CN113980136A - Chimeric antigen receptor containing CD3 epsilon intracellular domain with Y/F mutation and application thereof - Google Patents

Abstract

The present invention provides a chimeric antigen receptor comprising the intracellular domain of CD3 epsilon with a Y/F mutation, comprising: an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence; the end of the intracellular domain connected with the transmembrane domain is connected with a CD3 epsilon intracellular domain with Y/F mutation, and the CD3 epsilon intracellular domain with Y/F mutation refers to a Y/F mutant CD3 epsilon intracellular domain with phenylalanine from two tyrosine homomutations in the ITAM of the CD3 epsilon intracellular domain. The improvement of the survival ability, the reduction of the apoptosis level and the improvement of the proliferation ability of the T cell modified by the chimeric antigen receptor can obviously improve the persistence of the T cell, thereby obviously improving the anti-tumor ability of the T cell. The advantages can further improve the tumor treatment effect, prolong the disease remission time, reduce the recurrence rate and improve the survival time of patients in clinical application. Meanwhile, the modified T cells can down-regulate the level of expression cytokines IFN-gamma and TNF-alpha, and reduce the activation of macrophage monocyte to generate inflammatory cytokines such as IL-1 beta and IL-6.

Description

Chimeric antigen receptor containing CD3 epsilon intracellular domain with Y/F mutation and application thereof

Technical Field

The invention relates to the field of chimeric antigen receptors, in particular to a chimeric antigen receptor containing a CD3 epsilon intracellular domain with Y/F mutation and application thereof.

Background

Chimeric antigen receptors are abbreviated as CARs (chimeric antigen receptors), and T cells carrying CARs against specific tumor antigens are called CAR-T cells. CAR-T therapy is widely applied to the treatment of tumors, particularly to the immunotherapy of solid tumors, is a novel accurate targeted therapy for treating tumors, has good effect on clinical tumor treatment by optimizing and improving in recent years, and is a novel tumor immunotherapy method which has a very promising prospect, can be accurate, rapid and efficient and is possible to cure cancers.

However, CAR-T therapy still suffers from limitations such as poor cytokine storm, neurotoxic safety, and sustained proliferative capacity encountered in clinical treatment of tumors. A one-phase clinical trial was published in the Lancet magazine 2015 (clinical trial No.: NCT01593696, 2015. lace.T cells expressing CD19 clinical anticancer receptors for access lymphoblastic leukaemia in childreng and young adolescent adults-a phase 1 dose-evolution triple) showing that patients with relapsed or refractory acute lymphoid leukemia or non-Hodgkin lymphoma (age 1-30 years) were tested for their number in blood-T cells after receiving KTE-C19(anti-CD 1928Z) CAR-T therapy, and the number of CAR-T cells was found to reach peak at day 14, significant at days 28 and 42, and no CAR-T cells were tested in the total patient's blood at day 68. There was also a phase I clinical trial published in the New England journal of 2018 (clinical trial No.: NCT01044069, 2018.NEJ. Long-Term Follow-up of CD19 CAR Therapy in Acute lymphoblast Leukamia) results, which also showed that 53 patients with relapsed Acute lymphoid Leukemia had the median number of blood CAR-T cells detected after receiving anti-CD 1928Z CAR-T treatment on day 14. Both studies show that 28Z CAR-T cells have poor viability for sustained proliferation and that therapeutic efficacy is to be improved.

Disclosure of Invention

The CAR structure is designed to be modified, and the CAR-T cell survival ability can be improved, so that the anti-tumor effect is improved, the disease remission time is prolonged, the recurrence rate is reduced, and the survival time of a patient is prolonged. In view of the above-described drawbacks of the prior art, it is an object of the present invention to provide a chimeric antigen receptor comprising the intracellular domain of CD3 epsilon having a Y/F mutation and uses thereof.

To achieve the above and other related objects, the present invention provides, in a first aspect, a chimeric antigen receptor comprising the intracellular domain of CD3 epsilon having a Y/F mutation. The method comprises the following steps:

an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence;

the extracellular domain comprises an antigen recognition region and a hinge region;

the intracellular domain is connected with one end connected with the transmembrane domain, and the CD3 epsilon intracellular region with Y/F mutation is connected with one end connected with the transmembrane domain, and the CD3 epsilon intracellular region with Y/F mutation refers to a Y/F mutant CD3 epsilon intracellular region with two tyrosines mutated into phenylalanine in an Immunoreceptor Tyrosine Activation Motif (ITAM) of the CD3 epsilon intracellular region.

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

(1) a polynucleotide sequence encoding the chimeric antigen receptor comprising the intracellular domain of CD3 epsilon having a Y/F mutation of the preceding claim; and

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

in a third aspect, the invention provides a nucleic acid construct comprising a polynucleotide sequence as described above;

preferably, the nucleic acid construct is a vector;

more preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3 'LTR, a 5' LTR and a polynucleotide sequence as described above.

In a fourth aspect, the invention provides a lentiviral vector system comprising the nucleic acid construct described above and a lentiviral vector accessory component.

In a fifth aspect, the invention provides a genetically modified T cell or a pharmaceutical composition comprising a genetically modified T cell, wherein the cell comprises the polynucleotide sequence, or comprises the nucleic acid construct, or is infected with the lentiviral vector system.

In a sixth aspect, the invention provides the use of a chimeric antigen receptor comprising the intracellular region of CD3 epsilon having a Y/F mutation, a polynucleotide sequence as defined above, a nucleic acid construct as defined above or a lentiviral vector system as defined above, for the preparation of a product for any one or more of the following uses: (1) preparing T cells; (2) increasing the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation and/or survival of T cells; (5) improving the anti-tumor capacity of the T cells; (6) inhibit the secretion of the cytokines IFN-gamma and TNF-alpha by T cells.

The seventh aspect of the invention provides the use of a chimeric antigen receptor comprising the intracellular domain of CD3 epsilon having a Y/F mutation as described above, a polynucleotide sequence as described above, a nucleic acid construct as described above, a lentiviral vector system as described above or a genetically modified T cell as described above for the preparation of a product for the treatment of a tumor.

As described above, the chimeric antigen receptor of the present invention comprising the intracellular domain of CD3 epsilon having Y/F mutation and the use thereof have the following advantageous effects:

the improvement of the survival ability, the reduction of the apoptosis level and the improvement of the proliferation ability of the T cell modified by the chimeric antigen receptor can obviously improve the persistence of the T cell, thereby obviously improving the anti-tumor ability of the T cell. The advantages can further improve the tumor treatment effect, prolong the disease remission time, reduce the recurrence rate and improve the survival time of patients in clinical application. Meanwhile, the modified T cells can down-regulate the level of expression cytokines IFN-gamma and TNF-alpha, and reduce the activation of macrophage monocyte to generate inflammatory cytokines such as IL-1 beta and IL-6.

Drawings

anti-CD 1928Z CAR, anti-CD19E 28Z CAR and anti-CD19E_YFStructural schematic of 28Z CAR and CD3 epsilon intracellular domain amino acid sequence and CD3 epsilon intracellular domain amino acid sequence with Y/F mutation. Wherein FMC63 is a single chain antibody targeting CD19 antigen, the Hinge region (Hinge region) and transmembrane region (transmembrane region) of the receptor are derived from human CD 28;

anti-CD19E 28Z CAR, CD3 epsilon inserted behind the CD28 transmembrane region and in front of the CD28 intracellular region; anti-CD19E_YFIn the 28Z CAR, the amino acid sequence of the intracellular region of CD3 epsilon with the Y/F mutation was inserted after the CD28 transmembrane region and before the intracellular region of CD 28.

FIG. 1b anti-CD 1928Z CAR, anti-CD19E 28Z CAR and anti-CD19E_YFFlow pattern of 28Z CAR at upper membrane level after T cell expression.

FIG. 1c anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFCD4 in 28Z CAR-T cells⁺And CD8⁺Flow chart of cell proportion.

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFComparison of IL-2 cytokine levels after stimulation of 28Z CAR-T cells with Raji cells (CD19 antigen).

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFComparison of IFN- γ cytokine levels after stimulation of 28Z CAR-T cells with Raji cells (CD19 antigen).

FIG. 1f anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFComparison of TNF-alpha cytokine levels in 28Z CAR-T cells following stimulation with Raji cells (CD19 antigen).

FIG. 1g anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFProliferation of 28Z CAR-T cells following stimulation with Raji cells (CD19 antigen).

FIG. 1h.anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YF28Z CAR-T cells via Rapoptosis following aji cell (CD19 antigen) stimulation.

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFSurvival number of 28Z CAR-T cells after stimulation with Raji cells (CD19 antigen).

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YF28Z CAR-T cell Pair CD19⁺Tumor cell toxicity reaction.

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFSignal molecule serine/threonine protein kinase Erk (1/2) for promoting cell proliferation survival and anti-apoptosis after stimulation of 28Z CAR-T cells by Raji cells (CD19 antigen)^{Thr202/Tyr204}Phosphorylation (pErk (1/2)^{Thr202/Tyr204}) And (4) horizontal.

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFSignal molecule serine/threonine kinase AKT for promoting cell proliferation survival and resisting apoptosis of 28Z CAR-T cells after stimulation of Raji cells (CD19 antigen)^S473Phosphorylation (pAKT)^S473) And (4) horizontal.

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFSignal molecule ribosomal protein S6 for marking cell growth and proliferation after stimulation of 28Z CAR-T cells by Raji cells (CD19 antigen)^S235/236Phosphorylation (pS 6)^S235/236) And (4) horizontal.

Detailed Description

The chimeric antigen receptor of the invention, which comprises the intracellular domain of CD3 epsilon with Y/F mutation, comprises:

an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence;

the extracellular domain comprises an antigen recognition region and a hinge region;

the end of the intracellular domain connected with the transmembrane domain is connected with a CD3 epsilon intracellular region with Y/F mutation, and the CD3 epsilon intracellular region with Y/F mutation refers to a Y/F mutant CD3 epsilon intracellular region in which two tyrosines in a CD3 epsilon intracellular region Immunoreceptor Tyrosine Activation Motif (ITAM) are mutated into phenylalanine.

Further, the amino acid sequence of the intracellular domain of CD3 epsilon with the Y/F mutation is shown in SEQ ID NO: 1 is shown in the specification; specifically, the method comprises the following steps: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLFSGLNQRRI are provided.

The intracellular domain comprises a CD3 epsilon intracellular region (two tyrosine are mutated into phenylalanine in an ITAM motif of the intracellular region), a costimulatory signaling region intracellular region and a CD3 zeta intracellular segment which are connected in sequence.

In one embodiment, the co-stimulatory signaling region is selected from the intracellular segment of one or more of CD27, CD28, CD134, 4-1BB, OX40, or ICOS.

In a further preferred embodiment, said region of co-stimulatory signaling is selected from the intracellular segment of CD 28. The killing ability is stronger.

The amino acid sequence of the CD28 intracellular segment is shown as SEQ ID NO: 2, specifically:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS。

in one embodiment, the amino acid sequence of the intracellular segment of CD3 ζ is set forth in SEQ ID NO: 3, respectively. The method specifically comprises the following steps: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR are provided.

In one embodiment, the antigen recognition region is selected from a single chain antibody directed against a tumor surface antigen selected from one or more of CD19, mesothelin (mesothelin), CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibre Activation Protein (FAP), Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13R α 2, CD171, and GD 2.

Preferably, the target is selected from CD19 or mesothelin, and the targeting specificity is good.

The transmembrane domain is selected from the transmembrane region of one or more of CD28, CD4, CD8 alpha, OX40 or H2-Kb.

Preferably, the transmembrane domain is selected from the transmembrane region of CD 28. Specifically, the amino acid sequence of the transmembrane region of the CD28 is shown as SEQ ID NO: 4, respectively. Specifically, FWVLVVVGGVLACYSLLVTVAFIIFWV.

The hinge region is selected from one or more of a CD28 hinge region, a CD8 alpha hinge region, a CD4 hinge region, a hinge region of immunoglobulin IgG or a hinge region of immunoglobulin IgG coupled to a CH2CH3 region. I.e., IgG1 change or IgG1 change-CH 2CH 3.

Alternatively, the hinge region sequence is a CD28 hinge region (CD28 hinge). The amino acid structure is shown as SEQ ID NO: shown at 7. Specifically, IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

In one embodiment, the single chain antibody comprises a light chain variable region and a heavy chain variable region.

In one embodiment, the light chain variable region is linked to the heavy chain variable region by a linker sequence.

In one embodiment, the single chain antibody is selected from FMC 63.

FMC63 may be a commercially available product.

In one embodiment, the single chain antibody comprises the light chain variable region and the heavy chain variable region of monoclonal antibody FMC63, optionally linked by a linker.

In one embodiment, the antigen recognition region of the chimeric antigen receptor comprising the intracellular region of CD3 epsilon having the Y/F mutation is selected from FMC63, and the hinge region is selected from the group consisting of a CD28 hinge region; the transmembrane domain is selected from the CD28 transmembrane region; the intracellular domain comprises a CD3 epsilon intracellular domain with a Y/F mutation, an intracellular segment of CD28 and an intracellular segment of CD3 zeta linked in sequence.

In one embodiment, the chimeric antigen receptor comprising the intracellular domain of CD3 epsilon with a Y/F mutation (Anti-CD 19E)_YF28Z CAR) is as set forth in SEQ ID NO: 5, specifically:

SEQ ID NO：5(Anti-CD19 E _YF28Z CAR amino acid sequence): (5 '→ 3') starting from FMC63 scFv are linked in sequence to a CD28 hinge region, a CD28 transmembrane region, a CD3 epsilon intracellular region with Y/F mutation and a CD3 zeta intracellular region:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLFSGLNQRRIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

SEQ ID NO：5(Anti-CD19 E _YF28Z CAR amino acid sequence) is SEQ ID NO: 6(Anti-CD 19E _YF28Z CAR nucleotide sequence), SEQ ID NO: 6 the following: (5 '→ 3') gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacCTGTTCCCAACCCAGACTTTGAGCCCATCCGGAAAGGCCAGCGGGACCTGTTTTCTGgcctgaatcagagacgcatcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

The above-mentioned respective portions forming the chimeric antigen receptor of the present invention may be directly linked to each other, or may be linked via 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.

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. The signal peptide hypothesis states that mRNA encoding a secreted protein is first translated to synthesize a signal peptide with hydrophobic amino acid residues at the N-terminus, which is recognized by and bound to receptors on the endoplasmic reticulum membrane. The signal peptide reaches the lumen of endoplasmic reticulum through the pore canal formed by the protein in the membrane, and then is hydrolyzed by the signal peptidase on the surface of the lumen, and the new polypeptide can enter the lumen through the endoplasmic reticulum under the guidance of the signal peptide, and is finally secreted to the outside of the cell. The signal peptides related to CAR are of different origins, mainly signal peptides of CD8 and CD28, GM-CSF receptor. The amino-terminus or the carboxy-terminus of the fusion protein of the invention (i.e., the CAR) may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty 1. These tags can be used to purify proteins.

The present invention provides a polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence encoding a chimeric antigen receptor according to the foregoing; and

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

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.

The nucleic acid construct provided by the invention contains the polynucleotide sequence.

The nucleic acid construct further includes one or more control sequences operably linked to the polynucleotide sequences described above. The coding sequence of the CAR of the invention can be manipulated in a variety of ways to ensure expression of the protein. The nucleic acid construct may be manipulated prior to insertion into the vector, depending on the type of expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

Preferably, the nucleic acid construct is a vector.

Expression of the polynucleotide sequence encoding the CAR is typically achieved by operably linking the polynucleotide sequence encoding the CAR 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 sequence encoding the CAR of the 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. 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 functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers.

More preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3 'LTR, a 5' LTR and the polynucleotide sequence as described above.

An example of a suitable promoter is the 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 lentiviral 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, lentiviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a lentiviral 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.

The invention provides a lentiviral vector system comprising the nucleic acid construct and lentiviral vector auxiliary components.

The lentiviral accessory components include lentiviral packaging plasmids and cell lines.

The lentivirus vector system is formed by virus packaging of the nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The construction method of the lentivirus vector system is a method commonly used in the field.

The invention provides a method for activating T cells in vitro, which comprises the step of infecting the T cells by using the lentivirus.

Compared with other drug therapies, based on the self-proliferation and survival maintaining capacity of the CAR-T cells, the CAR-T cells can be maintained in vivo for a long time, can rapidly expand and kill tumor cells with targets when being stimulated by targeted specific antigens, and then can form specific memory cells such as effector memory cells (effector memory T cells) and central memory cells (central memory T cells).

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

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

The cancer that can be treated can be a non-solid tumor, such as a hematological tumor, e.g., leukemia and lymphoma. In particular, the diseases that can be treated with the CARs, their coding sequences, nucleic acid constructs, expression vectors, viruses, and CAR-T cells of the invention are preferably CD 19-mediated diseases, particularly CD 19-mediated hematologic tumors.

In particular, herein, "CD 19-mediated diseases" include, but are not limited to, leukemias and lymphomas, such as B-cell lymphomas, mantle cell lymphomas, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myelogenous leukemia.

The invention provides a genetically modified T cell comprising the polynucleotide sequence, or the nucleic acid construct, or infected with the lentiviral vector system, or a pharmaceutical composition comprising the genetically modified T cell.

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

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

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

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

In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressive agents. For example, treatment may be performed in conjunction with various radiotherapeutic agents, including: cyclosporin, azathioprine, methotrexate, mycophenolate mofetil, FK506, fludarabine, rapamycin, mycophenolic acid and the like. In further embodiments, the cell compositions of the invention are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation, T cell ablation therapy with chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.

Herein, "anti-tumor ability" 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 chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct and the lentiviral vector system are used for preparing any one or more of the following products: (1) preparing T cells; (2) increasing the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation and/or survival of T cells; (5) improving the anti-tumor capacity of the T cells; (6) inhibit the secretion of cytokines IFN-gamma and TNF-alpha of T cells.

The improvement of survival ability, the reduction of apoptosis level and the improvement of proliferation ability can obviously improve the persistence of T cells, thereby obviously improving the anti-tumor ability of the T cells.

Use of the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system, in a product that can also be used to prepare any one or more of the following: enhancing the proliferation survival ability, growth proliferation ability or continuous proliferation ability of the T cells or increasing the number of the T cells.

Use of a chimeric antigen receptor comprising the intracellular domain of CD3 epsilon with a Y/F mutation as described above, a polynucleotide sequence as described above, a nucleic acid construct as described above, a lentiviral vector system as described above or a genetically modified T cell as described above for the preparation of a product for the treatment of a tumor.

Optionally, the tumor is selected from one or more of leukemia or solid tumor.

Optionally, the tumor is selected from the group consisting of B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1

The anti-CD 1928Z CAR sequence includes FMC63 scFv, CD28 hinge region, CD28 transmembrane region, CD28 and CD3 zeta intracellular domain structures, i.e., the sequence shared by Rosenberg, s.a. laboratory at NCBI (GenBank: HM 852952.1); based on the anti-CD 1928Z CAR obtained by gene synthesis, the CAR structural sequence is designed as shown in figure 1a, and eGFP and CAR sequence are connected to the same Open Reading Frame (ORF) for encoding through T2A self-splicing peptide, so that the expression level of CAR can be indicated by eGFP expression quantity/fluorescence intensity. The intracellular region of CD3 epsilon with Y/F mutation (the cell is obtained by site-directed mutagenesis based on overlap extension PCR method and gene recombination ligation techniqueTwo tyrosines in the ITAM motif of the inner region are mutated to phenylalanine) is inserted behind the CD28 transmembrane region, and a novel CAR named anti-CD19E is constructed_YF28Z CAR。

anti-CD19 E_YFThe nucleotide sequence of the 28Z CAR is set forth in SEQ ID NO: shown in fig. 8. The method specifically comprises the following steps:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTatgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaTACCCCTACGACGTGCCCGACTACGCCgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacCTGTTCCCAACCCAGACTTTGAGCCCATCCGGAAAGGCCAGCGGGACCTGTTTTCTGgcctgaatcagagacgcatcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

by using E _YF28Z, anti-CD19E 28Z CAR, except that the intracellular region of CD3 epsilon with the Y/F mutation was replaced with the intracellular region of CD3 epsilon; the nucleotide sequence of anti-CD19E 28Z CAR is shown as SEQ ID NO: 9, specifically:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTatgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaTACCCCTACGACGTGCCCGACTACGCCgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaatcagagacgcatcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

the CAR was subcloned into the lentiviral expression plasmid, pHAGE vector, and the CAR expression plasmid was ligated with the packaging plasmids, pSPAX2 and pMD2G, according to 10: 7.5: 3.5 and then transferred into 293FT cells by calcium phosphate transfection to produce lentiviral particles. Expression of anti-CD 1928Z CAR, anti-CD19E 28Z CAR or anti-CD19E, respectively, by ultracentrifugation _YF28Z CAR lentivirus was concentrated to a small volume of high titer virus (-10)⁸IU/ml) and then transfected with primary T cells activated on a day with α CD3/α CD28 Ab-beads, respectively (MOI 10). Primary T cells were cultured in complete T cell medium (XIVO-15+ 1% P.S. +10ng/ml Human IL-7+10ng/ml Human IL-15+ 5% Human AB serum,10mM neutralized NAC) one day after virus transfection, four days after antibody stimulation, and 5 days after antibody stimulation, CAR-T cells with the same CAR expression level (indicated by eGFP fluorescence intensity) were sorted out and obtained as anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells, and anti-CD19E _YF28Z CAR-T cells. After a period of time of amplification, a series of indexes of CAR-T are identified, such as CAR upper membrane level, CD4 and CD8 subgroup proportion, in vitro cytokine secretion, in vitro killing capacity, in vitro proliferation capacity, in vitro apoptosis level, in vitro continuous survival capacity, in vivo anti-tumor capacity and the like.

Index detection method and result description:

the obtained anti-CD 1928Z CAR, anti-CD19E 28Z CAR and anti-CD19E_YFThe structure of the 28Z CAR is shown in FIG. 1a, as well as the CD3 epsilon intracellular domain amino acid sequence and the CD3 epsilon intracellular domain amino acid sequence with Y/F mutations. Wherein FMC63 is a single chain antibody targeting CD19 antigen, the Hinge region (Hinge region) and transmembrane region (transmembrane region) of the receptor are derived from human CD 28; anti-CD19E 28Z CAR, CD3 epsilon inserted behind the CD28 transmembrane region and in front of the CD28 intracellular region; anti-CD19E_YFIn the 28Z CAR, the amino acid sequence of the intracellular region of CD3 epsilon with the Y/F mutation was inserted after the CD28 transmembrane region and before the intracellular region of CD 28.

Flow detection of anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E using anti-mouse FMC63 scFv conjugated Alexa Fluor647 antibody_YFThe 28Z CAR-T cell anti-CD19 CAR receptor upper membrane level, flow chart results are shown in FIG. 1b, and show no difference between CAR positive rate and expression amount (MFI).

Flow detection of anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E with anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies _YF28Z CAR-T cells, flow chart results are shown in FIG. 1c, showing CD4 of the three⁺CAR-T cells and CD8⁺The proportion of CAR-T cells was similar.

10 Wannti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells, and anti-CD19E _YF28Z CAR-T cells were separately conjugated to CD19⁺Lymphoma cellsLine Raji follows 1: inoculating to a round bottom 96-well plate at a ratio of 1, mixing, centrifuging at room temperature (400g,1min) to promote cell-cell contact, and culturing in a 37 ℃ incubator for one day after complete culture. Adding 1x BFA to inhibit the discharge of cell factors 6 hours before sampling, washing a sample PBS once after sampling, fixing 4% PFA at room temperature for 5min, punching 0.1% TritonX-100 at room temperature for 5min, and detecting IL-2, IFN-gamma and TNF-alpha by using corresponding direct antibodies according to a conventional operation intracellular staining step. The results are shown in FIGS. 1 d-1 f, anti-CD19E compared to anti-CD19E 28Z CAR-T cells _YF28Z CAR-T cells via CD19⁺The obvious recovery and improvement of the cytokine levels of IL-2, IFN-gamma and TNF-alpha generated after the cell specific stimulation indicate that the ITAM basic sequence of the intracellular region of CD3 epsilon has an important inhibition effect on the generation of the cytokine. However, it is noteworthy that anti-CD19E_YFThe 28Z CAR-T cells still secreted significantly less IFN- γ, TNF- α cytokines than anti-CD 1928Z CAR-T cells (FIG. 1 e-FIG. 1 f). Can reduce macrophage monocyte activation to produce inflammatory cytokines such as IL-1 beta and IL-6.

10 Wannti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells, and anti-CD19E _YF28Z CAR-T cells were compared with the CD19+ lymphoma cell line Raji according to 1: inoculating to round bottom 96-well plate at a ratio of 1, mixing, centrifuging at room temperature (400g,1min) to promote cell-cell contact, and culturing in 37 deg.C incubator for a period of time. Samples were washed once with PBS after collection at different time points and the amount of Ki67 expressed was detected by flow-through Staining with anti-human Ki67-APC antibody according to the protocol of the Foxp3/Transcription Factor stabilizing Buffer Set kit. The results are shown in FIG. 1g, anti-CD19E _YF28Z CAR-T Via CD19⁺The proliferation speed after Raji cell stimulation is always faster than that of anti-CD 1928Z CAR-T cells and even than that of anti-CD19E 28Z CAR-T cells for a period of time, namely anti-CD19E _YF28Z CAR-T cells were the most proliferative.

10 Wannti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells, and anti-CD19E _YF28Z CAR-T cells were compared with the CD19+ lymphoma cell line Raji according to 1: inoculating to round bottom 96-well plate at a ratio of 1, mixing, centrifuging at room temperature (400g,1min) to promote cell-cell contact, and using total culture mediumIncubated at 37 ℃ for a period of time in an incubator. Samples were washed once with PBS after collection at different time points and stained according to the Annexin V Apoptosis Detection Kit APC Kit instructions and then flow-assayed for the expression level of Annexin V in CAR-T cells. The results are shown in FIG. 1h, anti-CD19E _YF28Z CAR-T cells and anti-CD19E 28Z CAR-T cells via CD19⁺After Raji cell stimulation, the level of apoptosis was significantly lower than that of anti-CD 1928Z CAR-T cells for a certain period of time.

10 Wannti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells, and anti-CD19E _YF28Z CAR-T cells were compared with the CD19+ lymphoma cell line Raji according to 1: inoculating to round bottom 96-well plate at a ratio of 1, mixing, centrifuging at room temperature (400g,1min) to promote cell-cell contact, and culturing in 37 deg.C incubator for a period of time. Samples were washed once with PBS after collection at different time points and stained for CAR with anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies⁺The number of T cells that survived. The results are shown in FIG. 1i, via CD19⁺anti-CD19E after Raji cell stimulation_YFThe highest number of 28Z CAR-T cells survived, followed by anti-CD19E 28Z CAR-T cells, and the lowest number of anti-CD 1928Z CAR-T cells survived, suggesting E_YFThe sustained survival of 28Z CAR-T cells was strongest.

anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells, and anti-CD19E _YF28Z CAR-T cell Pair CD19⁺Tumor cell toxicity reaction. Will CD19^-The K562 cells were resuspended in 1640 serum-free medium, then prestained with 1 XCellTraker deep red dye (37 ℃ water bath, 30min), washed once with 1640 serum-free medium, resuspended in complete T cell medium and then resuspended in CD19⁺K562_ CD19(IRES mCherry) cells were as follows 1: 1 mix to target cells. CAR-T cells were then mixed with the mixed target cells according to 3: 1,1: 1,1: mixing 3 proportion, spreading on round bottom 96-well plate, mixing, centrifuging at room temperature (400g,1min) to promote cell-cell contact, culturing in 37 deg.C incubator for one day, collecting sample, placing on ice, and detecting CD19 by flow method^-K562 cells (APC)⁺,mCherry^-) Percent and CD19⁺K562_ CD19 cell (APC)^-,mCherry⁺) Percent Change, of the test group“CD19^-K562 cells (APC)⁺,mCherry^-) percent/CD 19⁺K562_ CD19 cell (APC)^-,mCherry⁺) The percentage "is noted as N. N value "N" of mixed target cell sample well without CAR-T cells added₀＝CD19^-K562 cells (APC)⁺,mCherry^-) percent/CD 19⁺K562_ CD19 cell (APC)^-,mCherry⁺) Percentage "normalized" the survival ratio N/N is calculated₀The killing rate formula is 1-N/N₀". The results are shown in FIG. 1j for anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF28Z CAR-T cell three pairs of CD19⁺The killing toxicity of the tumor cells is equivalent.

10 Wannti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells, and anti-CD19E _YF28Z CAR-T cells were compared with the CD19+ lymphoma cell line Raji according to 1: 1 ratio in 1.5ml EP tubes, mix well, centrifuge at 4 ℃ (500g,2min) to promote cell-cell contact, use complete culture based on 37 ℃ water bath stimulation a series of time points. Adding equal volume of 8% PFA at different time points, fixing at room temperature for 5min, perforating with 0.1% TritonX-100 at room temperature for 5min, and performing conventional intracellular staining to obtain rabbit-derived pErk (1/2)^{Thr202/Tyr204}(FIG. 1k), Rabbit-derived pAKT^S473(FIG. 1l) and a primary anti-rabbit source pS6^S235/236(FIG. 1m), washed once, stained with secondary antibody, goat anti-rabbit IgG polyclonal antibody conjugated Alexa647, washed once and detected by flow. The results are shown in FIG. 1 k-FIG. 1m, anti-CD19E_YFpErk (1/2) following deliberate antigen stimulation of 28Z CAR-T cells^{Thr202/Tyr204}And pS6^S235/236The overall level is obviously higher than that of anti-CD 1928Z CAR-T and anti-CD19E 28Z CAR-T cells, anti-CD19E_YFAKT of 28Z CAR-T cells and anti-CD19E 28Z CAR-T cells^S473The phosphorylation level is obviously higher than that of anti-CD 1928Z CAR-T cells. Wherein in the signal transduction pathway after the chimeric antigen receptor CAR is activated by antigen, the signal molecule of the Raf/MAPK signal pathway is serine/threonine protein kinase Erk (1/2)^{Thr202/Tyr204}Phosphorylation levels and serine/threonine kinase AKT in the PI3K/AKT signaling pathway^S473The phosphorylation levels all indicate promoting cellsCell proliferation survival ability and anti-apoptosis ability, a signaling molecule ribosomal protein S6 downstream of the two pathways^S235/236The phosphorylation level marks the cell growth and proliferation level, and the phenotype of the three signal molecules is used for clarifying the anti-CD19E from the signal path level_YFThe 28Z CAR-T cells were most potent in proliferation survival.

The experimental data processing method comprises the following steps:

statistical analysis of data was performed using the software GraphPad Prism 8.0.2, and a statistical P <0.05 indicates that the difference is significant, which is specifically expressed as: p <0.05, P <0.01, P <0.001, P <0.0001, each set of data is presented as Mean ± SD.

FIG. 1 d-FIG. 1f single factor variance analysis plus multiple comparison tests with Sidak's;

FIG. 1g, FIG. 1h, FIG. 1 j-FIG. 1m two-factor variance analysis plus Sidak's multiple comparison test;

FIG. 1i two-factor variance analysis.

The experimental results are as follows:

(1)、anti-CD19 E_YFthe 28Z CAR-T cells were comparable to anti-CD19E 28Z CAR-T cells, anti-CD 1928Z CAR-T cells in membrane levels on the anti-CD19 CAR receptor and in CD4 and CD8 ratios;

(2) anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF28Z CAR-T cells are specifically stimulated by Raji cells in vitro, and then compared with anti-CD 1928Z CAR-T cells, anti-CD19E_YFThe obvious increase of IL-2, IFN-gamma and TNF-alpha cytokine levels produced by 28Z CAR-T cells indicates that the ITAM motif of the intracellular domain of CD3 epsilon plays an important role in the inhibition of cytokine production. However, it is noteworthy that anti-CD19E_YFThese cytokines secreted by 28Z CAR-T cells were still significantly less than anti-CD 1928Z CAR-T cells (fig. 1 e-fig. 1 f). Can reduce macrophage monocyte activation to produce inflammatory cytokines such as IL-1 beta and IL-6.

(3) anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFAfter 28Z CAR-T cells are specifically stimulated and amplified by Raji cells in vitro for a period of time, sampling is carried out at different time points to detect the proliferation capacity (namely Ki)67 expression level), anti-CD19E compared to anti-CD 1928Z CAR-T cells and anti-CD19E 28Z CAR-T cells_YFThe 28Z CAR-T cell has the best proliferation capacity;

(4) anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF28Z CAR-T cells were expanded by Raji cell specific stimulation in vitro for a period of time, and then sampled at different time points to detect the level of apoptosis (Annexin V), anti-CD19E 28Z CAR-T cells and anti-CD19E_YFThe 28Z CAR-T cell apoptosis level is obviously lower than that of anti-CD 1928Z CAR-T cells;

(5) anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFAfter 28Z CAR-T cells are subjected to Raji cell-specific stimulation and expanded for a period of time, sampling at different time points to detect the expanded CAR⁺T cell number, anti-CD19E over time_YFThe number of 28Z CAR-T cells continued to be significantly higher than anti-CD 1928Z CAR-T cells and anti-CD19E 28Z CAR-T cells;

(6) anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E_YFIn vitro killing experiment of 28Z CAR-T cells incubated with CD19+ K562 CD19-K562, anti-CD19E_YFThe killing ability of 28Z CAR-T cells was comparable to anti-CD 1928 ZCAR-T cells and anti-CD19E 28ZCAR-T cells.

(7) anti-CD 1928Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF28Z CAR-T cells were expanded by Raji cell-specific stimulation for a period of time, and the signal molecule serine/threonine protein kinase Erk (1/2) was detected by sampling at different time points^{Thr202/Tyr204}Serine/threonine kinase AKT^S473And ribosomal protein S6^{S235 /236}The level of phosphorylation. anti-CD19E_YFpErk (1/2) following deliberate antigen stimulation of 28Z CAR-T cells

^{Thr202/Tyr204}And pS6^S235/236The overall level is obviously higher than that of anti-CD 1928Z CAR-T cells and anti-CD19E 28Z CAR-T cells, and the anti-CD19E_YFAKT of 28Z CAR-T cells and anti-CD19E 28Z CAR-T cells^S473The phosphorylation level is obviously higher than that of anti-CD1928Z CAR-T cells. In a signal transduction pathway of the combined chimeric antigen receptor CAR after antigen activation, a Raf/MAPK signal pathway signal molecule Erk (1/2)^{Thr202/Tyr204}Phosphorylation levels and AKT in PI3K/AKT signaling pathway^S473The phosphorylation level indicates the ability of promoting cell proliferation survival and anti-apoptosis, and the common signal molecule ribosomal protein S6 is downstream of the two paths^S235/236The phosphorylation level marks the cell growth and proliferation level, and the phenotype of the three signal molecules is used for clarifying the anti-CD19E from the signal path level_YFThe 28Z CAR-T cells were most potent in proliferation survival.

anti-CD19 E_YFComparison of the in vivo anti-subcutaneous tumor capacity of 28Z CAR-T cells with anti-CD19E 28Z cells and anti-CD 1928Z CAR-T cells, protocol was as follows:

sterile procedure A3X 10^7/ml Raji cell-PBS resuspension was prepared and 3 million ul Raji cells were subcutaneously inoculated into the left dorsal side of each 6 week old B-NDG female mouse at 3 million time points designated day 0. After 6 days, when Raji subcutaneous tumors grew to 6-7mm in diameter, mice were randomly divided into three groups (E)_YF28Z, E28Z, 28Z, vector), then 100ul 8 million T cells were injected into the tail vein of each mouse, after which the size of the tumor was measured periodically with a vernier caliper, and tumor data and mouse survival were recorded, tumor area ═ length x width. And (4) experimental conclusion: anti-CD19E_YFThe 28Z CAR-T cells have anti-tumor capacity.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

<110> center of outstanding innovation in molecular cell science of Chinese academy of sciences

<120> a chimeric antigen receptor comprising intracellular domain of CD3 epsilon with Y/F mutation and use thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 55

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala

1 5 10 15

Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro

20 25 30

Asn Pro Asp Phe Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Phe Ser

35 40 45

Gly Leu Asn Gln Arg Arg Ile

50 55

<210> 2

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 3

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 4

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 5

<211> 522

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

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

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

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

225 230 235 240

Val Thr Val Ser Ser Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro

245 250 255

Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly

260 265 270

Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe

275 280 285

Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu

290 295 300

Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Asn Arg Lys Ala Lys

305 310 315 320

Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly

325 330 335

Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Phe Glu Pro

340 345 350

Ile Arg Lys Gly Gln Arg Asp Leu Phe Ser Gly Leu Asn Gln Arg Arg

355 360 365

Ile Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

370 375 380

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

385 390 395 400

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Ala Leu His Met Gln Ala Leu Pro Pro Arg

515 520

<210> 6

<211> 1569

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggactaagt tggaaataac aggctccacc tctggatccg gcaagcccgg atctggcgag 360

ggatccacca agggcgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca 420

cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc 480

tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa 540

accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag 600

agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt 660

gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca 720

gtcaccgtct cctcagcggc cgcaattgaa gttatgtatc ctcctcctta cctagacaat 780

gagaagagca atggaaccat tatccatgtg aaagggaaac acctttgtcc aagtccccta 840

tttcccggac cttctaagcc cttttgggtg ctggtggtgg ttgggggagt cctggcttgc 900

tatagcttgc tagtaacagt ggcctttatt attttctggg tgaagaatag aaaggccaag 960

gccaagcctg tgacacgagg agcgggtgct ggcggcaggc aaaggggaca aaacaaggag 1020

aggccaccac ctgttcccaa cccagacttt gagcccatcc ggaaaggcca gcgggacctg 1080

ttttctggcc tgaatcagag acgcatcagg agtaagagga gcaggctcct gcacagtgac 1140

tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1200

ccaccacgcg acttcgcagc ctatcgctcc agagtgaagt tcagcaggag cgcagacgcc 1260

cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1320

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1380

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1440

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1500

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1560

cctcgctaa 1569

<210> 7

<211> 39

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 8

<211> 2442

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga 720

agcggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggacct 780

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 840

atcccatacc cctacgacgt gcccgactac gccgacatcc agatgacaca gactacatcc 900

tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 960

agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 1020

catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 1080

gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 1140

cagggtaata cgcttccgta cacgttcgga ggggggacta agttggaaat aacaggctcc 1200

acctctggat ccggcaagcc cggatctggc gagggatcca ccaagggcga ggtgaaactg 1260

caggagtcag gacctggcct ggtggcgccc tcacagagcc tgtccgtcac atgcactgtc 1320

tcaggggtct cattacccga ctatggtgta agctggattc gccagcctcc acgaaagggt 1380

ctggagtggc tgggagtaat atggggtagt gaaaccacat actataattc agctctcaaa 1440

tccagactga ccatcatcaa ggacaactcc aagagccaag ttttcttaaa aatgaacagt 1500

ctgcaaactg atgacacagc catttactac tgtgccaaac attattacta cggtggtagc 1560

tatgctatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagc ggccgcaatt 1620

gaagttatgt atcctcctcc ttacctagac aatgagaaga gcaatggaac cattatccat 1680

gtgaaaggga aacacctttg tccaagtccc ctatttcccg gaccttctaa gcccttttgg 1740

gtgctggtgg tggttggggg agtcctggct tgctatagct tgctagtaac agtggccttt 1800

attattttct gggtgaagaa tagaaaggcc aaggccaagc ctgtgacacg aggagcgggt 1860

gctggcggca ggcaaagggg acaaaacaag gagaggccac cacctgttcc caacccagac 1920

tttgagccca tccggaaagg ccagcgggac ctgttttctg gcctgaatca gagacgcatc 1980

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 2040

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 2100

tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 2160

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 2220

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 2280

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 2340

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 2400

acctacgacg cccttcacat gcaggccctg ccccctcgct aa 2442

<210> 9

<211> 2442

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga 720

agcggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggacct 780

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 840

atcccatacc cctacgacgt gcccgactac gccgacatcc agatgacaca gactacatcc 900

tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 960

agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 1020

catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 1080

gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 1140

cagggtaata cgcttccgta cacgttcgga ggggggacta agttggaaat aacaggctcc 1200

acctctggat ccggcaagcc cggatctggc gagggatcca ccaagggcga ggtgaaactg 1260

caggagtcag gacctggcct ggtggcgccc tcacagagcc tgtccgtcac atgcactgtc 1320

tcaggggtct cattacccga ctatggtgta agctggattc gccagcctcc acgaaagggt 1380

ctggagtggc tgggagtaat atggggtagt gaaaccacat actataattc agctctcaaa 1440

tccagactga ccatcatcaa ggacaactcc aagagccaag ttttcttaaa aatgaacagt 1500

ctgcaaactg atgacacagc catttactac tgtgccaaac attattacta cggtggtagc 1560

tatgctatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagc ggccgcaatt 1620

gaagttatgt atcctcctcc ttacctagac aatgagaaga gcaatggaac cattatccat 1680

gtgaaaggga aacacctttg tccaagtccc ctatttcccg gaccttctaa gcccttttgg 1740

gtgctggtgg tggttggggg agtcctggct tgctatagct tgctagtaac agtggccttt 1800

attattttct gggtgaagaa tagaaaggcc aaggccaagc ctgtgacacg aggagcgggt 1860

gctggcggca ggcaaagggg acaaaacaag gagaggccac cacctgttcc caacccagac 1920

tatgagccca tccggaaagg ccagcgggac ctgtattctg gcctgaatca gagacgcatc 1980

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 2040

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 2100

tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 2160

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 2220

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 2280

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 2340

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 2400

acctacgacg cccttcacat gcaggccctg ccccctcgct aa 2442

Claims (14)

1. A chimeric antigen receptor comprising the intracellular domain of CD3 epsilon having a Y/F mutation, comprising:

an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence;

the extracellular domain comprises an antigen recognition region and a hinge region;

the end of the intracellular domain connected with the transmembrane domain is connected with a CD3 epsilon intracellular domain with Y/F mutation, and the CD3 epsilon intracellular domain with Y/F mutation refers to a Y/F mutant CD3 epsilon intracellular domain in which two tyrosines in an immunoreceptor tyrosine activation motif of the CD3 epsilon intracellular domain are mutated into phenylalanine.

2. The chimeric antigen receptor comprising an intracellular region of CD3 epsilon having a Y/F mutation of claim 1, further comprising one or more of the following characteristics:

(1) the amino acid sequence of the intracellular region of CD3 epsilon with Y/F mutation is shown as SEQ ID NO: 1 is shown in the specification;

(2) the intracellular domain comprises a CD3 epsilon intracellular domain with a Y/F mutation, a costimulatory signaling region, and a CD3 zeta intracellular segment connected in sequence.

3. The chimeric antigen receptor comprising an intracellular region of CD3 epsilon having a Y/F mutation according to claim 2, wherein said region of costimulatory signaling is selected from the intracellular segment of one or more of CD27, CD28, CD134, 4-1BB, OX40 and ICOS.

4. The chimeric antigen receptor comprising an intracellular region of CD3 epsilon having a Y/F mutation according to claim 3, further comprising one or more of the following characteristics:

(1) the amino acid sequence of the intracellular segment of the CD28 is shown as SEQ ID NO: 2 is shown in the specification;

(2) the amino acid sequence of the intracellular segment of CD3 zeta is shown in SEQ ID NO: 3, respectively.

5. The chimeric antigen receptor comprising an intracellular region of CD3 epsilon having a Y/F mutation of claim 1, further comprising one or more of the following characteristics:

a. the antigen recognition region is selected from a single chain antibody directed against a tumor surface antigen selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, fibrolast activation protein, cryptic-3, CEA, EGFRvIII, PSMA, Her2, IL13R α 2, CD171, and GD 2;

b. the transmembrane domain is selected from the transmembrane region of one or more of CD28, CD4, CD8 alpha, OX40 or H2-Kb.

c. The hinge region is selected from one or more of a CD28 hinge region, a CD8 alpha hinge region, a CD4 hinge region, a hinge region of immunoglobulin IgG or a hinge region of immunoglobulin IgG coupled to a CH2CH3 region.

6. The chimeric antigen receptor comprising an intracellular region of CD3 epsilon having a Y/F mutation according to claim 5, further comprising one or more of the following characteristics:

d. the single chain antibody is selected from FMC 63;

e. the amino acid sequence of the transmembrane region of the CD28 is shown as SEQ ID NO: 4, respectively.

f. The amino acid structure of the CD28 hinge region is shown as SEQ ID NO: shown at 7.

7. The chimeric antigen receptor comprising the intracellular domain of CD3 epsilon having the Y/F mutation of any one of claims 1 to 6, further comprising one or more of the following characteristics:

1) the antigen recognition region of the chimeric antigen receptor comprising an intracellular region of CD3 epsilon with a Y/F mutation is selected from FMC63, and the hinge region is selected from a CD28 hinge region; the transmembrane domain is selected from the CD28 transmembrane region; the intracellular domain comprises a CD3 epsilon intracellular domain with a Y/F mutation, an intracellular segment of CD28 and an intracellular segment of CD3 zeta linked in sequence.

2) The amino acid sequence of the chimeric antigen receptor containing the intracellular region of CD3 epsilon with Y/F mutation is shown as SEQ ID NO: 5, respectively.

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

(1) a polynucleotide sequence encoding the chimeric antigen receptor of any one of claims 1 to 7 comprising the intracellular region of CD3 epsilon having a Y/F mutation; and

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

9. The polynucleotide sequence of claim 8, wherein the polynucleotide sequence is as set forth in SEQ ID NO: and 6.

10. A nucleic acid construct comprising the polynucleotide sequence of any one of claims 8-9;

preferably, the nucleic acid construct is a vector;

more preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3 'LTR, a 5' LTR and a polynucleotide sequence according to any one of claims 8 to 9.

11. A lentiviral vector system comprising the nucleic acid construct of claim 10 and a lentiviral vector accessory component.

12. A genetically modified T cell comprising a polynucleotide sequence according to any one of claims 8 to 9, or comprising a nucleic acid construct according to claim 10, or infected with a lentiviral vector system according to claim 11.

13. Use of a chimeric antigen receptor comprising the intracellular region of CD3 epsilon with a Y/F mutation as defined in any one of claims 1 to 7, a polynucleotide sequence as defined in any one of claims 8 to 9, or a nucleic acid construct as defined in claim 10, or a lentiviral vector system as defined in claim 11, for the preparation of a product for any one or more of the following uses: (1) preparing T cells; (2) increasing the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation and/or survival of T cells; (5) improving the anti-tumor capacity of the T cells; (6) inhibit the cytokine IFN-gamma level and TNF-alpha secretion of T cells.

14. Use of a chimeric antigen receptor comprising the intracellular region of CD3 epsilon with a Y/F mutation as defined in any one of claims 1 to 7, a polynucleotide sequence as defined in any one of claims 8 to 9, or a nucleic acid construct as defined in claim 10, or a lentiviral vector system as defined in claim 11 in or a genetically modified T cell as defined in claim 12 for the preparation of a product for the treatment of tumors.

Images