CN108165568B

CN108165568B - Method for culturing CD19CAR-iNKT cells and application

Info

Publication number: CN108165568B
Application number: CN201611116333.6A
Authority: CN
Inventors: 黄飞; 何凤; 赵晓楠; 金涛; 王海鹰; 史子啸
Original assignee: Shanghai Hrain Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co.,Ltd.
Priority date: 2016-12-07
Filing date: 2016-12-07
Publication date: 2020-12-08
Anticipated expiration: 2036-12-07
Also published as: CN108165568A

Abstract

The present invention relates to targeting CD19CAR-iNKT cells and uses thereof. Specifically, the invention provides a polynucleotide sequence, an activation culture and application thereof, wherein the polynucleotide sequence is selected from: (1) comprises a coding sequence of anti-CD 19 single-chain antibody, a coding sequence of human CD8 alpha hinge region, a coding sequence of human CD8 transmembrane region, a coding sequence of human 41BB intracellular region and a coding sequence of human CD3 zeta intracellular region which are connected in sequence; and (2) the complement of the polynucleotide sequence of (1). The invention also provides a related fusion protein, a vector containing the coding sequence, and applications of the fusion protein, the coding sequence and the vector. The invention also provides activation of iNKT cells, a culture method and an application range thereof.

Description

Method for culturing CD19CAR-iNKT cells and application

Technical Field

The invention belongs to the field of chimeric antigen receptors, and particularly relates to a targeted CD19CAR-iNKT cell and application thereof.

Background

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 CAR-T cells are rapidly depleted in the patient and have poor persistence, so that CAR-T cells that have not yet reached apoptosis when they have been exposed to a large number of tumor cells can trigger an anti-tumor cytotoxic effect, but less cytokine secretion, but their short survival in vivo cannot trigger a persistent anti-tumor effect [ Cancer Res 2007, 67 (22): 11029-11036].

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 CARs 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, so that T cells can be continuously activated and proliferated, cytokines can be continuously secreted, and the ability of T cells to kill tumor cells is more remarkable, i.e. a 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.

Fourth generation CAR-T cells are supplemented with cytokines or co-stimulatory ligands, e.g. fourth generation CARs can produce IL-12, which can modulate the immune microenvironment-increase the activation of T cells, while activating innate immune cells to function to eliminate target antigen negative cancer cells, thus achieving a bi-directional regulatory effect [ Expert Opin biol ther, 2015; 15(8): 1145-54].

At present, the clinical findings of CAR-T immunotherapy in solid tumors are not optimistic. Firstly, the immunosuppression of the tumor microenvironment is not favorable for the migration, colonization and functional performance of T cells; secondly, the heterozygosity of the T cells is large, so that the prepared CAR-T cells have large heterogeneity and inconsistent functions, the anti-tumor effect is influenced, and the risk of inducing toxicity is increased. Although studies have suggested to boost the anti-tumor function of CAR-T cells by limiting the composition of T cell subsets, the survival of these CAR-T cells in vivo and the ability to infiltrate tumor tissue remains limited.

Natural Killer T (NKT) cells are a special class of lymphocytes that mediate innate and adaptive immunity, and are considered to be class 4 lymphocytes [ Nat Rev immunol., 2010; 10:272-277]. It expresses both T cell and NK cell surface molecular markers and shows the biological characteristics of both T cell and NK cell. According to currently accepted systematic nomenclature, these are classified into type I, type II and type III NKT cells, each with different functional characteristics. Type I NKT cells, i.e., iNKT (innovative natural killer T) cells, are considered to be classical NKT cells. iNKT cells express a constant TCR α chain (TCRV α 14-J α 18 in mice, TCRV α 24-J α 18 in humans), the pairing of which is diverse, but with limited variability compared to the TCR β chain of traditional MHC-restricted T cells. iNKT cells recognize lipid antigens presented by non-polymorphic CD1d molecules, where α GalCer is a classical antigen that activates iNKT cells. The iNKT cell can quickly secrete a large amount of cell factors after being stimulated by antigen, regulates innate immunity and adaptive immunity, and plays an important role in the aspects of body infection resistance, tumor and autoimmune disease.

Research shows that iNKT cells can migrate to tumor tissues under the induction of chemokines produced by tumor cells and tumor-associated macrophages, and can kill tumor cells, and also act on tumor-associated macrophages through CD1d dependence, kill such tumor growth promoting cells, or inhibit their tumor promoting functions [ J Clin invest, 2012; 122:2221-2233].

Since the receptor portion of iNKT cells is relatively constant. it is inevitable that iNKT cells, once activated by recognizing the glycolipid antigen α GalCer, produce high concentrations of cytokines and participate in diverse immune regulation. In fact, α GalCer itself is an anti-tumor agent. α GalCer-activated NKT cells play an important killing role against those metastatic cancers in cooperation with IL-12 in lung and liver. On the other hand, NKT cells are activated after α GalCer is used. The activated NKT cells release a large amount of IFN-gamma; IFN-gamma in turn activates NK cells in vivo. The aim of controlling the tumor is achieved due to the mobilizing of the tumor killing effect of NK cells. The cellular and molecular mechanisms by which α GalCer induces anti-tumor also include upregulation of CD40L and the expression of cytotoxic molecules. CD40L on the surface of NKT cells activates CD40 on the surface of DC cells, resulting in IL-12 production by DC cells. IL-12 secreted by DC cells in turn activates NKT cells. Activated NKT cells produce IFN-y. IFN-gamma can also activate NK cells and cytotoxic Cells (CTL) of CD8+, thereby mediating the anti-tumor effect [ Cancer Sci, 2006,97(9):807-812 ]. iNKT cells are present in intermediate states of natural and acquired immunity. Participate in various immune response reactions. iNKT cells are very attractive as a new technology for immunotherapy. At the same time, the potential toxicity of iNKT cells, both autologous and allogeneic, is greatly reduced compared to T cells due to the non-polymorphism of the CD1d molecule. Therefore, CAR-iNKT cell immunotherapy has a huge application space.

The chimeric antigen receptor modified iNKT cell targeting CD19 shows good functions in an in vitro function test, and lays a foundation for the future application in clinical experiments or the commercialization process of allogeneic CAR-iNKT cells.

Disclosure of Invention

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

(1) comprises a coding sequence of anti-CD 19 single-chain antibody, a coding sequence of human CD8 alpha hinge region, a coding sequence of human CD8 transmembrane region, a coding sequence of human 41BB intracellular region and a coding sequence of human CD3 zeta intracellular region which are connected 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 prior to the coding sequence for the anti-CD 19 single chain antibody. In one or more embodiments, the amino acid sequence of the signal peptide is as set forth in amino acids 1-21 of SEQ ID NO 2.In one or more embodiments, the light chain variable region of the anti-CD 19 single chain antibody has the amino acid sequence as shown in amino acids 22-128 of SEQ ID NO. 2.In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single chain antibody is as shown in amino acids 144-263 of SEQ ID NO. 2.In one or more embodiments, the amino acid sequence of the human CD8 α hinge region is depicted as amino acids 264-310 of SEQ ID NO 2.In one or more embodiments, the amino acid sequence of the transmembrane region of human CD8 is as shown in SEQ ID NO 2 at amino acids 311-332. In one or more embodiments, the amino acid sequence of the intracellular domain of human 41BB is as shown in amino acids 333-380 of SEQ ID NO 2.In one or more embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 381-491 of SEQ ID NO 2.

In one or more embodiments, the coding sequence for the signal peptide preceding the coding sequence for the anti-CD 19 single chain antibody is as set forth in nucleotide sequences 1-63 of SEQ ID NO. 1.In one or more embodiments, the light chain variable region encoding sequence of the anti-CD 19 single chain antibody is as shown in SEQ ID NO. 1, nucleotide sequences 64-384. In one or more embodiments, the coding sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is as shown in the nucleotide sequences at positions 430-789 of SEQ ID NO. 1.In one or more embodiments, the coding sequence for the human CD8 α hinge region is as shown in nucleotide sequence 790-930 of SEQ ID NO. 1.In one or more embodiments, the coding sequence for the transmembrane region of human CD8 is as shown in SEQ ID NO 1, nucleotide sequence 931 and 996. In one or more embodiments, the coding sequence of the intracellular region of human 41BB is as shown in the nucleotide sequence at position 997-1140 of SEQ ID NO. 1.In one or more embodiments, the coding sequence for the intracellular domain of human CD3 ζ is as set forth in nucleotide sequences SEQ ID No. 1, position 1144-1476.

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

(1) a fusion protein comprising an anti-CD 19 single-chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human 41BB intracellular region, and a human CD3 ζ intracellular region, which are linked in this order; 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 activating iNKT cells;

preferably, the anti-CD 19 single chain antibody is anti-CD 19 monoclonal antibody FMC 63.

In one or more embodiments, the fusion protein further comprises a signal peptide at the N-terminus of the anti-CD 19 single chain antibody. In one or more embodiments, the amino acid sequence of the signal peptide is as set forth in amino acids 1-21 of SEQ ID NO 2.In one or more embodiments, the light chain variable region of the anti-CD 19 single chain antibody has the amino acid sequence as shown in 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-CD 19 single chain antibody can be shown as amino acids 148-264 of SEQ ID NO. 2.In one or more embodiments, the amino acid sequence of the human CD8 α hinge region is depicted as amino acids 265 and 311 of SEQ ID NO 2.In one or more embodiments, the amino acid sequence of the transmembrane region of human CD8 is depicted as amino acids 312-333 of SEQ ID NO 2.In one or more embodiments, the amino acid sequence of the intracellular domain of human 41BB is as shown in amino acids 334-381 of SEQ ID NO 2.In one or more 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.

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 iNKT 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.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a genetically modified iNKT 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 iNKT cell.

The eighth aspect of the present invention is a method for activating a genetically modified iNKT cell, a culture method and use thereof

In a ninth aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct, retrovirus, or genetically modified INKT cell as described herein, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a CD 19-mediated disease.

In one or more embodiments, the CD 19-mediated disease is leukemia, lymphoma.

Drawings

FIG. 1 is a schematic representation of a CD19-CAR retroviral expression vector (CD 19-BBz). SP: a signal peptide; VL: a light chain variable region; and Lk: joint (G)₄S)₃(ii) a VH: a heavy chain variable region; h: a CD8 a hinge region; TM: the CD8 transmembrane domain.

FIG. 2 is a partial sequencing peak plot of the CD19-CAR retroviral expression vector (CD 19-BBz).

Figure 3 CAR positive rate of iNKT cells was shown by flow cytometry 5 days after retroviral infection of iNKT cells.

FIG. 4 shows IL-2 secretion from CD9CAR iNKT prepared for 14 days in coculture with target cells for 5 hours.

Figure 5 is the secretion of INF γ by co-culture of CD19CAR iNKT with target cells for 5 hours, prepared for 14 days.

Figure 6 is a graph of the killing effect on tumor cells after preparing 14 days of CD19CAR iNKT cells and co-culturing with target cells for 4 hours.

Detailed Description

The present invention provides a Chimeric Antigen Receptor (CAR) that targets CD 19. The CAR comprises fragments of a sequentially linked anti-CD 19 single chain antibody, human CD8 α hinge region, human CD8 transmembrane region, human 41BB intracellular region, human CD3 ζ intracellular region.

anti-CD 19 single chain antibodies suitable for use in the present invention may be derived from a variety of anti-CD 19 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, FMC63, SJ25C 1.In certain embodiments, the monoclonal antibody is a monoclonal antibody having clone number FMC 63. In certain embodiments, the light chain variable region of the anti-CD 19 single chain antibody has the amino acid sequence shown as amino acid residues 22-128 of SEQ ID NO. 2.In other embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single chain antibody is as shown in amino acid residues 144-263 of SEQ ID NO: 2.

The amino acid sequence of the human CD8 alpha hinge region suitable for use in the present invention can be shown as amino acids 264 and 310 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 311-332 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 333-380 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 381-491 of SEQ ID NO 2.

The above-mentioned portions forming the fusion protein of the invention, e.g. the anti-CD 19 single chainThe variable regions of the light chain and the heavy chain of the antibody, the human CD8 α hinge region, the human CD8 transmembrane region, 41BB, and the human CD3 ζ intracellular region, and the like, may be directly linked to each other, or may be linked by a linker sequence. The linker sequence may be one known in the art to be suitable for use with antibodies, for example, a G and S containing linker sequence. Typically, the linker contains one or more motifs which repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent in the linker sequence with no intervening amino acid residues between the repeats. The linker sequence may comprise 1, 2,3, 4 or 5 repeat motifs. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is usually 2 to 20, such as 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (a), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), and the like. As an example, the linker may consist of the amino acid sequence of any one of SEQ ID NO 7-18. In certain embodiments, the anti-CD 19 single chain antibody of the invention consists of (GGGGS) between the light chain variable region and the heavy chain variable region_nAnd (b) connecting, wherein n is an integer of 1-5.

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, B, gE and Ty 1. These tags can be used to purify proteins.

The invention also includes mutants of the CAR represented by the CAR sequence shown as amino acid sequence 22-491 of SEQ ID NO 2. These mutants include: an amino acid sequence having 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 retaining the biological activity of the CAR (e.g., activating iNKT cells). Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI.

Mutants also include: an amino acid sequence having one or several mutations (insertions, deletions or substitutions) in the amino acid sequence shown in positions 22-491, the amino acid sequence shown in positions 1-491 of SEQ ID NO:2, while still retaining the biological activity of the CAR. The number of mutations usually means within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, conservative substitutions with amino acids of similar or similar properties are not typically used in the art to alter the function of a protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, substitution of one or more sites with another amino acid residue from the same side chain species in the polypeptide of the invention will not substantially affect its activity.

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 1473 of SEQ ID NO. 1, or as set forth in nucleotides 1 to 1473 of SEQ ID NO. 1.

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.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV 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. Further, inducible promoters are also contemplated. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter during periods of expression and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline 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.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

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 iNKT cells, the virus containing a retroviral vector as described herein and corresponding packaging genes, such as gag, pol and vsvg.

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

In certain embodiments, upon obtaining iNKT cells, activation may be stimulated with an appropriate amount of α Galcer (e.g., 3-8 μ g/ml, e.g., 5 μ g/ml), followed by culturing in a medium containing an appropriate amount of IL2 (e.g., 30-80 IU/ml, e.g., 50IU/ml) for use.

Thus, in certain embodiments, the invention provides a genetically modified iNKT 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.

The CAR-iNKT cells of the invention can undergo robust in vivo iNKT cell expansion and persist at high levels in the blood and bone marrow for extended amounts of time and form specific memory iNKT cells. Without wishing to be bound by any particular theory, the CAR-iNKT 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 anti-tumor immune response elicited by CAR-iNKT cells may be an active or passive immune response. Additionally, the CAR-mediated immune response may be part of an adoptive immunotherapy step, in which the CAR-iNKT cells induce an immune response specific to 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-iNKT cells of the invention are preferably CD 19-mediated diseases.

The CAR-modified iNKT 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, the pharmaceutical compositions of the invention may comprise CAR-iNKT 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 iNKT 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. iNKT cell compositions 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 iNKT cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the iNKT cell composition of the present invention is preferably administered by intravenous injection. Compositions of iNKT cells can be injected directly into the tumor, lymph node or site of infection.

In some embodiments of the invention, the CAR-iNKT 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 combined with radiation or chemotherapeutic agents known in the art for the treatment of CD19 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 adopts the gene sequence of an anti-CD 19 antibody (particularly scFV derived from clone number FMC 63), searches the gene sequence information of human CD8 alpha hinge region, human CD8 transmembrane region, human 41BB intracellular region and human CD3 zeta intracellular region from NCBI GenBank database, synthesizes the gene segment of chimeric antigen receptor anti-CD 19scFv-CD8 hinge region-CD 8TM-41BB-CD3 zeta in a whole gene and inserts the gene segment into a retrovirus vector. The recombinant plasmid packages the virus in 293T cells, infects iNKT cells, and allows iNKT cells to express the chimeric antigen receptor. The invention discloses a method for transforming iNKT cells modified by chimeric antigen receptor genes, which is based on a retrovirus transformation method. The method has the advantages of high transformation efficiency, stable expression of exogenous genes, and capability of shortening the time for culturing iNKT cells in vitro to reach clinical level number. On the surface of the transgenic iNKT cell, the transformed nucleic acid is expressed by transcription and translation. The CAR-iNKT cell prepared by the invention has strong killing function on specific tumor cells, and the killing efficiency exceeds 65% under the condition that the effective target ratio is 10: 1.

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 CD19scFv-CD8 alpha-41 BB-CD3 zeta Gene

The sequence information of human CD8 transmembrane region, human 41BB intracellular region and human CD3 zeta intracellular region gene is searched from NCBI website database, the cloning number of the anti-CD 19 single-chain antibody is FMC63, and the sequences are subjected to codon optimization on website http:// sg.

The sequences are connected in sequence by adopting overlapping PCR according to the sequences of anti-CD 19scFv, human CD8 alpha hinge region gene, human CD8 transmembrane region gene, human 41BB intracellular region gene and human CD3 zeta intracellular region gene, and different enzyme cutting sites are introduced at the connection positions of the sequences to form a complete CD19-CAR gene sequence.

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 (DH 5. alpha.).

The recombinant plasmid is sent to Shanghai Biotechnology Limited company for sequencing, and the sequencing result is compared with the sequence of the synthesized mCD19-CAR to verify whether the sequence is correct. The sequencing primer is as follows:

sense of justice AGCATCGTTCTGTGTTGTCTC

Antisense TGTTTGTCTTGTGGCAATACAC

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: retroviral packaging

1. Day 1 293T cells should be less than 20 passages, but overgrown. Plating with 0.6 x 10 cells/ml, adding 10ml DMEM medium to 10cm dish, mixing well, culturing at 37 degrees overnight.

2. On day 2, 293T cells are transfected to a confluence of about 90% (usually, plating for about 14-18 h); plasmid complexes were prepared with 12.5ug of retrointerbone, 10ug of Gag-pol, 6.25ug of VSVg, CaCl for each plasmid₂ 250ul，H₂O is 1ml, and the total volume is 1.25 ml; in anotherHBS with the same volume as the plasmid complex was added to the tube, 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 with pre-warmed fresh medium.

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

Example 3: activation of iNKT cells

1. After PBMC isolation, iNKT cells were harvested using the iNKT sorting kit from Miltenyi Biotec (Cat.130-091-) -221 and resuspended in iNKT medium [ X-VIVO ]^M15(LONZA) + 1% diabody (GIBCO) + 1% GlutaMax (GIBCO) + 1% HEPES (GIBCO) + 2% N-acetyl-cysteine (Melinant) + 5% inactivated human AB serum (GEMINI) +200IU/ml IL-2 (Beijing Erlu)]。

2. The PBMC after being irradiated by 40Gy gamma rays is taken, centrifuged and resuspended in iNKT culture medium to suspend cells, 5 mu g/ml alpha Galcer (Avanti) is added, and after uniform mixing, the cells are put into an incubator at 37 ℃ and 5% CO2 for pretreatment for 2 h.

3. After pretreatment, iNKT: the PBMC after gamma irradiation were mixed at a ratio of 1:5, cultured at 37 ℃ in an incubator containing 5% CO2, and IL-2 was added every other day at a concentration of 200 IU/ml.

After 4.10 days, iNKT cells were activated twice in the same manner.

Example 4: retroviral infection of iNKT cells

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

On day 3 after the second activation culture of iNKT cells, the coated 24-well plate was removed, the coating solution was aspirated and added with hbss (life) containing 2% bsa (excell) 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.

3. Adding the virus solution into each well, adding 2ml of virus solution into each well, centrifuging at 32 ℃ for 2000g, and centrifuging for 2 h.

4. The supernatant was discarded, and activated iNKT cells were added to each well of 24-well plates at 5X 10⁵Volume 1ml, culture medium iNKT medium, 30 ℃, 1000g, centrifugation for 10 min.

5. After centrifugation, the plates were incubated at 37 ℃ in a 5% CO2 incubator.

6. 24h after infection, the cell suspension was aspirated and centrifuged at 1500rpm, 4 ℃ for 5 min.

7. After cell infection, the density of the cells was observed every day, and iNKT medium was supplemented at appropriate times to maintain the density of iNKT cells at 5 × 10⁵Cells were expanded at around/ml.

8. CAR-iNKT cells infected with CD19-41BB-CAR retrovirus, named CD19CAR-iNKT cells, were thus obtained.

Example 5: flow cytometry for detecting proportion of iNKT cells after infection and expression of surface CAR protein

And respectively centrifuging and collecting the CAR-iNKT cells and NT-iNKT cells (a control group) 72 hours after infection, washing with PBS (phosphate buffer solution) for 1 time, then discarding the supernatant, adding corresponding antibodies, washing with PBS (phosphate buffer solution) after keeping out of the sun for 30min, resuspending, and finally detecting the CAR positive rate by a flow cytometer. The antibody is anti-mouse IgG F (ab') antibody (Jackson Immunoresearch).

The detection result of the embodiment is shown in fig. 3, the proportion of iNKT cells is above 90%, and the purity is high; the proportion of CAR-iNKT cells was 65.8% 120 hours after infection of iNKT cells with CD19-41BB-CAR retrovirus.

Example 6: IL-2 secretion assay after coculture of CAR-iNKT cells with target cells

1. Prepared CAR-iNKT cells were taken and resuspended in Lonza medium to adjust the cell concentration to 1X 10⁶/mL。

2. The experimental group contained 2X 10 target cells (Raji) or negative control cells (K562) per well ⁵2, CAR-T cells 2X 10⁵200. mu.l of Lonza medium; mixing completely and adding into 96-well plate; adding BD GolgiPlug (containing BFA, and adding 1 μ l BD GolgiPlug to 1ml cell culture medium), mixing, and incubating at 37 deg.C for 5 hr; after the incubation, the cells were collected as experimental groups.

3. The cells were washed with 1mL of PBS per tube, centrifuged at 300g for 5min and the supernatant carefully aspirated or decanted.

4, PBS washing cells, adding 250 μ l/EP tube Fixation/Permeabilization solution, and incubating at 4 deg.C for 20min to fix cells and rupture membranes; using 1 XBD Perm/Wash^TMbuffer washes cells 2 times, 1 mL/time.

5. Staining with intracellular factor, collecting appropriate amount of IL-2 cytokine fluorescent antibody or negative control, and performing BD Perm/Wash^TMDiluting to 50 μ l with buffer, resuspending the fixed and ruptured cells with the antibody diluent, incubating at 4 deg.C in the dark for 30min, 1 XBD Perm/Wash^TMbuffer 1 mL/wash cells 2 times, then use PBS heavy suspension.

6. Flow cytometry detection

The results of this example show in FIG. 4 that the percentage of IL-2 secretion by CD19-41BB-CAR-iNKT cells under the action of CD19 negative K562 cells was 52.78% ((36.80% -2.49%)/CAR⁺Rate), the percentage of IL-2 secreted by CD19-41BB-CAR-iNKT cells under the action of CD19 positive Raji cells was 75.08% ((52.5% -3.10%)/CAR⁺Rate).

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

5. Staining with intracellular factor, taking appropriate amount of IFN-gamma cytokine fluorescent antibody or negative control, and performing BD Perm/Wash^TMDiluting to 50 μ l with buffer, resuspending the fixed and ruptured cells with the antibody diluent, incubating at 4 deg.C in the dark for 30min, 1 XBD Perm/Wash^TMbuffer 1 mL/wash cells 2 times, then use PBS heavy suspension.

6. Flow cytometry detection

The results of this example show in FIG. 5 that the percentage of INF- γ secretion by CD19-41BB-CAR-iNKT cells is 9.20% ((6.64% -0.60%)/CAR in CD19 negative K562 cells⁺Rate), the percentage of INF- γ secreted by CD19-41BB-CAR-iNKT cells under the action of CD19 positive Raji cells was 66.69% ((44.6% -0.72%)/CAR⁺Rate).

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

K562 cells (not containing CD19 target protein, negative control cells for target cells) resuspended in serum-free medium (1640)

In (1), the cell concentration is adjusted to 1X 10⁶Perml, the fluorescent dye BMQC (2,3,6,7-tetrahydro-9-bromomethyl-1H,5Hquinolizino (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 cells were washed twice and resuspended in fresh cytotoxic Medium at a density of 1X 10⁶/ml。

Raji cells (expressing CD19 target protein, as 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。

10. iNKT cells were washed and suspended in cytotoxic medium at a concentration of 5X 10⁶/ml。

CAR-iNKT and NT-iNKT, according to effector cell: the target cells were cultured in 5ml sterile test tubes (BD Biosciences) at a ratio of 10:1, 3:1, 1:1, with triplicate wells per group. In each co-culture group, the target cells were Raji cells (50. mu.l) and the negative control cells were K562 cells (50. mu.l) of 50,000. A panel was set up to contain only Raji target cells and K562 negative control cells.

12. The co-cultured cells were incubated at 37 ℃ for 4 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 ratio of viable Raji target cells to viable K562 negative control cells after coculture of iNKT cells and target cells was determined using 7AAD negative viable cell gating.

16. For each set of co-cultured iNKT cells and target cells:

17. the% cytotoxic killer cells is 100-the% calibrated target cell survival, i.e. (ratio of Raji viable cell number when no effector cells were present-Raji viable cell number when effector cells were present)/K562 viable cell number.

The detection result of the embodiment shows that in the figure 6, the killing rate of CD19-41BB-CAR-iNKT cells to CD19 positive Raji cells reaches 65% under the condition that the effective target ratio is 10: 1.

Sequence listing

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

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20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

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Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

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Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

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

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

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275 280 285

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

290 295 300

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

305 310 315 320

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325 330 335

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340 345 350

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355 360 365

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

370 375 380

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

385 390 395 400

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Claims

1. A genetically modified iNKT cell or a pharmaceutical composition comprising the genetically modified iNKT cell, wherein the cell comprises a fusion protein comprising, in sequence, an anti-CD 19 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,

wherein the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown as the amino acids at the 22 nd to 128 th positions of SEQ ID NO. 2, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is shown as the amino acids at the 144 nd and 263 nd positions of SEQ ID NO. 2, and the amino acid sequence of the human CD8 alpha hinge region is shown as the amino acids at the 264 th and 310 nd 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 311-332 position of SEQ ID NO. 2; the amino acid sequence of the intracellular region of the human 41BB is shown as the 333-380 amino acid of SEQ ID NO. 2; the amino acid sequence of the intracellular domain of human CD3 zeta is shown as amino acid 381-491 of SEQ ID NO 2.

2. The iNKT cell or the pharmaceutical composition of claim 1, wherein the fusion protein further comprises a signal peptide at the N-terminus of the anti-CD 19 single chain antibody.

3. The iNKT cell or pharmaceutical composition of claim 2, wherein the signal peptide has the amino acid sequence as set forth in amino acids 1 to 21 of SEQ ID NO 2.

4. A genetically modified iNKT cell or a pharmaceutical composition comprising the genetically modified iNKT cell, wherein the cell comprises a polynucleotide whose sequence comprises (1) a sequence encoding a single chain antibody to CD19, a sequence encoding a human CD8 α hinge region, a sequence encoding a human CD8 transmembrane region, a sequence encoding a human 41BB intracellular region and a sequence encoding a human CD3 ζ intracellular region, linked in that order, and/or (2) a sequence complementary to (1) the sequences;

wherein, the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown as the amino acid at the 22 nd-128 th site of SEQ ID NO. 2, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is shown as the amino acid at the 144 nd-263 nd site of SEQ ID NO. 2, and the amino acid sequence of the human CD8 alpha hinge region is shown as the amino acid at the 264 th-310 nd site 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 311-332 position of SEQ ID NO. 2; the amino acid sequence of the intracellular region of the human 41BB is shown as the 333-380 amino acid of SEQ ID NO. 2; the amino acid sequence of the intracellular domain of human CD3 zeta is shown as amino acid 381-491 of SEQ ID NO 2.

5. The iNKT cell or pharmaceutical composition of claim 4, wherein the polynucleotide further comprises a coding sequence for a signal peptide prior to the coding sequence for the single chain antibody against CD 19.

6. The iNKT cell or pharmaceutical composition of claim 5, wherein the signal peptide has the amino acid sequence as set forth in amino acids 1 to 21 of SEQ ID NO 2.

7. The iNKT cell or pharmaceutical composition of claim 4,

the coding sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown as the nucleotide sequence of the 64 th to 384 th positions of SEQ ID NO. 1; the coding sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is shown as the nucleotide sequence of the 430 th and 789 th positions of SEQ ID NO 1; the coding sequence of the human CD8 alpha hinge region is shown as the nucleotide sequence at the 790 nd and 930 th positions of SEQ ID NO. 1; the coding sequence of the transmembrane region of the human CD8 is shown as the nucleotide sequence of No. 931 and No. 996 of SEQ ID NO 1; the coding sequence of the human 41BB intracellular region is shown as the nucleotide sequence at position 997-1140 of SEQ ID NO. 1; the coding sequence of the intracellular region of human CD3 zeta is shown in the nucleotide sequence at position 1141-1473 of SEQ ID NO. 1.

8. A genetically modified iNKT cell or a pharmaceutical composition comprising the genetically modified iNKT cell, said cell comprising a nucleic acid construct comprising a polynucleotide whose sequence comprises (1) a coding sequence for a single chain antibody to CD19, a coding sequence for a human CD8 α hinge region, a coding sequence for a human CD8 transmembrane region, a coding sequence for a human 41BB intracellular region, and a coding sequence for a human CD3 ζ intracellular region, joined in that order, and/or (2) a sequence complementary to (1) the sequences;

9. The iNKT cell or pharmaceutical composition of claim 8, wherein the polynucleotide further comprises a coding sequence for a signal peptide prior to the coding sequence for the single chain antibody against CD 19.

10. The iNKT cell or the pharmaceutical composition of claim 9, wherein the signal peptide has the amino acid sequence as set forth in amino acids 1 to 21 of SEQ ID No. 2.

11. The iNKT cell or pharmaceutical composition of claim 8,

12. The iNKT cell or pharmaceutical composition of claim 8, wherein the nucleic acid construct is a vector.

13. The iNKT cell or the pharmaceutical composition of claim 8, wherein the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR.

14. A genetically modified iNKT cell or a pharmaceutical composition comprising the genetically modified iNKT cell, wherein the cell is infected with a retrovirus comprising a polynucleotide having a sequence comprising (1) a coding sequence for a single-chain antibody against CD19, a coding sequence for a hinge region of human CD8 a, a coding sequence for a transmembrane region of human CD8, a coding sequence for an intracellular region of human 41BB, and a coding sequence for an intracellular region of human CD3 ζ, linked in that order, and/or (2) a complementary sequence of (1) the sequences,

15. The iNKT cell or pharmaceutical composition of claim 14, wherein the polynucleotide further comprises a coding sequence for a signal peptide prior to the coding sequence for the single chain antibody against CD 19.

16. The iNKT cell or the pharmaceutical composition of claim 15, wherein the signal peptide has the amino acid sequence as set forth in amino acids 1 to 21 of SEQ ID No. 2.

17. The iNKT cell or pharmaceutical composition of claim 14,

18. The iNKT cell or pharmaceutical composition of claim 14, wherein the nucleic acid construct is a vector.

19. The iNKT cell or the pharmaceutical composition of claim 14, wherein the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR.

20. Use of a fusion protein comprising an anti-CD 19 single-chain antibody, a human CD8 a 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, a polynucleotide comprising the coding sequence of said fusion protein, a nucleic acid construct comprising said polynucleotide, or a retrovirus containing said polynucleotide, for the preparation of an agent for activating or culturing iNKT cells,

21. The use of claim 20, wherein said fusion protein further comprises a signal peptide at the N-terminus of said anti-CD 19 single chain antibody.

22. The use of claim 20, wherein the signal peptide has the amino acid sequence shown as amino acids 1-21 of SEQ ID No. 2.

23. Use of the iNKT cell or the pharmaceutical composition of any one of claims 1-19 in the manufacture of a medicament for treating a CD 19-mediated disease.

24. The use of claim 23, wherein the CD 19-mediated disease is leukemia or lymphoma.