CN113980136B

CN113980136B - Chimeric antigen receptor comprising CD3 epsilon intracellular region with Y/F mutation and application thereof

Info

Publication number: CN113980136B
Application number: CN202010733636.2A
Authority: CN
Inventors: 许琛琦; 周秋萍; 吴微; 徐心怡; 何星
Original assignee: Center for Excellence in Molecular Cell Science of CAS
Current assignee: Center for Excellence in Molecular Cell Science of CAS
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2023-08-22
Anticipated expiration: 2040-07-27
Also published as: CN113980136A

Abstract

The present invention provides a chimeric antigen receptor comprising a CD3 epsilon intracellular region having a Y/F mutation, comprising: an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence; one 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 mutation type CD3 epsilon intracellular region in which two tyrosine in the CD3 epsilon intracellular region ITAM are mutated into phenylalanine. The chimeric antigen receptor modified T cell has the advantages of improving the survival capability, reducing the apoptosis level and improving the proliferation capability, and can obviously improve the persistence of the T cell, thereby obviously improving the anti-tumor capability of the T cell. The advantages can further improve the tumor treatment effect, prolong the disease release 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 the expressed cytokines IFN-gamma and TNF-alpha, and reduce the activation of macrophage and monocyte to produce inflammatory cytokines such as IL-1 beta and IL-6.

Description

Chimeric antigen receptor comprising CD3 epsilon intracellular region 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 comprising a CD3 epsilon intracellular region with Y/F mutation and application thereof.

Background

The chimeric antigen receptor is abbreviated as CAR (chimeric antigen receptor), and T cells carrying a CAR against a specific tumor antigen are called CAR-T cells. The CAR-T therapy has wide application in tumor treatment, especially solid tumor immunotherapy, is a novel accurate targeted therapy for treating tumors, achieves good effect on clinical tumor treatment through optimization and improvement in recent years, and is a novel tumor immunotherapy method which has very good prospect, can be accurate, rapid and efficient, and can possibly cure cancers.

However, CAR-T therapies still have many limitations, such as cytokine storm, neurotoxicity safety, and poor sustained proliferation ability, encountered in the clinical treatment of tumors. The journal of lancets in 2015 released a one-stage clinical trial (clinical trial number: NCT01593696, 2015.Lacet.T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults-alpha phase 1dose-escalation three) showing that patients with recurrent or refractory acute lymphoblastic leukemia or non-hodgkin's lymphoma (age 1-30 years), examined the number of blood CAR-T cells in vivo after KTE-C19 (anti-CD 19 28Z) CAR-T treatment, found that CAR-T cell numbers reached peak on day 14, and then significantly decreased on days 28, 42, with no CAR-T cells detectable in all patients' blood on day 68. There was also a result of a one-phase clinical trial published in the new England journal of 2018 (clinical trial number: NCT01044069, 2018.NEJ. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia), which also indicated that 53 patients with recurrent acute lymphoblastic leukemia, the median number of detection in blood CAR-T cells after treatment with anti-CD19 28Z CAR-T occurred on day 14. Both studies showed that the sustained proliferative viability of 28Z CAR-T cells was poor and the efficacy was improved.

Disclosure of Invention

The design of the transformation aiming at the CAR structure can be considered from the point of improving the continuous survival capability of the CAR-T cells, so that the anti-tumor effect is improved, the disease release time is prolonged, the recurrence rate is reduced, and the survival period 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 a CD3 epsilon intracellular region with Y/F mutations and uses thereof.

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

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

the extracellular domain includes 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 are mutated into phenylalanine in an immunoreceptor tyrosine activation motif (Immunoreceptor tyrosine-based activation motif, ITAM) of the CD3 epsilon intracellular region.

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

(1) A polynucleotide sequence encoding a chimeric antigen receptor comprising a CD3 epsilon intracellular region with a Y/F mutation as defined in the preceding claims; and

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

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

preferably, the nucleic acid construct is a vector;

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

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

In a fifth aspect, the invention provides a genetically modified T cell or a pharmaceutical composition comprising the 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 described above.

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

In a seventh aspect, the invention provides the use of the aforementioned chimeric antigen receptor comprising a CD3 epsilon intracellular region with a Y/F mutation, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system, or the aforementioned genetically modified T cell for the preparation of a tumor therapeutic product.

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

the chimeric antigen receptor modified T cell has the advantages of improving the survival capability, reducing the apoptosis level and improving the proliferation capability, and can obviously improve the persistence of the T cell, thereby obviously improving the anti-tumor capability of the T cell. The advantages can further improve the tumor treatment effect, prolong the disease release 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 the expressed cytokines IFN-gamma and TNF-alpha, and reduce the activation of macrophage and monocyte to produce inflammatory cytokines such as IL-1 beta and IL-6.

Drawings

FIG. 1a. Anti-CD 19Z 28 CAR, anti-CD 19E 28Z CAR and anti-CD 19E _YF The structure of a 28Z CAR is schematically represented as a CD3 epsilon intracellular domain amino acid sequence and a CD3 epsilon intracellular domain amino acid sequence with Y/F mutations. Wherein FMC63 is a single chain antibody targeting the CD19 antigen, the Hinge (Hinge region) and transmembrane (transmembrane region) regions of the receptor are derived from human CD28;

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

FIG. 1b. Anti-CD 19Z CAR 28 anti-CD19 E28Z CAR and anti-CD19E _YF Flow chart of the upper membrane level of the 28Z CAR after T cell expression.

FIG. 1c anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF CD4 in 28Z CAR-T cells ⁺ And CD8 ⁺ Flow chart of cell ratio.

FIG. 1d. Anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF Comparison of IL-2 cytokine levels in 28Z CAR-T cells stimulated with Raji cells (CD 19 antigen).

FIG. 1E. Anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF Comparison of IFN-gamma cytokine levels after 28Z CAR-T cells were stimulated with Raji cells (CD 19 antigen).

FIG. 1f anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF Comparison of TNF- α cytokine levels after 28Z CAR-T cells were stimulated with Raji cells (CD 19 antigen).

FIG. 1g anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF Proliferation of 28Z CAR-T cells after stimulation with Raji cells (CD 19 antigen).

FIG. 1h anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF Apoptosis of 28Z CAR-T cells after stimulation with Raji cells (CD 19 antigen).

FIG. 1i. anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF Number of survival of 28Z CAR-T cells after stimulation with Raji cells (CD 19 antigen).

FIG. 1j anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF 28Z CAR-T cell pair CD19 ⁺ Tumor cytotoxicity response.

FIG. 1k. Anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF Signal molecule serine/threonine protein kinase Erk (1/2) for promoting cell proliferation survival and resisting apoptosis of 28Z CAR-T cells stimulated by Raji cells (CD 19 antigen) ^{Thr202/Tyr204} Phosphorylation (pErk (1/2) ^{Thr202/Tyr204} ) Horizontal.

FIG. 1l anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF Signal molecule serine/threonine kinase AKT for promoting cell proliferation survival and resisting apoptosis of 28Z CAR-T cells after being stimulated by Raji cells (CD 19 antigen) ^S473 Phosphorylation (pAKT) ^S473 ) Horizontal.

FIG. 1m anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF Labeled cell growth proliferation signal molecule ribosomal protein S6 of 28Z CAR-T cells stimulated by Raji cells (CD 19 antigen) ^S235/236 Phosphorylation (pS 6) ^S235/236 ) Horizontal.

Detailed Description

The chimeric antigen receptor comprising a CD3 epsilon intracellular region with Y/F mutation according to the present invention comprises:

one end of the intracellular domain connected with the transmembrane domain is connected with a CD3 epsilon intracellular region with Y/F mutation, wherein the CD3 epsilon intracellular region with Y/F mutation refers to a Y/F mutant CD3 epsilon intracellular region in which two tyrosines in an immunoreceptor tyrosine activation motif (Immunoreceptor tyrosine-based activation motif, ITAM) of the CD3 epsilon intracellular region are mutated into phenylalanine.

Further, the amino acid sequence of the CD3 epsilon intracellular region with Y/F mutation is shown in SEQ ID NO:1 is shown in the specification; specific: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLFSGLNQRRI.

The intracellular domain comprises a CD3 epsilon intracellular region with a Y/F mutation (two tyrosine mutations in the ITAM motif of the intracellular region are amphetamine), a costimulatory signaling region intracellular region and a CD3 zeta intracellular segment which are connected in sequence.

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

In a further preferred embodiment, the costimulatory signaling region is selected from the group consisting of 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 CD3 zeta intracellular segment has the amino acid sequence as set forth in SEQ ID NO: 3. The method comprises the following steps: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

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, fibroblast Activation Protein (FAP), glypican-3, CEA, EGFRvIII, PSMA, her2, IL13 ra 2, CD171, and GD 2.

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

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 CD28 is shown in SEQ ID NO: 4. Specifically, FWVLVVVGGVLACYSLLVTVAFIIFWV.

The hinge region is selected from one or more of a CD28 hinge region, a CD8 a hinge region, a CD4 hinge region, an immunoglobulin IgG hinge region, or an immunoglobulin IgG hinge region coupled to a CH2CH3 region. I.e. IgG1 range or IgG1 range-CH 2CH3.

Alternatively, the hinge region sequence is a CD28 hinge region (CD 28 hinge). The amino acid structure is shown in SEQ ID NO: shown at 7. Specifically, IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

In one embodiment, the single chain antibody contains 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 FMC63.

FMC63 is commercially available.

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

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

In one embodiment, the chimeric antigen receptor (Anti-CD 19E) comprising a CD3 epsilon intracellular region with a Y/F mutation _YF 28Z CAR) has the amino acid sequence as set forth in SEQ ID NO:5, specifically:

SEQ ID NO：5(Anti-CD19 E _YF 28Z CAR amino acid sequence): (5 '. Fwdarw.3') starting from FMC63 scFv, the linkage is a CD28 hinge region, a CD28 transmembrane region, a CD 3. Epsilon. Intracellular region with Y/F mutation, and a CD 3. Zeta. Intracellular region in this order:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLFSGLNQRRIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

SEQ ID NO：5(Anti-CD19 E _YF 28Z CAR amino acid sequence) is SEQ ID NO:6 (Anti-CD 19E) _YF 28Z CAR nucleotide sequence), SEQ ID NO:6 is as follows: (5 '. Fwdarw.3') gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacCTGTTCCCAACCCAGACTTTGAGCCCATCCGGAAAGGCCAGCGGGACCTGTTTTCTGgcctgaatcagagacgcatcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

The above-described individual parts forming the chimeric antigen receptor of the invention may be directly linked to each other or may be linked by a linker sequence. The linker sequences may be linker sequences suitable for antibodies as known in the art, such as G and S containing linker sequences. Typically, a linker contains one or more motifs that repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are contiguous in the linker sequence with no amino acid residues inserted between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motif compositions. The length of the linker may be 3 to 25 amino acid residues, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a glycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is usually 2 to 20, for example 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), etc.

It will be appreciated that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed amino acid sequence, without affecting the activity of the sequence of interest. To construct fusion proteins, facilitate expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside of the host cell, or facilitate purification of recombinant proteins, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, for example, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. The signal peptide hypothesis suggests that mRNA encoding a secreted protein is first synthesized at translation as a signal peptide with a hydrophobic amino acid residue at the N-terminus, which is recognized by and bound to a receptor on the endoplasmic reticulum membrane. The signal peptide reaches the lumen of the endoplasmic reticulum through the channel formed by the protein in the membrane, is then hydrolyzed by the signal peptidase on the surface of the lumen, and due to its guidance, the nascent polypeptide can enter the lumen through the endoplasmic reticulum membrane and finally be secreted extracellularly. The signal peptides of the CAR are of different origins, mainly the signal peptides of the CD8 and CD28, GM-CSF receptors. The amino-or 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 Ty1. 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 the chimeric antigen receptor of the preceding claim; and

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

The polynucleotide sequences of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The invention also includes degenerate variants of the polynucleotide sequence encoding a fusion protein, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

The polynucleotide sequences described herein can generally be obtained using PCR amplification methods. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

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

The nucleic acid construct further comprises one or more regulatory sequences operably linked to the aforementioned polynucleotide sequence. The coding sequences of the CARs of the invention can be manipulated in a variety of ways to ensure expression of the protein. The nucleic acid construct may be manipulated according to the expression vector or requirements prior to insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The regulatory sequence may be a suitable 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 that exhibits 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 regulatory 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 sequences may also be suitable leader sequences, untranslated regions of mRNA that are important for host cell translation. 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 of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

Polynucleotide sequences encoding the CARs 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 as a viral vector. Viral vector technology is well known in the art. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, 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 aforementioned polynucleotide sequences.

One 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 levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when expressed for a period of time and switching off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both of a selectable marker gene or reporter gene to facilitate identification and selection of the expressing cell 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 single 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 the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

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

Methods for introducing genes into cells and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the 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 for introducing the polynucleotide into a host cell 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 transferring genes into mammalian cells. For example, lentiviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into lentiviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

The invention provides a lentiviral vector system, which comprises the nucleic acid construct and lentiviral vector auxiliary components.

The lentiviral adjunct ingredients include lentiviral packaging plasmids and cell lines.

The slow virus vector system is formed by virus packaging the nucleic acid construct with the aid of slow virus packaging plasmids and cell lines. The lentiviral vector system construction method is a method commonly used in the art.

The invention provides a method for ex vivo activation of T cells, comprising the step of infecting the T cells with a lentivirus as described above.

Based on the ability of CAR-T cells to self-proliferate and remain viable for longer periods of time in vivo than other drug therapies, CAR-T cells can rapidly expand and kill targeted tumor cells upon stimulation with specific antigens, and can then form specific memory cells, such as effector memory cells (effector memory T cell) and central memory cells (central memory T cell).

The invention also includes a class of cell therapies in which T cells are genetically modified to express a CAR as described herein, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to persistent 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, diseases treatable with the CAR, coding sequences thereof, nucleic acid constructs, expression vectors, viruses and CAR-T cells of the invention are preferably CD19 mediated diseases, in particular CD19 mediated hematological tumors.

In particular, herein, "CD19 mediated diseases" include, but are not limited to, leukemias and lymphomas, such as B-cell lymphomas, mantle cell lymphomas, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myelogenous leukemia.

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

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 the relevant cytokine or cell population. Briefly, the pharmaceutical compositions of the invention may comprise a CAR-T cell 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 composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number 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", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be administered at 10 ⁴ To 10 ⁹ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Dosage of individual cells/kg body weight. T cell compositions may also be administered multiple times at these doses. 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 one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are 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 may 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 immunosuppressants. For example, treatment may be performed in combination with various radiotherapeutic agents, including: cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, fludarabine, rapamycin, mycophenolic acid, and the like. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablation therapy with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH.

Herein, "antitumor ability" refers to a biological effect that can be represented by a decrease in tumor volume, a decrease in tumor cell number, a decrease in metastasis number, 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 to 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.

Use of the chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, the lentiviral vector system described above for the preparation of a product for any one or more of the following uses: (1) preparing T cells; (2) increasing T cell viability; (3) inhibiting T cell apoptosis; (4) enhancing T cell proliferation and/or viability; (5) improving the anti-tumor capability of the T cells; (6) Inhibit the secretion of cytokines IFN-gamma and TNF-alpha of T cells.

The improvement of the viability, the reduction of the apoptosis level and the improvement of the proliferation capability can obviously improve the persistence of the T cells, thereby obviously improving the anti-tumor capability 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 for the preparation of a product that can also be used for any one or more of the following uses: enhancing T cell proliferation viability, growth proliferation or sustained proliferation or increasing T cell numbers.

Use of the aforementioned chimeric antigen receptor comprising a CD3 epsilon intracellular region with a Y/F mutation, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system, or the aforementioned genetically modified T cell in the preparation of a tumor therapeutic product.

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

Alternatively, the tumor is selected from the group consisting of B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myelogenous leukemia.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

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

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

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

Example 1

The anti-CD19 28Z CAR sequence includes FMC63 scFv, CD28 hinge region, CD28 transmembrane region, CD28 and CD3 zeta intracellular domain structures, i.e., rosenberg, S.A. sequence shared by the laboratory at NCBI (GenBank: HM 852952.1); the anti-CD19 28Z CAR is obtained through gene synthesis, and on the basis, a CAR structure sequence is designed as shown in figure 1a, and eGFP and the CAR sequence are connected in the same Open Reading Frame (ORF) for encoding through T2A self-shearing peptide, so that the expression level of the CAR can be indicated by eGFP expression quantity/fluorescence intensity. By adopting a site-directed mutagenesis technology and a gene recombination connection technology based on an overlap extension PCR method, a CD3 epsilon intracellular region with Y/F mutation (two tyrosine mutations in ITAM motif of the intracellular region are phenylalanine) is inserted behind a CD28 transmembrane region, and a novel CAR is constructed and named as anti-CD 19E _YF 28Z CAR。

anti-CD19 E _YF The nucleotide sequence of the 28Z CAR is shown in SEQ ID NO: shown at 8. The method comprises the following steps:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTatgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaTACCCCTACGACGTGCCCGACTACGCCgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacCTGTTCCCAACCCAGACTTTGAGCCCATCCGGAAAGGCCAGCGGGACCTGTTTTCTGgcctgaatcagagacgcatcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

by E _YF 28Z, constructing anti-CD19 E28Z CAR, except that the CD3 epsilon intracellular region with Y/F mutation was replaced with a CD3 epsilon intracellular region sequence; the nucleotide sequence of the anti-CD 19E 28Z CAR is shown in SEQ ID NO:9, specifically:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTatgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaTACCCCTACGACGTGCCCGACTACGCCgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggacctgtattctggcctgaatcagagacgcatcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

the CAR was subcloned into the lentiviral expression plasmid pHAGE vector, and the CAR expression plasmid was combined with the packaging plasmids pSPAX2 and pMD2G according to 10:7.5:3.5, and transferred into 293FT cells by transfection with calcium phosphate to produce lentiviral particles. The anti-CD 19Z CAR, the anti-CD 19E 28Z CAR or the anti-CD 19E are expressed by a super-high speed centrifugation method _YF The 28Z CAR lentivirus was concentrated to a small volume high titer virus (-10) ⁸ IU/ml) and then transfected with primary T cells activated by αcd3/αcd28ab-beads for one day (moi=10), respectively. During the culture of primary T cells in T cell complete medium (XIVO-15+1% P.S. +10ng/ml human IL-7+10ng/ml human IL-15+5%Human AB serum,10mM neutralized NAC), virus transfection was removed one day after virus transfection, and after four days of antibody stimulation, CAR-T cells with the same CAR expression level (indicated by eGFP fluorescence intensity) were selected on day 5 after antibody stimulation, and obtained T-cells were anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E, respectively _YF 28Z CAR-T cells. After a period of amplification, a series of index identifications are carried out on the CAR-T, 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 viability, in vivo anti-tumor capacity and the like.

Index detection method and result description:

obtained anti-CD 19Z CAR, anti-CD 19E 28Z CAR and anti-CD 19E _YF 28Z CAR structure and CD3 epsilon intracellular domain amino groupThe acid sequence and the amino acid sequence of the intracellular domain of CD3 epsilon with Y/F mutation are shown in FIG. 1 a. Wherein FMC63 is a single chain antibody targeting the CD19 antigen, the Hinge (Hinge region) and transmembrane (transmembrane region) regions of the receptor are derived from human CD28; in anti-CD 19E 28Z CAR, CD3 epsilon is inserted behind the CD28 transmembrane region and in front of the CD28 intracellular region; anti-CD 19E _YF In the 28Z CAR, the amino acid sequence of the CD3 ε intracellular domain with Y/F mutation was inserted after the CD28 transmembrane domain and before the CD28 intracellular domain.

Detection of anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E by anti-mouse FMC63 scFv conjugated Alexa Fluor647 antibody flow _YF The upper membrane level of anti-CD19 CAR receptor in 28Z CAR-T cells, flow chart results are shown in fig. 1b, showing no difference in CAR positive rate and expression amount (MFI) of the three.

Detection of anti-CD 19Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E by anti-human CD4-APC and anti-human CD8-PE-Cy7 antibody flow _YF The results of the 28Z CAR-T cell and the flow chart are shown in FIG. 1c, showing CD4 of the three ⁺ CAR-T cells and CD8 ⁺ The ratio of CAR-T cells was similar.

10-thousand anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF 28Z CAR-T cells and CD19, respectively ⁺ Lymphoma cell line Raji was according to 1:1 in a round bottom 96-well plate, mixing, centrifuging (400 g,1 min) at room temperature, and collecting samples after culturing in a 37 ℃ incubator for one day. Adding 1 XBFA to inhibit cytokine excretion 6 hours before sample collection, washing the sample with PBS once after sample collection, fixing 4% PFA for 5min at room temperature, punching 0.1% TritonX-100 for 5min at room temperature, and detecting IL-2, IFN-gamma and TNF-alpha by using corresponding direct standard antibodies according to the conventional operation of intracellular staining steps. The results are shown in FIGS. 1 d-1 f, for anti-CD19E compared to anti-CD19E 28Z CAR-T cells _YF 28Z CAR-T cell warp CD19 ⁺ The levels of IL-2, IFN-gamma and TNF-alpha cytokines produced after specific stimulation of the cells were significantly increased, indicating that the ITAM motif of the intracellular domain of CD3 epsilon has an important inhibitory effect on cytokine production. But notably anti-CD19E _YF IFN-gamma, TNF-alpha cells secreted by 28Z CAR-T cellsThe factor was still significantly less than anti-CD19 28Z CAR-T cells (FIGS. 1 e-1 f). Can reduce macrophage monocyte activation to produce inflammatory cytokines such as IL-1 beta and IL-6.

10-thousand anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF 28Z CAR-T cells were isolated from CD19+ lymphoma cell line Raji according to 1:1 ratio was inoculated in a round bottom 96 well plate, mixed well, centrifuged (400 g,1 min) at room temperature for contact between the cells, and the whole culture was based on culture in an incubator at 37℃for a period of time. Samples were washed once in PBS after harvest at different time points, and the expression level of Ki67 was flow-detected after staining with anti-human Ki67-APC antibody according to the Foxp3/Transcription Factor Staining Buffer Set kit instructions. The results are shown in FIG. 1g, anti-CD 19E _YF 28Z CAR-T warp CD19 ⁺ Proliferation rate after Raji cell stimulation is always faster than anti-CD 19Z 28Z CAR-T cells, even than anti-CD 19E 28Z CAR-T cells, i.e. anti-CD 19E _YF 28Z CAR-T cells proliferate most.

10-thousand anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF 28Z CAR-T cells were isolated from CD19+ lymphoma cell line Raji according to 1:1 ratio was inoculated in a round bottom 96 well plate, mixed well, centrifuged (400 g,1 min) at room temperature for contact between the cells, and the whole culture was based on culture in an incubator at 37℃for a period of time. Samples were washed once in PBS after collection at different time points and stained according to Annexin V Apoptosis Detection Kit APC kit instructions for the flow-through detection of the expression level of CAR-T cell Annexin V. The results are shown in FIG. 1h, anti-CD 19E _YF 28Z CAR-T cells and anti-CD19E 28Z CAR-T cells via CD19 ⁺ After Raji cell stimulation, apoptosis levels were significantly lower than anti-CD19 28Z CAR-T cells for a period of time.

10-thousand anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF 28Z CAR-T cells were isolated from CD19+ lymphoma cell line Raji according to 1:1 ratio was inoculated in a round bottom 96 well plate, mixed well, centrifuged (400 g,1 min) at room temperature for contact between the cells, and the whole culture was based on culture in an incubator at 37℃for a period of time. After sampling at different time points, the sample was washed once with PBS and then with antDetection of CAR by i-human CD4-APC and anti-human CD8-PE-Cy7 antibody staining ⁺ T cell survival number. The results are shown in FIG. 1i, via CD19 ⁺ anti-CD19E after Raji cell stimulation _YF The number of survival of 28Z CAR-T cells was the greatest, followed by anti-CD19E 28Z CAR-T cells, and the number of survival of anti-CD19 28Z CAR-T cells was the least, indicating E _YF The continuous viability of 28Z CAR-T cells was the strongest.

anti-CD19 28Z CAR-T cells, anti-CD19E 28Z CAR-T cells and anti-CD19E _YF 28Z CAR-T cell pair CD19 ⁺ Tumor cytotoxicity response. CD19 ^- K562 cells were resuspended in 1640 serum-free medium, then pre-stained with 1x CellTraker deep red dye (37℃water bath, 30 min), then 1640 serum-free medium washed once, resuspended in T-cell complete medium and then incubated with CD19 ⁺ K562_cd19 (IRES mCherry) cells were isolated according to 1:1 into target cells. CAR-T cells were then mixed with the mixed target cells according to 3:1,1:1,1:3 proportion mixing and plating on a round bottom 96-well plate, mixing uniformly, centrifuging (400 g,1 min) at room temperature, contacting the cells, completely culturing, collecting sample after culturing in a culture box at 37 ℃ for one day, placing ice, and detecting CD19 in a flow mode ^- K562 cells (APC) ⁺ ,mCherry ^- ) Percentage and CD19 ⁺ K562_CD19 cells (APC) ^- ,mCherry ⁺ ) Percent change, "CD 19" of the experimental group ^- K562 cells (APC) ⁺ ,mCherry ^- ) percentage/CD 19 ⁺ K562_CD19 cells (APC) ^- ,mCherry ⁺ ) The percentage is denoted as N. N value "N" in wells of mixed target cell samples without CAR-T cells ₀ ＝CD19 ^- K562 cells (APC) ⁺ ,mCherry ^- ) percentage/CD 19 ⁺ K562_CD19 cells (APC) ^- ,mCherry ⁺ ) Percentage "normalized, survival ratio N/N was calculated ₀ The killing rate formula is "Lysis (%) =1-N/N ₀ ". As shown in FIG. 1j, anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF CD19 pairs of 28Z CAR-T cells ⁺ Tumor cell killing toxicity is equivalent.

10-thousand anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF 28ZCAR-T cells were individually associated with the cd19+ lymphoma cell line Raji according to 1:1 ratio was inoculated into 1.5ml EP tube, mixed well, centrifuged (500 g,2 min) at 4℃to promote cell-cell contact, and the whole culture was stimulated based on 37℃water bath for a series of time points. Adding equal volume of 8% PFA at different time points, fixing at room temperature for 5min, then punching at room temperature for 5min with 0.1% TritonX-100, and performing intracellular staining according to conventional procedures to obtain anti-staining Rabbit source pErk (1/2) ^{Thr202/Tyr204} (FIG. 1 k), rabbit pAKT ^S473 (FIG. 1 l) and primary antibody to rabbit pS6 ^S235/236 (FIG. 1 m), washing once, and coupling the secondary anti-goat anti-rabbit IgG polyclonal antibody with Alexa647, and performing flow detection after washing once. The results are shown in FIG. 1 k-FIG. 1m, anti-CD 19E _YF pErk (1/2) after deliberate antigen stimulation of 28Z CAR-T cells ^{Thr202/Tyr204} And pS6 ^S235/236 The overall level is significantly higher than anti-CD 19Z CAR-T and anti-CD 19E 28Z CAR-T cells, anti-CD 19E _YF AKT of 28Z CAR-T cells and anti-CD 19E 28Z CAR-T cells ^S473 The phosphorylation levels were significantly higher than for anti-CD19 28Z CAR-T cells. In the signal transduction pathway of chimeric antigen receptor CAR after antigen activation, the signal molecule serine/threonine protein kinase Erk (1/2) of Raf/MAPK signal pathway ^{Thr202/Tyr204} Phosphorylation levels and serine/threonine kinase AKT in the PI3K/AKT signaling pathway ^S473 The phosphorylation levels represent the signal molecule ribosomal protein S6 downstream of both pathways, which promotes cell proliferation viability and anti-apoptotic capacity ^S235/236 The phosphorylation level marks the proliferation level of cell growth, and the phenotype of the three signal molecules is used for elucidating anti-CD 19E from the signal path level _YF The proliferation viability of 28Z CAR-T cells was strongest.

The experimental data processing method comprises the following steps:

statistical analysis of data using software GraphPad Prism 8.0.2, statistical P <0.05 indicated that the differences were significant, specifically expressed as: * P <0.05, < P <0.01, < P <0.001, < P <0.0001, < data in each group are shown as mean±sd.

FIGS. 1 d-1 f are single factor variance analysis plus Sidak's multiple comparison test;

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

FIG. 1i two-factor variant analysis.

Experimental results:

(1)、anti-CD19 E _YF the 28Z CAR-T cells are equivalent to the anti-CD 19E 28Z CAR-T cells, the anti-CD19 CAR receptor upper membrane level of the anti-CD19 28Z CAR-T cells, and the ratio of CD4 and CD 8;

(2) anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF After the 28Z CAR-T cells are subjected to Raji cell in-vitro specific stimulation, compared with anti-CD19 CD 28Z CAR-T cells, anti-CD 19E _YF The significant reversion of IL-2, IFN-gamma and TNF-alpha cytokine levels produced by 28Z CAR-T cells suggests that the ITAM motif of the CD3 epsilon intracellular domain has an important inhibitory effect on cytokine production. But notably anti-CD 19E _YF These cytokines were still significantly less secreted by 28Z CAR-T cells than anti-CD19 28Z CAR-T cells (FIGS. 1 e-1 f). Can reduce macrophage monocyte activation to produce inflammatory cytokines such as IL-1 beta and IL-6.

(3) anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF After 28Z CAR-T cells are subjected to Raji cell in-vitro specific stimulation and amplification for a period of time, the proliferation capacity (namely Ki67 expression level) is sampled and detected at different time points, and compared with anti-CD19 28Z CAR-T cells and anti-CD 19E 28Z CAR-T cells, anti-CD 19E _YF The 28Z CAR-T cells have the best proliferation capacity;

(4) anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF After 28Z CAR-T cells are subjected to Raji cell in-vitro specific stimulation and amplification for a period of time, sampling and detecting apoptosis levels (expressed by Annexin V) at different time points, and anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF The apoptosis level of the 28Z CAR-T cells is obviously lower than that of the anti-CD19 28Z CAR-T cells;

(5) anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF After 28Z CAR-T cells are amplified by Raji cell specific stimulation for a period of time, the amplified CAR is sampled and detected at different time points ⁺ T cell number, over timeanti-CD 19E _YF The number of 28Z CAR-T cells persisted significantly higher than anti-CD19 28Z CAR-T cells and anti-CD 19E 28Z CAR-T cells;

(6) anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF In an in vitro killing experiment of 28Z CAR-T cells incubated with CD19+K562:CD19-K562, anti-CD 19E _YF The killing capacity of the 28Z CAR-T cells is equivalent to that of the anti-CD19 28ZCAR-T cells and the anti-CD 19E 28ZCAR-T cells.

(7) anti-CD19 28Z CAR-T cells, anti-CD 19E 28Z CAR-T cells and anti-CD 19E _YF After 28Z CAR-T cells are subjected to Raji cell specific stimulation and amplification for a period of time, detection signal molecules serine/threonine protein kinase Erk (1/2) are sampled at different time points ^{Thr202/Tyr204} Serine/threonine kinase AKT ^S473 And ribosomal protein S6 ^S235 ^/236 Phosphorylation level. anti-CD 19E _YF pErk (1/2) after deliberate antigen stimulation of 28Z CAR-T cells

^{Thr202/Tyr204} And pS6 ^S235/236 The overall level is significantly higher than that of anti-CD 19E 28Z CAR-T cells and anti-CD 19E 28Z CAR-T cells, while anti-CD 19E _YF AKT of 28Z CAR-T cells and anti-CD 19E 28Z CAR-T cells ^S473 The phosphorylation levels were significantly higher than for anti-CD19 28Z CAR-T cells. In the signal transduction pathway of the complex chimeric antigen receptor CAR after antigen activation, raf/MAPK signal pathway signal molecule Erk (1/2) ^{Thr202/Tyr204} Phosphorylation levels and AKT in the PI3K/AKT signaling pathway ^S473 The phosphorylation level indicates that the cell proliferation and survival ability and anti-apoptosis ability are promoted, and the common signal molecule ribosomal protein S6 downstream of the two paths ^S235/236 The phosphorylation level marks the proliferation level of cell growth, and the phenotype of the three signal molecules is used for elucidating anti-CD 19E from the signal path level _YF The proliferation viability of 28Z CAR-T cells was strongest.

anti-CD19 E _YF Comparison of the anti-subcutaneous tumor ability of 28Z CAR-T cells with anti-CD 19E 28Z cells and anti-CD19 28Z CAR-T cells in vivo, the experimental protocol is as follows:

Sterile manipulation preparation of 3x 10A 7/ml Raji cellsPBS resuspension 100ul Raji cells were inoculated subcutaneously 3 million on the left dorsal part of each 6 week old B-NDG female mouse, time point being day0. After 6 days, when Raji subcutaneous tumors grew to a diameter of 6-7mm, mice were randomly grouped into three groups (E _YF 28Z group, E28Z group, vector group) and then 100ul of 8 million T cells were intravenously injected into each mouse tail, after which tumor size was measured periodically with vernier calipers, tumor data and mouse survival were recorded, tumor area = length x width. Conclusion of experiment: anti-CD 19E _YF The 28Z CAR-T cell has anti-tumor capability.

While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Sequence listing

<110> China academy of sciences molecular cell science Excellent innovation center

<120> a chimeric antigen receptor comprising a CD3 epsilon intracellular region with Y/F mutation and uses 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

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

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

the intracellular domain comprises the sequence shown in SEQ ID NO:1, a Y/F mutated CD3 epsilon intracellular region as set forth in SEQ ID NO:2 and a CD28 intracellular domain co-stimulatory signaling region as set forth in SEQ ID NO:3, a cd3ζ intracellular segment shown in figure 3;

The antigen recognition region is selected from FMC63;

the hinge region is selected from the group consisting of a CD28 hinge region;

the transmembrane domain is selected from the group consisting of the CD28 transmembrane region.

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

a. the amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO:4 is shown in the figure;

b. the amino acid structure of the CD28 hinge region is shown in SEQ ID NO: shown at 7.

3. The chimeric antigen receptor comprising a CD3 epsilon intracellular region with a Y/F mutation of claim 1, wherein said chimeric antigen receptor comprising a CD3 epsilon intracellular region with a Y/F mutation has the amino acid sequence set forth in SEQ ID NO: shown at 5.

4. A polynucleotide molecule selected from the group consisting of:

a polynucleotide sequence encoding a chimeric antigen receptor according to any one of claims 1-3 comprising a CD3 epsilon intracellular region with a Y/F mutation.

5. The polynucleotide molecule of claim 4, wherein said polynucleotide sequence is set forth in SEQ ID NO: shown at 6.

6. A nucleic acid construct comprising the polynucleotide molecule of any one of claims 4-5.

7. The nucleic acid construct of claim 6, wherein the nucleic acid construct is a vector.

8. The nucleic acid construct of claim 7, which is a lentiviral vector comprising a replication origin, a 3'LTR, a 5' LTR and a polynucleotide molecule according to any of claims 4 to 5.

9. A lentiviral vector system comprising the nucleic acid construct of any one of claims 6 to 8 and a lentiviral vector accessory ingredient.

10. A genetically modified T cell comprising the polynucleotide molecule of any one of claims 4 to 5, or comprising the nucleic acid construct of any one of claims 6 to 8, or infected with the lentiviral vector system of claim 9.

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

12. Use of a chimeric antigen receptor comprising a CD3 epsilon intracellular region with a Y/F mutation according to any one of claims 1-3, a polynucleotide molecule according to any one of claims 4-5, or a nucleic acid construct according to any one of claims 6-8, or a lentiviral vector system according to claim 9, or a genetically modified T cell according to claim 10, in the preparation of a tumor therapeutic product.