibody, the hinge region of human IgG4, the transmembrane region of human CD28, 41BB, the intracellular domain of human CD3ζ etc., may be linked one to another directly or via linkers. The linkers may be any of those used in antibodies, like those comprising G and S. Usually, linkers comprise repeats of one or more motifs. Examples of the motif include GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent one another in a linker, without in-between amino acid residues. The linker may comprise 1, 2, 3, 4 or 5 repeats of a motif. The linker may be 3-25 amino acid residues, for example, 3-15, 5-15, 10-20 amino acid residues in length. In some embodiments, the linker is a poly (glycine) linker. There is no limit on the number of glycine residues in the linker, while the number is usually in the range of 2-20, for example, 2-15, 2-10 and 2-8. Apart from glycine and serine, the linker may further comprises some additional amino acid residues, like alanine (A) , leucine (L) , threonine (T) , glutamic acid (E) , phenylalanine (F) , arginine (R) , glutamine (Q) , etc. In some embodiments, in the anti-BCMA single chain antibody, the light chain variable region and the heavy chain variable region are linked via (GGGGS) _n, wherein n is an integer from 1 to 5.

In some embodiments, the CAR of the present disclosure may further comprises in its amino acid sequence a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV of the receptor, the signal peptide thereof and a linker.

It should be understood that due to restriction sites designed for cloning, the expressed amino acid sequence will include one or more irrelevant residues at the end (s) , which will not interfere the activity of the sequence of interests. To construct a fusion protein, to increase recombinant expression, to enable spontaneous secretion to outside of the hosts or to facilitate purification, the protein may need to include some additional amino acids at the N-terminal, the C-terminal of some other region of the fusion protein as appropriate. For example, the additional amino acids include but are not limited to a linker peptide, a signal peptide, a leader, a terminal extension. Accordingly, the fusion protein (i.e., the CAR) of the present disclosure may further include at the N-or the C-terminal one or more polypeptide fragment (s) as protein tag (s) . Any suitable tags are useful in the present disclosure. 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 Ty1. These tags are useful in protein purification.

The present disclosure further includes variants of the CAR having the sequence of amino acids 22-723 as set forth in SEQ ID NO: 2, the CAR having the sequence of amino acids 1-723 as set forth in SEQ ID NO: 2 or the CAR having the amino acid sequence of SEQ ID NO: 2. The variant includes a amino acid sequence that has a sequence identity of at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%to the specified CAR and the same biological activity (e.g., of T-cell activation) as the specified CAR. Sequence identity can be calculated, for example, using BLASTp from NCBI.

Variants also include those having one or a plurality of mutation (s) (insertion, deletion or substitution) in the sequence of amino acids 22-723 as set forth in SEQ ID NO: 2, the sequence of amino acids 1-723 as set forth in SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 and remaining the biological activity of the CAR. The "plurality" normally refers to a number raging from 1 to 10, such as from 1 to 8, from 1 to 5 or from 1 to 3. The substitution is preferably a conservative one. For instance, conservative substitution between amino acids close or similar in property is known as will not change the effect of the protein or polypeptide. "Amino acids close or similar in property" include, for example, a family of amino acid residues having similar side chains. These families include, for example, amino acids with a basic side chain (e.g., lysine, arginine, histidine) , amino acids with an acidic side chain (e.g., aspartic acid, glutamic acid) , amino acids with an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , amino acids with a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine valine, phenylalanine, methionine, tryptophan) , amino acids with a β-branched side chain (e.g., threonine, proline, isoleucine) and amino acids with an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine) . Accordingly, in the polypeptides according to the present disclosure, substitution with another member of the same family of side chain will not cause any substantive change in activity.

The present disclosure includes polynucleotide sequences encoding the fusion proteins according to the present disclosure. The polynucleotide sequences of the present disclosure may be in the form of DNA or RNA. The term "DNA" includes cDNA, genomic DNA or artificial synthetic DNA. The DNA may be a single-strand or a doubled-strand DNA. The DNA may be the coding strand or the non-coding strand. The present disclosure further includes degenerate variants of the polynucleotide sequence encoding the fusion protein, i.e., different nucleotide sequences that encode the same amino acid sequence.

The polynucleotide sequences according to the present disclosure can be prepared by PCR amplification. Specifically, the sequence can be amplified using primers designed according to the given nucleotide sequence (particularly the Open Reading Frames) and commercially available cDNA libraries or self-made cDNA libraries as templates. For longer sequences, two or more runs will be needed, and fragments from each run are then assembled into the correct sequence. For example, in some embodiments, the polynucleotide sequence encoding the fusion protein of the present disclosure has the sequence of nucleotides 64-2169 as set forth in SEQ ID NO: 1 or the sequence of nucleotides 1-2169 as set forth in SEQ ID NO: 1.

The present disclosure further includes a nucleic acid construct, which comprises the polynucleotide sequence according to the present disclosure operably linked to one or more regulatory sequence (s) . The polynucleotide sequence according to the present disclosure can be manipulated in various ways to ensure a successful expression of the fusion protein (CAR) . Before being inserted into a vector, the nucleic acid construct may be adaptively processed according to the selected expression vector. The recombinant DNA techniques useful to modify polynucleotide sequences are already known.

The regulatory sequence may be a proper promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter may be any of the nucleotide sequences that exhibit transcription activity in the host, which includes mutated, truncated or hybrid promoters, and which may be obtained from the gene of an extracellular or intracellular polypeptide that is homologous or heterologous to the host. A regulatory sequence may also be a transcription terminator sequence as appropriate, which terminates transcription upon recognition by the host cell. The terminator sequence is operably linked to the 3’-end of the nucleotide sequence encoding the polypeptide. Any terminator functional in a selected host is useful in the present disclosure. The regulatory sequence may also be a leader sequence as appropriate, which is a untranslated region of mRNA important for translation in the host cell. The leader sequence is operably linked to the 5′-end of the nucleotide sequence encoding the polypeptide. Any leader sequence functional in a selected host is useful in the present disclosure.

In some embodiments, the nucleic acid construct is a vector. Typically, the polynucleotide sequence according to the present disclosure is operably link to the promoter, and the construct is incorporated into an expression vector to obtain an effective expression of the polynucleotide sequence according to the present disclosure. The vector may be one suitable for replication in and integration into an eukaryotic cell. Typically, a cloning vector comprises a transcription terminator, a translation terminator, an initiation region and a promoter to modulate the desired expression of a nucleic acid sequence.

The polynucleotide sequence according to the present disclosure may be cloned into various types of vectors. For example, it can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus or a cosmid. Further, the vector may be an expression vector. The expression vector may be delivered into the cell in form of a virus vector. Viral vector technology is already known and has been described in, for example, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Sambrook et al., 2001, New York) and many other virology and molecular biology manuals. Viruses that are useful as vectors include but are not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and lentivirus.

Typically, a suitable vector comprises a replication origin, promoter sequence, convenient restriction site and one or more selectable markers that are functional in at least one organism (e.g., WO 01/96584; WO01/29058; and US patent No. 6,326,193) .

For instance, in some embodiments, used in the present disclosure is a retrovirus vector, which comprises an origin of replication, a 3’LTR, a 5’LTR, the polynucleotide sequence according to the present disclosure, and optionally a selectable marker.

An example of suitable promoters is the immediate early promoter of cytomegalovirus (CMV) . This is a strong constitutive promoter that is capable of driving high expression of whatever polynucleotide sequence operably linked to it. Another example of suitable promoters is extension growth factor-1α (EF-1α) . Still, other constitutive promoter sequences may also be used, including but not limited to: simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, EB virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, such as actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, also contemplated are inducible promoters. Inducible promoters provide a molecular switch, which can turn on the expression of the polynucleotide sequence operably linked to the inducible promoter as desired, and turn off when the expression is not desired. Examples of inducible promoter include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter.

For assessment of expression of the CAR polypeptide or part of it, the expression vector to be introduced into cells may further comprise a selectable marker gene or a reporter gene or both, such that expression cells can be identified or selected from the cell population transfected or infected with the virus vector. In some other aspects, the selectable marker may be carried on a separate DNA sequence for use in co-transfection. Both the selectable marker and the reporter genes may be flanked by one or more regulatory sequences for expression in host cells. Useful selectable markers include for example antibiotics resistance genes, like neo, etc.

Reporter genes are used to identify potentially transfected cells and to assess functionality of the regulatory sequences. After DNA being transferred into the recipient cells, the reporter gene may be detected at an appropriate time point. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein. Suitable expression systems are already known and can be prepared using existing techniques or commercially obtained.

Methods for transferring genes into and for expressing genes in cells are already known. Vectors can be conveniently delivered into host cells, like mammalian, bacterial, yeast or insect cells, using various methods as known to a person in the art. For instance, an expression vector may be transferred into the host cell using a physical, chemical or biological means.

Physical methods of transferring polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods of transferring polynucleotides into host cells involve DNA and RNA vectors. Chemical methods of transferring polynucleotides into host cells involve colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres and beads; and lipid-based systems, including oil in water emulsion, micelle, mixed micelles and liposomes.

Biological methods of transferring polynucleotides into host cells involve virus vectors, especially retrovirus vector, which has been widely used in gene integration into mammalian cells, like human cells. Additional virus vectors may be those derived from lentivirus, poxvirus, simple herpes virus I, adenovirus, adeno-associated virus, etc. Many virus-based systems have been developed for use in gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. A selected gene may be inserted into a vector and then packaged into a retrovirus particle using techniques as known to a person in the art. The recombinant virus may then be isolated and transferred into cells from a subject in vivo or ex vivo. There are many retrovirus systems as known to a person in the art. In some examples, adenovirus vectors may be used. There are many adenovirus vectors as known to a person in the art. In one embodiment, a lentivirus vector is used.

Accordingly, in some embodiments, the present disclosure further provides a retrovirus useful in T-cell activation, wherein the virus comprises the retrovirus vector according to the present disclosure and corresponding package genes, like gag, pol and vsvg.

T-cells useful in the present disclosure can be those of any origins and of any types. For example, T-cells may be those from PBMC from a patient having B-cell malignant tumor.

In some embodiments, before use, the obtained T-cells may first be stimulated using an appropriate amount (such as 30-80ng/ml, e.g., 50ng/ml) of an anti-CD3 antibody, and then cultured in a medium supplemented with an appropriate amount (such as 30-80IU/ml, e.g., 50IU/ml) of IL2.

Accordingly, in some embodiments, the present disclosure provides a genetically modified T-cell, which comprises the polynucleotide sequence according to the present disclosure or the retrovirus vector according to the present disclosure, or is infected with the retrovirus vector according to the present disclosure, or is prepared by the method according to the present disclosure, or stably expresses the fusion protein according to the present disclosure.

The CAR-T-cells according to the present disclosure may undergo robust in vivo T-cell expansion, sustain in blood and bone marrow for a prolonged time period, and form specific memory T-cells. Without being bound to any particular theory, after encountering and depleting the target cells expressing the antigen substitute, the CAR-T-cells according to the present disclosure can differentiate into the central memory status in vivo.

The present disclosure further includes a kind of cell therapy, wherein T-cells are genetically modified to express the CAR and optionally the tEGFR according to the present disclosure, and the CAR-T-cells are infused into a recipient in need of such a therapy. The infused cells kill tumor cells in the recipient. Unlike antibody therapy, CAR-T-cells are capable of in vivo replication and production, which leads to a long-term sustained control of tumor.

The CAR-T-cells-mediated anti-tumor immune response may be an active or a passive one. Additionally, the CAR-mediated immune response may be part of an adoptive immunotherapy, wherein the CAR-T-cells induce an immune response with a specificity defined by the antigen-binding part of the CAR.

Accordingly, the diseases that can be treated using the CAR, the sequence encoding same, the nucleic acid construct, the expression vector, the virus and the CAR-T-cells according to the present disclosure are preferably diseases mediated by BCMA.

The CAR-modified T-cells according to the present disclosure may be used alone or in form of a pharmaceutical composition and in combination with a diluent and/or other components like relevant cytokine (s) or cell population (s) . Briefly, the pharmaceutical composition according to the present disclosure may comprise the CAR-T-cells according to the present disclosure in combination with one or more pharmaceutically or physiologically acceptable carrier (s) , diluent (s) or excipient (s) . The composition may comprise a buffer solution, such as a neutral buffered saline, sulfate buffered saline, etc; a carbohydrate, such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid, such as glycine; an antioxidant; a chelator, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide) ; and a preservative.

The pharmaceutical composition according to the present disclosure may be administered in a manner as appropriate for the disease that is to be treated or prevented. The amount and frequency of administration will be determined by known factors, like the medical condition of the patient and the classification and severity of the disease.

When referring to "an immunologically effective amount" , "an anti-tumor effective amount" , "a tumor-inhibition effective amount" or "a therapeutically effective amount" , the exact amount at which the composition according to the present disclosure is to be administered will be determined by a physician on an individual basis with considerations including patient's (subject's) age, body weight, tumor size, degree of invasion or metastasis. Typically, the pharmaceutical composition comprising the T-cell may be administered at an dosage ranging from 10 ⁴ to 10 ⁹ cells/kg bodyweight, preferably 10 ⁵ to 10 ⁶ cells/kg bodyweight. The T-cell composition may also be administered multiple times by repeating the specified dosage. The cells may be administered using conventional infusion techniques as known in immunotherapy (see for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988) . The optimal dosage and regimen for a particular patient may be conveniently determined by monitoring the patient's signs of disease and making adjustment accordingly.

The composition may be administered in any way as convenient, like aerosol, injection, swallowing, infusion, implantation or transplantation. The composition may be administered to a patient subcutaneously, intradermally, intratumorally, intraductally, intraspinally, intramuscularly, intravenously or intraperitoneally. In one example, the T-cell composition according to the present disclosure is administered via intradermal or subcutaneous injection. In another example, the T-cell composition is preferably administered via intravenous injection. The T-cell composition may be directly injected into the tumor, lymph nodes or sites of infection.

In some examples according to the present disclosure, the CAR-T-cells or the composition may be supplied in combination with an additional therapy. The additional therapy may include but is not limited to chemotherapy, radiation and immunosuppressants. For instance, the additional therapy may be any of the radio-or chemo-therapies known as useful in diseases mediated by BCMA.

The term "anti-tumor effect" , as used herein, refers to a biological effect that is characterized in decreased tumor size, reduced number of tumor cells, reduced metastasis, increased life expectancy or improvement in any physiological symptoms associated with cancer.

The terms "patient" , "subject" , "individual" are used herein as exchangeable and all refer to a living organism, like an mammalian, in which the immune response can be induced. Examples include but are not limited to human beings, dogs, cats, mice, rats and corresponding transgenic species.

The present invention may provide inter alia the following advantages. The present invention advantageously synthesizes the whole gene sequence of the chimeric antigen receptor, which may be expressed by BCMA scFV-CD19scFV-41BB-CD3ζ, using the gene sequences of BCMA scFV+CD19 scFV, and sequences of the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB and the intracellular domain of human CD3ζ from NCBI GenBank. The whole gene sequence is then incorporated into a retrovirus vector (RV) , which enables delivery of the nucleic acid sequence of interests, i.e., the sequence encoding CAR. The recombinant plasmids are packaged into viruses in 293T-cells, to infect T-cells to express the chimeric antigen receptor. In one embodiment according to the invention, the chimeric antigen receptor-genetically modified T lymphocytes are transformed using a retrovirus-based process, which advantageously provides inter alia a high efficiency of transformation, a stable expression of the exogenous gene and a shortened period of in vitro culturing before reaching a clinical-grade number of T lymphocytes. The transferred nucleic acid is transcribed and expressed on surface of the transgenic T lymphocytes. The CAR expression retroviruses according to the invention were used to prepare CAR-T-cells according to the Retronectin method. CAR-T-cells of 3 days post-infection are assayed for infection efficiency for CAR by flowmetry. CAR-T-cells of 5 days post-infection were incubated in vitro with CD19-positive or BCMA-positive tumor cells (K562-CD19, MM. 1S, MM. 1S-CD19) for 5 hours before detection of CD107a expression and IFNγ secretion. CAR-T-cells of 5 days post-infection were incubated in vitro with CD19-positive or BCMA-positive tumor cells (K562-CD19, MM. 1S, MM. 1S-CD19) for 5 hours before detection of specific killing of tumor cells by the CAR-T-cells (cytotoxicity) . Accordingly, the CD19-BCMA-BBz CART according to the present disclosure is potentially useful in treatment of multiple myeloma.

The inventions will be described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. The inventions should never be construed as being limited to these following examples, but intended to include any and all variations that are obvious from the teachings provided herein. The methods and reagents used in the examples are those regular ones, unless otherwise indicated.

Example 1: Gene sequence of BCMA scFV-CD19 scFV-41BB-CD3ζ

1.1 Gene sequences of the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB, and the intracellular domain of human CD3ζ were obtained from the database on the website of NCBI. The anti-BCMA single chain antibody is derived from Clone C11D5.3, and the anti-CD19 single chain antibody from Clone FMC63. These sequences were codon-optimized at http: //sg. idtdna. com/site to ensure a better expression in human cells without change in the amino acid sequence encoded thereby.

The information of amino acid sequences and gene sequences are compiled in the SEQUENCE LISTING (SEQ ID NOs. 1-2) .

The specified sequences were linked in sequence, with distinct restriction sites introduced at each of the adjunctions between sequences, whereby to generate the complete sequence of BCMA -CD19-BBz.

1.2 Sequencing of the recombinant plasmids

The obtained recombinant plasmid was sequenced by Sangon Biotech (Shanghai) Co., Ltd. . The result was aligned to the to-be-synthesized BCMA -CD19-BBz sequence to confirm the correct sequence. The sequencing primers are:

forward: AGCATCGTTCTGTGTTGTCTC (SEQUNCE ID NO. 3)

reverse: TGTTTGTCTTGTGGCAATACAC (SEQUNCE ID NO. 4)

The plasmid constructed according to this example is schematically depicted in Fig. 1.

Example 2: Construction of viral vector comprising the CAR molecule

The nucleotide sequence of the CAR molecule prepared according to Example 1 was double digested with NotI (NEB) and EcoRI (NEB) , and inserted into the retrovirus RV vector at the NotI-EcoRI site using T4 ligase (NEB) . The vector was then transferred into the competent Escherichia coli. strain (DH5α) . After being confirmed correct by sequencing, the plasmids were purified using the plasmid purification kit from Qiagen. The purified plasmids were transferred into 293T-cells using the calcium phosphate-method for retrovirus packaging.

Example 3: Retrovirus packaging

1. Day 1: The 293T-cells should be within the 20 ^th passage and not over-confluent. The cells were plated at 0.6×10 ⁶ cells/ml on a 10cm dish containing 10ml DMEM medium. The cells were mixed well until uniform, and cultured at 37℃ over night;

2. Day 2: Transfection was conducted on 293T-cells grown to 90%confluence (normally, about 14-18 hrs after plating) ; A plasmid complex was prepared, which comprised RV-BCMA-CD19-BBz 12.5ug, Gag-pol 10ug, VSVg 6.25ug, CaCl ₂ 250ul, H ₂O 1ml, in a total volume of 1.25ml; Into a separate tube filled with HBS of the same volume as the plasmid complex, the plasmid complex was added with vortexing for 20s; The obtained mixture was gently added along the vessel's wall into the petri dish of 293T, incubated at 37℃ for 4hrs. Then the medium was removed and, after washing once with PBS, replaced with pre-warmed fresh medium;

3. Day 4: 48hrs after transfection, the supernatant was collected and filtered through a 0.45um filter, then divided into aliquots and stored at -80℃; To the cells, pre-warmed fresh DMEM medium was supplemented.

Example 4: Retrovirus infection of human T-cells

1. CD3+T-cells were purified using Ficcol solution (Tian Jin Hao Yang Biological Manufacture Co., Ltd) and conditioned in X-VIVO (LONZA) medium supplemented with 5%AB serum till cell density of 1×10 ⁶/mL. The cells were inoculated at 1ml/well onto a plate pre-treated with 50ng/ml anti-human CD3 antibody (Beijing T&L Biotechnology Co. Ltd) and 50ng/ml CD28 antibody (Beijing T&L Biotechnology Co. Ltd) , followed by addition of 100IU/ml IL-2 (Beijing SL Pharmaceutical Co. Ltd) . After cultivation under stimulation for 48 hours, the cells were infected with the viruses.

2. At the second day of the T-cell cultivation and activation, a 24-well non-tissue treated plates (corning) were prepared by coating with 250μl/well of Retronectin (Takara) diluted in PBS to the final concentration of 15μg/ml. The plates were kept in dark at 4℃overnight for use.

3. Two days after the T-cell cultivation and activation, two coated 24-well plates were taken out, the coating solution was pipetted off, and HBSS supplemented with 2%BSA was added at 500μl/well to block at room temperature for 30min. Then, the blocking solution was pipetted off and the plates were washed twice with HBSS supplemented with 2.5%HEPES.

4. Each well was added with 2ml virus solution/well of the virus solution, and the plate was then centrifuged at 2000g, 32℃ for 2 hrs.

5. Supernatant being discarded, into each well on the 24-well plates, 1×10 ⁶ activated T-cells were added in a volume of 1ml, wherein the medium was the T-cell culturing medium supplemented with 200IU/ml of IL-2; the plates were centrifuged at 1000g, 30℃ for 10min.

6. After centrifugation, the plates were incubated in an incubator at 37℃, 5%CO ₂.

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

8. Since the infection, cell density was measured every day; T-cell culturing medium supplemented with 100IU/ml of IL-2 was added as appropriate to maintain T-cell density at around 5×10 ⁵/ml and to effect cell expansion.

Example 5: Flow cytometric assay of CAR expression on surface of T-lymphocytes

The BCMA-CD19-BBz cells were collected by centrifugation 72 hours after the infection The cells were washed once with PBS and the supernatant was discarded. The cells were then exposed to corresponding antibodies in dark for 30min, washed again with PBS, re-suspended and assayed via flow cytometry. CAR ⁺ was detected using anti-mouse IgG F (ab') antibody (Jackson Immunoresearch) .

As exhibited in Fig. 2, the percentage of BCMA-CD19-tEGFR CART positive is at least 50%.

Example 6: Expression of CD19 and BCMA in target cells

The target cell MM. 1S-CD19 expresses both CD19 and BCMA. The cell line was constructed in-lab.

As exhibited in Fig. 3, MM. 1S-CD19 expresses CD19 at the efficiency of 99.6%.

Example 7: CD107a expression by CAR-T-cells co-incubated with target cells

1. Onto a V-bottom 96-well plate was added CART/NT-cells (2×10 ⁵ cells) and target cells (K562-CD19, MM. 1S, MM. 1S-CD19, 2×10 ⁵ cells) /control cells (K562, 2×10 ⁵ cells) re-suspended to 200μl in IL-2-free X-VIVO complete medium supplemented with BD GolgiStop (containing monesin, 1μl BD GolgiStop/1ml medium) . 2ul/well anti-CD107a antibody (1: 50) was added. After incubation at 37℃ for 4 hours, cells were collected.

2. Medium was removed by centrifugation, pellets were washed once with PBS and centrifuged at 400g, 4℃ for 5 minutes. Supernatant being discarded, into each tube was added an appropriate amount of antibodies specific for surface molecules CAR, CD3 respectively. The mixture was re-suspended into 100μl and incubated on ice in dark for 30 min.

3. Each tube was washed once with PBS (3mL) , followed by centrifugation at 400g, 5 minutes. The supernatant was carefully pipetted off.

After re-suspending in PBS, CAR, CD3, CD107a were detected by flow cytometry.

As exhibited in Fig. 4, CD19-BCMA CART-cells express CD107a at a percentage around 80%when co-incubated with single-target target cells (K562-CD19, MM. 1S) , around 80%with the dual-target target cell (MM. 1S-CD19) , and almost null with the control (K562) . The BCMA-CD19 dual-target CART's activity in vitro on the single-target target cells MM. 1S and K562-CD19 is comparable to the single-target BCMA CART or the CD19 CART in terms of CD107a expression.

Example 8: INF-γ secretion by CAR-T-cell co-incubated with target cells

1. Prepared CAR-T-cells were re-suspended in the Lonza medium, wherein cell density was adjusted to 1×10 ⁶/mL.

2. The test groups comprised in each well 2×10 ⁵ target cells (K562-CD19, MM. 1S, MM. 1S-CD19) or negative control cells (K562) , 2×10 ⁵ CAR-T-cells and 200μl Lonza medium free of IL-2. The mixture was added onto a 96-well plate. BD GolgiPlug (with monesin, 1μl BD GolgiPlug/1ml cell culture) was added and mixed well. The mixture was then incubated at 37℃ for 5 hours. The cells were collected as the test group.

3. Each tube was washed once with 1mL PBS, then centrifuged at 300g for 5 min. Supernatant being discarded, into each tube was added an appropriate amount of antibodies specific for surface molecules CAR, CD3 respectively. The mixture was re-suspended into 100μl and incubated on ice in dark for 30 min.

4. After washing with PBS, Fixation/Permeabilization solution was added at 250μl/EP tube followed by incubation at 4℃ for 20 min to fix the cells and to disrupt cell membrane. The cells were washed twice with 1× BD Perm/Wash ^TM buffer, 1mL each time.

5. The cells were stained for intracellular cytokines: certain amount of IFN-γ cytokine fluorescent antibody or negative control was diluted in BD Perm/Wash ^TM buffer to 50μl. Cells after fixation and membrane disruption were re-suspended in this diluted antibody solution, and incubated at 4℃ in dark for 30min, washed twice with 1×BD Perm/Wash ^TM buffer 1mL/time, and re-suspended in PBS.

6. Detection of CAR, CD3, IFN-γ was carried out by flow cytometry assay.

As exhibited in Fig. 5, CD19-BCMA CART-cells express IFN-γ at a percentage around 70%when co-incubated with single-target target cells (K562-CD19, MM. 1S) , around 70%with the dual-target target cell (MM. 1S-CD19) , and almost null with the control (K562) . The BCMA-CD19 dual-target CART's activity in vitro on the single-target target cells MM. 1S and K562-CD19 is comparable to the single-target BCMA CART or the CD19 CART in terms of IFN-γ expression

Example 9: Tumor-specific killing by CAR-T-cells incubated with target cells

1. K562 cells (CD19 or BCMA free, serving as the negative control) were re-suspended in serum-free medium (1640) , with cell concentration adjusted to 1×10 ⁶/ml, followed by addition of Fluorescent dye BMQC (2, 3, 6, 7-tetrahydro-9-bromomethyl-1H, 5H-quinolizino (9, 1-gh) coumarin) to the final concentration of 5μM.

2. After mixed to uniform, the mixture was incubated at 37℃ for 30min.

3. After centrifugation at room temperature, 1500rpm for 5min and the supernatant being discarded, the cells were re-suspended in the cytotoxicity medium (phenol red-free 1640+5%AB serum) and incubated at 37℃ for 60min.

4. The cells were washed twice with fresh cytotoxicity medium and re-suspended in fresh cytotoxicity medium to 1×10 ⁶ cells/ml.

5. MM. 1S-CD19 cells (expressing CD19 and BCMA, serving as target cells) were re-suspended in PBS supplemented with 0.1%BSA, and cell concentration was adjusted to 1×10 ⁶ cells/ml.

6. Fluorescent dye CFSE (carboxyfluorescein diacetate succinimidyl ester) was added to the final concentration of 1μM.

7. After mixed to uniform, the mixture was incubated at 37℃ for 10min.

8. At the end of incubation, an equal volume of FBS was added and incubated under room temperature for 2min to end the labeling reaction.

9. The cells were washed and re-suspended in the fresh cytotoxicity medium to 1×10 ⁶ cells/ml.

10. The effector T-cells were washed and re-suspended in the cytotoxicity medium and the concentration was adjusted to 5×10 ⁶ cells/ml.

11. In all experiments, cytotoxicity of the CD19-BCMA-BBz CAR-infected T-cells (CAR-T cell) was compared with cytotoxicity of the uninfected negative control effector T-cells (NT-cells) , both coming from the same patient.

12. The CD19-BCMA-BBz CAR-T and negative control effector T-cells were incubated at the T-cell : target cell ratios =5: 1, 1: 1 in 5ml sterile tubes (BD Biosciences) . In each of the groups of co-incubation, the target cells were 100,000 (50μl) in amount, and the negative control cells were K562 cells (100,000 cells, 50μl) . At the same time, another group is designed to merely comprise target cells and K562 negative control cells.

13. The cells were co-incubated at 37℃ for 5hrs.

14. At the end of incubation, 7-AAD (7-aminoactinomycin D) was added according to instruction immediately after the cells were washed with PBS, and then incubated on ice for 30min.

15. The cells were directly loaded onto the flow cytometer without washing, and the data were analyzed using Flow Jo.

16. The analysis was gated by 7AAD-negative living cells to detect the percentage of living target cells and the percentage of living negative control cells after co-incubation of the T-cells and the target cells.

cytotoxicity killing %= (1- (living target cells in presence of effector /living K562 cells in presence of effector) / (living target cells in absence of effector /living K562 cells in absence of effector) ) ×100%.

As exhibited in Fig. 6, at the effector: target ratio (E: T) of 5: 1, CD19-BCMA CART killed over 80%of the target cells MM. 1S-CD19.

Claims

A polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence comprising the followings linked in sequence: sequences encoding an anti-BCMA and an anti-CD19 single chain antibodies, a sequence encoding the hinge region of human IgG4, a sequence encoding the transmembrane region of human CD28, a sequence encoding the intracellular domain of human 41BB, a sequence encoding the intracellular domain of human CD3ζ and optionally a sequence encoding a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV; and

(2) a complementary sequence of the polynucleotide sequence of (1) .
The polynucleotide sequence of claim 1, characterized in that

a sequence encoding a signal peptide lies ahead of said sequence encoding the anti-BCMA single chain antibody and has the sequence of nucleotides 1-63 as set forth in SEQ ID NO: 1; and/or

the sequence encoding the light chain variable region of said anti-BCMA single chain antibody has the sequence of nucleotides 64-396 as set forth in SEQ ID NO: 1; and/or

the sequence encoding the heavy chain variable region of said anti-BCMA single chain antibody has the sequence of nucleotides 442-792 as set forth in SEQ ID NO: 1; and/or

the sequence encoding the heavy chain variable region of said anti-CD19 single chain antibody has the sequence of nucleotides 853-1212 as set forth in SEQ ID NO: 1; and/or

the sequence encoding the light chain variable region of said anti-CD19 single chain antibody has the sequence of nucleotides 1267-1587 as set forth in SEQ ID NO: 1; and/or

the sequence encoding said hinge region of human IgG4 has the sequence of nucleotides 1588-1623 as set forth in SEQ ID NO: 1; and/or

the sequence encoding the transmembrane region of human CD28 has the sequence of nucleotides 1627-1707 as set forth in SEQ ID NO: 1; and/or

the sequence encoding the intracellular domain of human 41BB has the sequence of nucleotides 1708-1833 as set forth in SEQ ID NO: 1; and/or

the sequence encoding the intracellular domain of human CD3ζ has the sequence of nucleotides 1834-2169 as set forth in SEQ ID NO: 1;

wherein, said fragment of EGFR comprises or consists of the extracellular domain III, the extracellular domain IV and the transmembrane region of EGFR; more preferably, the polynucleotide sequence of said fragment has the sequence of nucleotides 2314-3318 as set forth in SEQ ID NO: 1; preferably, said polynucleotide sequence further comprises a sequence encoding the signal peptide of the α chain of GM-CSF receptor, wherein said signal peptide of the α chain of GM-CSF receptor is positioned N terminal to said fragment of EGFR; preferably, the polynucleotide sequence of the signal peptide of the α chain of GM-CSF receptor has the sequence of nucleotides 2248-2313 as set forth in SEQ ID NO: 1.
A fusion protein selected from the group consisting of:

(1) a fusion protein comprising the followings linked in sequence: an anti-BCMA single chain antibody, an anti-CD19 single chain antibody, the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB and the intracellular domain of human CD3ζ, as well as optionally a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV; and

(2) a fusion protein derived from (1) , comprising one or more substitution (s) , deletion (s) or addition (s) in the amino acid sequence of (1) while retaining the activity of T-cell activation;

preferably, said anti-CD19 single chain antibody is of the anti-CD19 monoclonal antibody FMC63;

preferably, said anti-BCMA single chain antibody is of the anti-BCMA monoclonal antibody C11D5.3.
The fusion protein of claim 3, wherein, said fusion protein comprises one or more of the following features:

said fusion protein further comprises a signal peptide positioned N-terminal to said anti-BCMA single chain antibody, wherein said signal peptide preferably has the sequence of amino acids 1-21 as set forth in SEQ ID NO: 2;

the light chain variable region of said anti-BCMA single chain antibody has the sequence of amino acids 22-132 as set forth in SEQ ID NO: 2;

the heavy chain variable region of said anti-BCMA single chain antibody has the sequence of amino acids 148-264 as set forth in SEQ ID NO: 2;

the heavy chain variable region of said anti-CD19 single chain antibody has the sequence of amino acids 285-404 as set forth in SEQ ID NO: 2;

the light chain variable region of said anti-CD19 single chain antibody has the sequence of amino acids 423-529 as set forth in SEQ ID NO: 2;

said hinge region of human IgG4 has the sequence of amino acids 530-541 as set forth in SEQ ID NO: 2;

said transmembrane region of human CD28 has the sequence of amino acids 532-569 as set forth in SEQ ID NO: 2;

said intracellular domain of human 41BB has the sequence of amino acids 570-611 as set forth in SEQ ID NO: 2;

said intracellular domain of human CD3ζ has the sequence of amino acids 612-723 as set forth in SEQ ID NO: 2;

wherein, said signal peptide of the α chain of GM-CSF receptor and said intracellular domain of human CD3ζ are linked by a linker having the sequence of amino acids 750-771 as set forth in SEQ ID NO: 2; and/or

said fragment of EGFR has the sequence of amino acids 772-1106 set forth in SEQ ID NO: 2.
A nucleic acid construct, comprising the polynucleotide sequence according to claim 1 or 2;

preferably, said nucleic acid construct is a vector;

more preferably, said nucleic acid construct is a retrovirus vector comprising an origin of replication, a 3’ -LTR, a 5’ -LTR and the polynucleotide sequence according to claim 1 or 2.
A retrovirus, comprising the nucleic acid construct, preferably the vector and more preferably the retrovirus vector according to claim 5.
A genetically modified T-cell or a pharmaceutical composition comprising said genetically modified T-cell, wherein said cell comprises the polynucleotide sequence according to claim 1 or 2 or the nucleic acid construct according to claim 5, or is infected with the retrovirus according to claim 6, or stably expresses the fusion protein and optionally the fragment of EGFR comprising the extracellular domain III and the extracellular domain IV according to claim 3 or 4.
Use of the polynucleotide sequence of claim 1 or 2, the fusion protein of claim 3 or 4, the nucleic acid construct of claim 5 or the retrovirus of claim 6 for producing activated T-cells.
Use of the polynucleotide sequence according to claim 1 or 2, the fusion protein according to claim 3 or 4, the nucleic acid construct according to claim 5, the retrovirus according to claim 6 or the genetically modified T-cell or the pharmaceutical composition according to claim 7 for manufacturing a medicament for treating a disease mediated by BCMA;

wherein said disease mediated by BCMA is multiple myeloma.