Chimeric antigen receptor and application thereof

Abstract

To provide a chimeric antigen receptor including a CD3ε intracellular region having Y / F mutation or including a motif of a CD3ε intracellular basic amino acid rich region, and an application thereof.SOLUTION: A structure of a chimeric antigen receptor including a CD3ε intracellular region having Y / F mutation includes an extracellular domain, a transmembrane domain and an intracellular domain which are sequentially coupled. To one end coupled to the transmembrane domain in the intracellular domain, the CD3ε intracellular region having the Y / F mutation is coupled. The CD3ε intracellular region having the Y / F mutation is a Y / F mutation type CD3ε intracellular region in which two tyrosines in an ITAM in the CD3ε intracellular region are mutated to phenylalanines. T cells modified with the chimeric antigen receptor have improved viability, decreased apoptosis level, and increased proliferation ability, and downregulate levels of expression of cytokines IFN-γ and TNF-α.SELECTED DRAWING: Figure 1a

Description

[Technical field]

[0001] The present invention relates to the field of chimeric antigen receptors, specifically to chimeric antigen receptors comprising a CD3ε intracellular domain with a Y / F mutation or comprising a motif of the CD3ε intracellular basic amino acid-rich domain, and uses thereof. [Background technology]

[0002] Chimeric antigen receptor is abbreviated as CAR, and T cells equipped with CAR that targets a specific tumor antigen are called CAR-T cells. CAR-T therapy has been widely applied in tumor treatment (especially immunotherapy for solid tumors). This therapy has become a new type of precision targeting therapy in tumor treatment, and in recent years, through optimization and improvement, it has achieved good results in clinical tumor treatment. In other words, CAR-T therapy is a new type of tumor immunotherapy that is very promising, accurate, rapid, efficient, and can cure cancer.

[0003] However, CAR-T therapy still has many limitations, such as the cytokine storm encountered in clinical tumor treatment, the safety of neurotoxicity, and poor sustained proliferation ability. In 2015, one phase I clinical trial (clinical trial number: NCT01593696, 2015.Lacet.T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults-a phase 1 dose-escalation trial) was published in The Lancet. According to this announcement, after administering KTE-C19 (anti-CD19 28Z) CAR-T therapy to patients (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukemia or non-Hodgkin's lymphoma, the number of CAR-T cells in the blood of the body was detected, and the number of CAR-T cells peaked on the 14th day, but then there was a significant decline on the 28th and 42nd days, and by the 68th day, CAR-T cells could no longer be detected in the blood of any patient. Furthermore, in 2018, the results of one Phase I clinical trial (clinical trial number: NCT01044069, 2018.NEJ.Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia) were published in the New England Journal of Medicine. This presentation also showed that after administering anti-CD19 28Z CAR-T therapy to 53 patients suffering from relapsed acute lymphoblastic leukemia, the number of CAR-T cells in the blood in the body was detected, and the median number appeared on day 14. Both of these studies indicate that 28Z CAR-T cells have relatively poor sustained proliferation and survival ability, and that improvements in the therapeutic effect are desirable. Summary of the Invention [Problem to be solved by the invention]

[0004] When designing modifications to the CAR structure itself, consideration should be given to improving the sustained viability of CAR-T cells in order to improve antitumor effects, extend disease remission periods, reduce relapse rates, and extend patient survival. In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a chimeric antigen receptor comprising a CD3ε intracellular domain having a Y / F mutation or comprising a motif of the CD3ε intracellular basic amino acid-rich domain, and uses thereof. [Means for solving the problem]

[0005] To achieve the above and other related objects, the present invention provides in a first aspect In accordance with the present invention, there is provided a chimeric antigen receptor comprising a CD3ε intracellular domain having a Y / F mutation. The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, which are linked in order. The extracellular domain comprises an antigen recognition region and a hinge region.

[0006] A CD3ε intracellular domain having a Y / F mutation is linked to one end of the intracellular domain linked to the transmembrane domain. The CD3ε intracellular domain having a Y / F mutation refers to a Y / F mutant CD3ε intracellular domain in which both of the two tyrosines in the immunoreceptor tyrosine-based activation motif (ITAM) of the CD3ε intracellular domain are mutated to phenylalanine.

[0007] In a second aspect, the present invention provides a polynucleotide sequence selected from (1) a polynucleotide sequence encoding a chimeric antigen receptor comprising a CD3ε intracellular domain having a Y / F mutation as described in the first aspect of the present invention, and (2) a complementary sequence of the polynucleotide sequence described in (1).

[0008] In a third aspect, the present invention provides a nucleic acid construct comprising a polynucleotide sequence as set out in the second aspect of the invention.

[0009] Preferably, the nucleic acid construct is a vector.

[0010] More preferably, the nucleic acid construct is a lentiviral vector and comprises an origin of replication, a 3'LTR, a 5'LTR and a polynucleotide sequence as described in the second aspect of the invention.

[0011] In a fourth aspect, the present invention provides a lentiviral vector system comprising the nucleic acid construct and ancillary lentiviral vector components as described in the third aspect of the invention.

[0012] In a fifth aspect, the present invention provides a genetically modified T cell, or a pharmaceutical composition comprising said genetically modified T cell, characterized in that said cell comprises a polynucleotide sequence as defined in the second aspect of the invention, or comprises a nucleic acid construct as defined in the third aspect of the invention, or is infected with a lentiviral vector system as defined in the fourth aspect of the invention.

[0013] In a sixth aspect, the present invention provides an application of a chimeric antigen receptor comprising a CD3ε intracellular domain having a Y / F mutation as described in the first aspect of the present invention, a polynucleotide sequence as described in the second aspect of the present invention, a nucleic acid construct as described in the third aspect of the present invention, or a lentiviral vector system as described in the fourth aspect of the present invention, in the manufacture of a product having any one or more of the following uses: (1) generating T cells; (2) improving the viability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation and / or viability of T cells; (5) improving the anti-tumor ability of T cells; and (6) suppressing the level of the cytokine IFN-γ and inhibiting the secretion of TNF-α from T cells.

[0014] In a seventh aspect, the present invention provides the use of a chimeric antigen receptor comprising a CD3ε intracellular domain carrying a Y / F mutation as defined in the first aspect of the invention, a polynucleotide sequence as defined in the second aspect of the invention, a nucleic acid construct as defined in the third aspect of the invention, a lentiviral vector system as defined in the fourth aspect of the invention, or a genetically modified T cell as defined in the fifth aspect of the invention in the manufacture of a tumor therapeutic product.

[0015] As described above, the chimeric antigen receptor comprising the CD3ε intracellular domain having the Y / F mutation of the present invention and uses thereof have the following beneficial effects.

[0016] The chimeric antigen receptor modified T cells of the present invention can significantly improve the persistence of T cells by improving their survival ability, reducing the apoptosis level, and improving their proliferation ability, and can significantly improve the anti-tumor ability of T cells. This advantage can further improve the efficacy of tumor treatment, extend the remission period of the disease, reduce the relapse rate, and extend the survival time of patients in clinical applications. In addition, the modified T cells can down-regulate the expression levels of cytokines IFN-γ and TNF-α, and reduce the production of inflammatory cytokines such as IL-1β and IL-6 caused by the activation of macrophages and monocytes.

[0017] To achieve the above and other related objects, the present invention provides, in an eighth aspect, a chimeric antigen receptor comprising a CD3ε intracellular basic amino acid rich region motif, the chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, which are linked in order.

[0018] The extracellular domain includes an antigen recognition region and a hinge region.

[0019] The intracellular domain has a CD3ε intracellular basic amino acid rich region motif linked to one end linked to the transmembrane domain.

[0020] In a ninth aspect, the present invention provides a polynucleotide sequence selected from (1) a polynucleotide sequence encoding a chimeric antigen receptor comprising the CD3ε intracellular basic amino acid rich region motif as described in the eighth aspect of the present invention, and (2) a complementary sequence of the polynucleotide sequence described in (1).

[0021] In a tenth aspect, the present invention provides a nucleic acid construct comprising a polynucleotide sequence as set out in the ninth aspect of the present invention.

[0022] Preferably, the nucleic acid construct is a vector.

[0023] More preferably, said nucleic acid construct is a lentiviral vector and comprises an origin of replication, a 3'LTR, a 5'LTR and a polynucleotide sequence as set out in the ninth aspect of the invention.

[0024] In an eleventh aspect, the present invention provides a lentiviral vector system comprising the nucleic acid construct and ancillary lentiviral vector components as described in the tenth aspect of the invention.

[0025] In a twelfth aspect, the present invention provides a genetically modified T cell, or a pharmaceutical composition comprising said genetically modified T cell, characterized in that said cell comprises a polynucleotide sequence as defined in the ninth aspect of the invention, or comprises a nucleic acid construct as defined in the tenth aspect of the invention, or is infected with a lentiviral vector system as defined in the eleventh aspect of the invention.

[0026] In a thirteenth aspect, the present invention provides an application of a chimeric antigen receptor comprising a motif of CD3ε intracellular basic amino acid rich region as described in the eighth aspect of the present invention, a polynucleotide sequence as described in the ninth aspect of the present invention, a nucleic acid construct as described in the tenth aspect of the present invention, or a lentiviral vector system as described in the eleventh aspect of the present invention in the manufacture of a product having any one or more uses of: (1) generating T cells; (2) improving the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation ability of T cells; (5) improving the anti-tumor ability of T cells; and (6) suppressing the level of cytokine IFN-γ from T cells.

[0027] In a fourteenth aspect, the present invention relates to a method for the treatment of a CD3ε intracellular basal cytoplasmic reticulum (CD3ε) as described in the eighth aspect of the present invention. The present invention provides an application of a chimeric antigen receptor comprising an amino acid rich region motif, a polynucleotide sequence as defined in the ninth aspect of the invention, a nucleic acid construct as defined in the tenth aspect of the invention, a lentiviral vector system as defined in the eleventh aspect of the invention, or a genetically modified T cell as defined in the twelfth aspect of the invention in the manufacture of a tumor therapeutic product.

[0028] As described above, the chimeric antigen receptor comprising the motif of the CD3ε intracellular basic amino acid-rich region of the present invention and uses thereof have the following beneficial effects:

[0029] The T cells modified with the chimeric antigen receptor containing the motif of CD3ε intracellular basic amino acid rich region of the present invention can significantly improve the persistence of T cells and the antitumor ability of T cells by improving the survival ability, reducing the apoptosis level, and improving the proliferation ability. This advantage can further improve the tumor treatment effect, extend the remission period of the disease, reduce the relapse rate, and extend the survival time of patients in clinical applications. In addition, the modified T cells can downregulate the expression level of the cytokine IFNγ and reduce the production of inflammatory cytokines such as IL-1β and IL-6 caused by the activation of macrophages and monocytes. [Brief description of the drawings]

[0030] [Figure 1a] FIG. 1a shows the schematic structure of anti-CD19 28Z CAR, anti-CD19 E28Z CAR, and anti-CD19 EYF28Z CAR, as well as the amino acid sequence of the CD3ε intracellular region and the amino acid sequence of the CD3ε intracellular region with a Y / F mutation. In the figure, FMC63 is a single-chain antibody targeting the CD19 antigen, and the hinge region and transmembrane region of the receptor are derived from human CD28. In anti-CD19 E28Z CAR, CD3ε is inserted behind the CD28 transmembrane region and in front of the CD28 intracellular region. In addition, in anti-CD19 EYF28Z CAR, the amino acid sequence of the CD3ε intracellular region with a Y / F mutation is inserted behind the CD28 transmembrane region and in front of the CD28 intracellular region. [Figure 1b] Figure 1b is a flow cytometry graph of the membrane level of anti-CD19 28Z CAR, anti-CD19 E28Z CAR and anti-CD19 EYF28Z CAR after expression in T cells. [Figure 1c] Figure 1c is a flow cytometry graph of the CD4+ and CD8+ cell percentages in anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells. [Figure 1d] Figure 1d shows a comparison of IL-2 cytokine levels after stimulation of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 1e] Figure 1e is a comparison of IFN-γ cytokine levels after stimulation of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 1f]Figure 1f shows a comparison of TNF-α cytokine levels after stimulation of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 1g] Figure 1g shows the proliferation of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells after stimulation with Raji cells (CD19 antigen). [Figure 1h] Figure 1h shows the apoptosis status of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells after stimulation with Raji cells (CD19 antigen). [Figure 1i] Figure 1i shows the survival rates of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells after stimulation with Raji cells (CD19 antigen). [Figure 1j] Figure 1j shows the toxic responses of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells against CD19+ tumor cells. [Figure 1k] Figure 1k shows the phosphorylation level of the signal molecule serine / threonine protein kinase Erk(1 / 2)Thr202 / Tyr204 (pErk(1 / 2)Thr202 / Tyr204), which promotes cell proliferation, survival, and anti-apoptosis, after stimulation of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 1l]Figure 1l shows the phosphorylation (pAKTS473) levels of the signal molecule serine / threonine kinase AKTS473, which promotes cell proliferation, survival, and anti-apoptosis, after stimulation of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 1m] Figure 1m shows the phosphorylation (pS6S235 / 236) levels of the signal molecule ribosomal protein S6S235 / 236, which marks cell growth and proliferation, after stimulation of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 2a] Figure 2a shows the schematic structure of anti-CD19 28Z CAR and anti-CD19 EBRS28Z CAR. FMC63 in the figure is a single chain antibody targeting CD19 antigen, and the hinge region and transmembrane region of the receptor are derived from human CD28. The motif of CD3ε intracellular basic rich region is inserted after the CD28 transmembrane region and before the CD28 intracellular region. [Figure 2b] Figure 2b is a flow cytometry graph of the membrane level of anti-CD19 28Z CAR and anti-CD19 EBRS28Z CAR after expression in T cells. [Figure 2c] Figure 2c is a flow cytometry graph of CD4+ and CD8+ cell percentages in anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells. [Figure 2d] Figure 2d is a comparison of IL-2 cytokine levels after stimulation of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 2e]Figure 2e is a comparison of IFN-γ cytokine levels after stimulation of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 2f] Figure 2f shows a comparison of TNF-α cytokine levels after stimulation of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 2g] Figure 2g shows the proliferation of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells after stimulation with Raji cells (CD19 antigen). [Figure 2h] Figure 2h shows the apoptosis status of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells after stimulation with Raji cells (CD19 antigen). [Figure 2i] Figure 2i shows the survival rate of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells after stimulation with Raji cells (CD19 antigen). [Figure 2j] Figure 2j shows the phosphorylation level of the signal molecule serine / threonine kinase AKTS473, which promotes cell proliferation, survival, and anti-apoptosis, after stimulation of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 2k] Figure 2k shows the phosphorylation level of the signal molecule ribosomal protein S6S235 / 236, which marks cell growth and proliferation, after anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells were stimulated with Raji cells (CD19 antigen). [Figure 2l] Figure 2l shows the toxic responses of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells against CD19+ tumor cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The chimeric antigen receptor comprising the CD3ε intracellular region carrying the Y / F mutation described in the present invention comprises an extracellular domain, a transmembrane domain and an intracellular domain, which are linked in order.

[0032] The extracellular domain includes an antigen recognition region and a hinge region.

[0033] A CD3ε intracellular domain having a Y / F mutation is linked to one end of the intracellular domain linked to the transmembrane domain. The CD3ε intracellular domain having a Y / F mutation refers to a Y / F mutant CD3ε intracellular domain in which both of the two tyrosines in the immunoreceptor tyrosine-based activation motif (ITAM) of the CD3ε intracellular domain are mutated to phenylalanine.

[0034] Furthermore, the amino acid sequence of the CD3ε intracellular domain having the Y / F mutation is as shown in SEQ ID NO:1, specifically, KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDFEPIRKGQRDLFSGLNQRRI.

[0035] The intracellular domain comprises a CD3ε intracellular region with a Y / F mutation (two tyrosines in the ITAM motifs of the intracellular region are mutated to phenylalanines), an intracellular region of a costimulatory signaling region, and a CD3ζ intracellular segment linked in sequence.

[0036] In one embodiment, the costimulatory signaling region is selected from one or more intracellular segments of CD27, CD28, CD134, 4-1BB, OX40, or ICOS.

[0037] In a further preferred embodiment, the costimulatory signaling region is selected from the CD28 intracellular segment, which results in stronger killing ability.

[0038] The amino acid sequence of the CD28 intracellular segment is as shown in SEQ ID NO:2, and specifically, is as follows: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

[0039] In one embodiment, the amino acid sequence of the CD3 zeta intracellular segment is As shown in O:3. Specifically, RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0040] In one embodiment, the antigen recognition region is selected from a single chain antibody that targets a tumor surface antigen, and the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein (FAP), Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, and GD2.

[0041] Preferably, it is selected from CD19 or mesothelin, which have good target specificity.

[0042] The transmembrane domain is selected from one or more transmembrane regions of CD28, CD4, CD8α, OX40, or H2-Kb.

[0043] Preferably, the transmembrane domain is selected from the transmembrane region of CD28. Specifically, the amino acid sequence of the transmembrane region of CD28 is as shown in SEQ ID NO:4. Specifically, it is FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0044] The hinge region is selected from one or more of the following: a CD28 hinge region, a CD8α hinge region, a CD4 hinge region, an immunoglobulin IgG hinge region, or an immunoglobulin IgG hinge region and a CH2CH3 region linked hinge region, i.e., it can be an IgG1 hinge or an IgG1 hinge-CH2CH3.

[0045] Optionally, the sequence of the hinge region is CD28 hinge region, and the amino acid structure is as shown in SEQ ID NO: 7, specifically, IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0046] In one embodiment, the single chain antibody comprises a light chain variable region and a heavy chain variable region.

[0047] In one embodiment, the light chain variable region and the heavy chain variable region are connected by a linker sequence.

[0048] In one embodiment, the single chain antibody is selected from FMC63.

[0049] Commercially available products can be used for the FMC63.

[0050] In one embodiment, the single chain antibody comprises the light chain variable region and the heavy chain variable region of monoclonal antibody FMC63, optionally connected by a linker sequence.

[0051] In one embodiment, the antigen recognition region of the chimeric antigen receptor comprising the CD3ε intracellular region having the Y / F mutation is selected from FMC63, the hinge region is selected from the CD28 hinge region, and the transmembrane domain is selected from the CD28 transmembrane region. The intracellular domain comprises the CD3ε intracellular region having the Y / F mutation, the intracellular segment of CD28, and the CD3ζ intracellular segment, which are linked in sequence.

[0052] In one embodiment, a chimeric antigen receptor (anti-CD19 E) comprising a CD3ε intracellular domain having the Y / F mutation is YF The amino acid sequence of 28Z CAR is as shown in SEQ ID NO:5, and specifically, is as follows:

[0053] SEQ ID NO:5(Anti-CD19 E YF Amino acid sequence of 28Z CAR): (5'→3') Starting from FMC63 scFv, the CD28 hinge region, the CD28 transmembrane region, the CD3ε intracellular region with a Y / F mutation, and the CD3ζ intracellular region are linked in that order. .

[0054] SEQ ID NO:5(Anti-CD19 E YF The nucleotide sequence corresponding to the amino acid sequence of 28Z CAR is SEQ ID NO:6 (Anti-CD19 E YF The nucleotide sequence of 28Z CAR is as follows (5'→3'): gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagt .

[0055] The chimeric antigen receptor containing the CD3ε intracellular basic amino acid rich region motif described in this invention comprises an extracellular domain, a transmembrane domain and an intracellular domain, which are linked in order.

[0056] The extracellular domain includes an antigen recognition region and a hinge region.

[0057] A CD3ε intracellular basic amino acid rich region motif (Basic Residue Rich Sequence), abbreviated as BRS motif, is linked to one end of the intracellular domain linked to the transmembrane domain.

[0058] Furthermore, the amino acid sequence of the motif of the CD3ε intracellular basic amino acid rich region is as shown in SEQ ID NO:10, specifically, KNRKAKAK.

[0059] The intracellular domain comprises, in sequence, a CD3ε intracellular basic amino acid rich region motif, a costimulatory signaling region and a CD3ζ intracellular segment.

[0060] In one embodiment, the costimulatory signaling region is selected from one or more intracellular segments of CD27, CD28, CD134, 4-1BB, OX40, or ICOS.

[0061] In a further preferred embodiment, the costimulatory signaling region is selected from the intracellular segment of CD28, which results in stronger killing ability.

[0062] The amino acid sequence of the intracellular segment of CD28 is as shown in SEQ ID NO:2, specifically, RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

[0063] In one embodiment, the amino acid sequence of the CD3 zeta intracellular segment is as shown in SEQ ID NO:3. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0064] In one embodiment, the antigen recognition region is selected from a single chain antibody that targets a tumor surface antigen, and the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein (FAP), Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, and GD2.

[0065] Preferably, the tumor surface antigen is selected from CD19 or mesothelin, which have good target specificity.

[0066] Further, the transmembrane domain is selected from one or more transmembrane regions of CD28, CD4, CD8α, OX40, or H2-Kb.

[0067] Preferably, the transmembrane domain is selected from the transmembrane region of CD28. Specifically, the amino acid sequence of the transmembrane region of CD28 is as shown in SEQ ID NO:4. Specifically, it is FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0068] The hinge region is selected from one or more of the following: a CD28 hinge region, a CD8α hinge region, a CD4 hinge region, an immunoglobulin IgG hinge region, or an immunoglobulin IgG hinge region and a CH2CH3 region linked hinge region, i.e., it can be an IgG1 hinge or an IgG1 hinge-CH2CH3.

[0069] Optionally, the sequence of the hinge region is CD28 hinge region, and the amino acid structure is as shown in SEQ ID NO: 7, specifically, IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0070] In one embodiment, the single chain antibody comprises a light chain variable region and a heavy chain variable region.

[0071] In one embodiment, the light chain variable region and the heavy chain variable region are connected by a linker sequence.

[0072] In one embodiment, the single chain antibody is selected from FMC63.

[0073] Commercially available products can be used for the FMC63.

[0074] In one embodiment, the single chain antibody comprises the light chain variable region and the heavy chain variable region of monoclonal antibody FMC63, optionally connected by a linker sequence.

[0075] The antigen recognition region of the chimeric antigen receptor containing the CD3ε intracellular basic amino acid rich region motif is selected from FMC63, the hinge region is selected from the CD28 hinge region, and the transmembrane domain is selected from the CD28 transmembrane region. The intracellular domain contains the CD3ε intracellular basic amino acid rich region motif, the CD28 intracellular segment, and the CD3ζ intracellular segment, which are linked in sequence.

[0076] In one embodiment, a chimeric antigen receptor (CAR) comprising a CD3ε intracellular basic amino acid rich region motif (Anti-CD19 E BRS 28Z CAR) is a fusion protein consisting of FMC63 scFv, CD28 hinge region, CD28 transmembrane domain, CD3εBRS motif and CD3ζ cell line. Includes intracellular region.

[0077] In one embodiment, a chimeric antigen receptor (CAR) comprising a CD3ε intracellular basic amino acid rich region motif (Anti-CD19 E BRS The amino acid sequence of 28Z CAR is SEQ ID NO: As shown in ID NO:11, specifically, as follows:

[0078] SEQ ID NO:11(Anti-CD19 E BRS Amino acid sequence of 28Z CAR): (5'→3') Starting from FMC63 scFv, the CD28 hinge region, the CD28 transmembrane region, the CD3ε BRS motif and the CD3ζ intracellular region are linked in that order. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKNRKAKAKRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0079] Anti-CD19 E BRS The nucleotide sequence corresponding to the amino acid sequence of 28Z CAR is SEQ ID NO:12 (Anti-CD19 E BRS The nucleotide sequence of 28Z CAR is as follows (5'→3'): gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaag gccaagaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

[0080] The above-mentioned portions forming the chimeric antigen receptor of the present invention may be linked to each other directly or via a linker sequence. The linker sequence may be a linker sequence applied to antibodies well known in the art, for example, a linker sequence containing G and S. Usually, the linker contains one or more motifs repeated before and after. For example, the motifs can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the linker sequence, and no amino acid residues are inserted between the repeats. The linker sequence can be configured to contain 1, 2, 3, 4, or 5 repeat motifs. The length of the linker can be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the linker sequence is a polyglycine linker sequence. There is no particular limit to the number of glycines in the linker sequence, and it is usually 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may contain other known amino acid residues (e.g., alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.).

[0081] It should be understood that gene cloning often requires the design of an appropriate enzyme cleavage site. In that case, one or more irrelevant residues are necessarily introduced at the end of the expressed amino acid sequence, but this does not affect the activity of the target sequence. In addition, in order to create a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or advantageously purify the recombinant protein, some amino acid must often be added to the N-terminus, C-terminus, or other appropriate region of the protein (for example, including but not limited to an appropriate linker peptide, signal peptide, leader peptide, terminal extension, etc.). A signal peptide is tentatively considered as follows: That is, a signal peptide having a hydrophobic amino acid residue at the N-terminus is synthesized first during translation of an mRNA encoding a secretory protein. The signal peptide is recognized by and binds to a receptor on the endoplasmic reticulum membrane. When the signal peptide reaches the lumen of the endoplasmic reticulum via a tunnel formed by a membrane protein, it is hydrolyzed by a signal peptidase located on the lumen surface. The induction of the signal peptide allows the nascent polypeptide to pass through the endoplasmic reticulum membrane and enter the lumen, and finally to be secreted outside the cell. There are different sources of the signal peptide of the CAR, mainly the signal peptides of CD8, CD28, and GM-CSF receptor. The N-terminus or C-terminus of the fusion protein of the present invention (i.e., the above-mentioned CAR) may further comprise one or more polypeptide fragments as a protein tag. Any suitable tag can be used herein. For example, the tag can 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 for protein purification.

[0082] The polynucleotide sequence provided by the present invention is selected from (1) a polynucleotide sequence encoding the above-mentioned chimeric antigen receptor, and (2) a complementary sequence of the polynucleotide sequence described in (1).

[0083] The polynucleotide sequences of the present invention may be in the form of DNA or RNA. DNA formats include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be the coding strand or the non-coding strand. The present invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins (i.e., nucleotide sequences that encode the same amino acid sequence but differ in portions of the nucleotide sequence).

[0084] Typically, the polynucleotide sequences described herein can be obtained by PCR amplification. Specifically, primers are designed based on the nucleotide sequences disclosed herein (particularly the open reading frame sequences) and the relevant sequences are obtained by amplification using a commercially available cDNA library or a cDNA library prepared by a common method known to those skilled in the art as a template. If the sequence is long, it is often necessary to carry out two or more PCR amplifications and then ligate the amplified fragments in the correct order.

[0085] The nucleic acid construct provided by the present invention comprises the above-described polynucleotide sequence.

[0086] Said nucleic acid construct further comprises one or more regulatory sequences operatively linked to said polynucleotide sequence. The coding sequence of the CAR described in the present invention can be manipulated in various ways to ensure the expression of said protein. Before inserting the nucleic acid construct into a vector, the nucleic acid construct may be manipulated according to the different expression vectors and requirements. Techniques for modifying polynucleotide sequences using recombinant DNA techniques are known in the art.

[0087] The regulatory sequence may be a suitable promoter sequence. Typically, the promoter sequence is operatively 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 mutated, truncated and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0088] The regulatory sequence may be a suitable transcription terminator sequence, i.e., a sequence that is recognized by a host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.

[0089] The regulatory sequence may be a suitable leader sequence, a non-translated region of an mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.

[0090] Preferably, the nucleic acid construct is a vector.

[0091] Typically, expression of the polynucleotide sequence encoding the CAR is achieved by operably linking the polynucleotide sequence encoding the CAR to a promoter and incorporating the construct into an expression vector, which may be suitable for replication and integration in eukaryotic cells. Exemplary cloning vectors include transcription and translation terminators, initiation sequences, and promoters capable of regulating expression of the desired nucleic acid sequence.

[0092] The polynucleotide sequence encoding the CAR of the present invention can be cloned into many types of vectors. For example, it can be cloned into a plasmid, a bacteriophage, a phage derivative, an animal virus, and a cosmid. Furthermore, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is 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. Usually, a suitable vector includes an origin of replication that is functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.

[0093] More preferably, the nucleic acid construct is a lentiviral vector and comprises an origin of replication, a 3'LTR, a 5'LTR and the polynucleotide sequence.

[0094] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that allows high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is the elongation factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may be used, including, but not limited to, the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, EB virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters (e.g., but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter). In addition, the use of inducible promoters may be considered. The use of inducible promoters provides a molecular switch. The molecular switch can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0095] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cells may contain either a selectable marker gene or a reporter gene, or both. This is convenient for identifying and selecting expressing cells from a group of cells that have been attempted to be transfected or infected with a viral vector. On the other hand, the selectable marker can be carried on a single DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences so that they can be expressed in the host cell. Useful selectable markers include antibiotic resistance genes, such as neo.

[0096] Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. After the DNA is introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are also known and can be produced by known techniques or obtained commercially.

[0097] Methods for introducing genes into cells and for expressing genes in cells have been known in the art. The vector can 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, an expression vector can be introduced into a host cell by physical, chemical or biological means.

[0098] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Chemical means for introducing a 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.

[0099] Biological methods for introducing polynucleotides into host cells include using viral vectors, particularly lentiviral vectors. This has become the most common method for introducing genes into mammalian (e.g., human) cells. Other viral vectors are available from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses and adeno-associated viruses. Numerous virus-based systems have been developed for introducing genes into mammalian cells, and lentiviruses, for example, provide a convenient platform for gene transfer systems. Using techniques known in the art, a selected gene can be introduced into a vector and packaged into lentiviral particles. The recombinant virus can then be isolated and introduced into target cells in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Numerous adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0100] The present invention provides a lentiviral vector system, which comprises the above-mentioned nucleic acid construct and auxiliary components of a lentiviral vector.

[0101] The lentiviral accessory components include lentiviral packaging plasmids and cell lines.

[0102] The lentiviral vector system is formed by packaging the nucleic acid construct into a virus with the aid of a lentiviral packaging plasmid and a cell line. The method for constructing the lentiviral vector system is a method commonly used in the art.

[0103] The present invention provides a method for in vitro activation of T cells, said method comprising the step of infecting said T cells with said lentivirus.

[0104] Compared with other drug therapies, CAR-T cells can be maintained in the body for a long time based on their self-proliferation and survival ability, and when stimulated by a specific target antigen, they can rapidly expand and kill the target tumor cells, and then form specific memory cells, such as effector memory cells and central memory cells.

[0105] The present invention further includes cell therapy, in which T cells are genetically engineered to express a CAR as described herein, and the CAR-T cells are infused into a recipient in need thereof. The infused cells are capable of killing tumor cells in the recipient. Unlike antibody therapy, CAR-T cells are capable of replicating in the body and have the long-term potential to control tumors in a sustained manner. It demonstrates long-term sustainability.

[0106] The anti-tumor immune response caused by CAR-T cells can be an active or passive immune response. In addition, the immune response induced by CAR can be part of an adoptive cell therapy step. CAR-T cells induce an immune response specific to the antigen-binding portion of the CAR.

[0107] The treatable cancer can be a non-solid tumor, such as a hematological tumor (e.g., leukemia or lymphoma). In particular, the diseases treatable using the CARs of the invention, their coding sequences, nucleic acid constructs, expression vectors, viruses and CAR-T cells are preferably CD19-driven diseases, in particular CD19-driven hematological tumors.

[0108] Specifically, "CD19-induced diseases" in this context include, but are not limited to, leukemias and lymphomas, such as B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0109] The present invention provides a genetically modified T cell or a pharmaceutical composition comprising said genetically modified T cell, said cell comprising said polynucleotide sequence, said nucleic acid construct or said lentiviral vector system.

[0110] The CAR-modified T cells of the present invention may be administered alone or in combination with diluents and / or other components (e.g., associated cytokines or cell populations) as a pharmaceutical composition. Briefly, a pharmaceutical composition of the present invention may include a combination of the CAR-T cells described herein with one or more pharma- ceutical or physiologically acceptable vectors, diluents or excipients. Such compositions may include buffers (e.g., neutral buffered saline, sulfate buffered saline, etc.), carbohydrates (e.g., glucose, mannose, sucrose or dextran, mannitol), proteins, polypeptides or amino acids (e.g., glycine), antioxidants, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and preservatives.

[0111] The pharmaceutical compositions of the present invention are administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will depend on factors such as the patient's condition, the type and severity of the patient's disease, etc.

[0112] When referring to an "immunologically effective amount," an "antitumor effective amount," a "tumor-suppressing effective amount," or a "therapeutic amount," the exact amount of the composition of the present invention to be administered can be determined by a physician. In doing so, the age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease of the patient (subject) are taken into consideration. Usually, the dosage of the pharmaceutical composition containing T cells described herein is 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6The T cell compositions may be administered in multiple doses at these doses. The cells may be administered by injection techniques known in immunotherapy. The optimal dosage and treatment plan for a particular patient can be readily determined by one skilled in the medical arts by observing the patient's symptoms of disease and adjusting treatment accordingly.

[0113] The subject compositions can be administered in any convenient manner, including by aerosolization, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous or intraperitoneal injection. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions can also be directly injected into a tumor, lymph node or site of infection.

[0114] In some embodiments of the present invention, the CAR-T cells of the present invention or compositions thereof can be combined with other therapies known in the art. Therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressants. For example, various radiation therapy preparations can be combined for treatment. These radiation therapy preparations include cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, FK506, fludarabine, rapamycin, and mycophenolic acid. In a further embodiment, the cell composition of the present invention is administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation or T cell ablation therapy using chemotherapy drugs (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (e.g., OKT3 or CAMPATH).

[0115] In this context, "anti-tumor ability" refers to a biological effect, which can be demonstrated 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.

[0116] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to a living organism (e.g., a mammal) in which an immune response can be generated, including, but not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0117] The chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, and the lentiviral vector system are used in the manufacture of products for any one or more of the following purposes: (1) generating T cells; (2) improving the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation and / or survival ability of T cells; (5) improving the anti-tumor ability of T cells; and (6) suppressing the level of the cytokine IFN-γ and inhibiting the secretion of TNF-α from T cells.

[0118] The improved survival ability, reduced apoptosis levels, and enhanced proliferation ability can significantly improve T cell persistence and thus significantly enhance the antitumor ability of T cells.

[0119] The chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, and the lentiviral vector system can further be used to manufacture a product for one or more of enhancing the proliferation / survival, growth / proliferation or sustained proliferation of T cells, or increasing the number of T cells.

[0120] The chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, the lentiviral vector system or the genetically modified T cell are applied in the manufacture of a tumor treatment product.

[0121] Optionally, the tumor is selected from one or more of a leukemia or a solid tumor.

[0122] Preferentially, said tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and acute myeloid leukemia.

[0123] Hereinafter, the embodiments of the present invention will be described through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Furthermore, the present invention can be implemented or applied by other different specific embodiments. In addition, each detail of this specification can be variously modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0124] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific implementations described below. The terms used in the examples are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. In addition, in the specification and claims of the present invention, unless otherwise specified in the text, the singular forms "one", "one" and "this" include the plural forms.

[0125] When a numerical range is given in the examples, it should be understood that any two endpoints of each numerical range and any numerical value between the two endpoints can be selected unless otherwise specified in the present invention. In addition, unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition, in addition to the specific methods, devices, and materials used in the examples, the present invention may be realized using any method, device, and material in the prior art that is similar or equivalent to the method, device, and material described in the examples of the present invention based on the understanding of the prior art by those skilled in the art and the description of the present invention.

[0126] Unless otherwise specified, the experimental methods, detection methods, and production methods disclosed in the present invention all employ common techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. EXAMPLES

[0127] The sequence of anti-CD19 28Z CAR contains the structure of FMC63 scFv, CD28 hinge region, CD28 transmembrane domain, CD28 and CD3ζ intracellular domain (i.e., the sequence shared by Rosenberg, SA laboratory at NCBI (GenBank: HM852952.1). Anti-CD19 28Z CAR was obtained by gene synthesis, and based on this, the sequence of the CAR structure shown in Figure 1a was designed. Then, eGFP and CAR sequences were linked to the same open reading frame (ORF) via the T2A self-cleaving peptide. Therefore, the expression level of CAR could be indicated by the expression amount of eGFP / fluorescence intensity. In addition, the overlap extension PCR method (gene splicing by overlap extension PCR) was used to design the CAR structure shown in Figure 1a. Using PCR-based site-directed mutagenesis and gene recombination ligation techniques, a new type of CAR was created by inserting the CD3ε intracellular domain with the Y / F mutation (two tyrosines in the ITAM motif of the intracellular domain are mutated to phenylalanines) into the rear of the CD28 transmembrane domain, and expressing anti-CD19 E YF It was named 28Z CAR.

[0128] Anti-CD19 E YF The nucleotide sequence of 28Z CAR is as shown in SEQ ID NO:8. ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC

[0129] E YF Anti-CD19 E28Z C was constructed in the same manner as 28Z, except that the CD3ε intracellular domain carrying the Y / F mutation was replaced with a sequence that corresponds to the CD3ε intracellular domain. The nucleotide sequence of anti-CD19 E28Z CAR is shown in SEQ ID NO:9, specifically as follows: .

[0130] CAR was subcloned into the lentivirus expression plasmid pHAGE vector, and the CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2G in a ratio of 10:7.5:3.5, respectively, and then transfected into 293FT cells using calcium phosphate transfection to generate lentivirus particles. Next, anti-CD19 28Z CAR, anti-CD19 E28Z CAR, or anti-CD19 E28Z CAR were transfected into 293FT cells using ultra-high speed centrifugation. YF The lentiviruses expressing 28Z CAR were concentrated to produce high titers of virus in small volumes (~10 8IU / ml) and then transfected into primary T cells (MOI=10) that had been activated with αCD3 / αCD28 Ab-beads for one day. Primary T cells were cultured in complete T cell medium (XIVO-15+1%PS+10ng / ml human IL-7+10ng / ml human IL-15+5% Human AB serum, 10mM neutralized NAC) with the virus transfected for one day, then removed, stimulated with antibody for four days, and then removed. CAR-T cells with the same CAR expression level (shown by eGFP fluorescence intensity) were then sorted on the fifth day after antibody stimulation, and the obtained T-cells were each transfected with anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF The 28Z CAR-T cells were then grown for a certain period of time, and a series of indicators were assessed for CAR-T, including CAR membrane level, CD4 and CD8 subset ratio, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro sustained viability, and in vivo antitumor ability.

[0131] Regarding the indicator detection method and results: The obtained anti-CD19 28Z CAR, anti-CD19 E28Z CAR and anti-CD19 E YF The structure of 28Z CAR, the amino acid sequence of the CD3ε intracellular domain, and the amino acid sequence of the CD3ε intracellular domain with Y / F mutation are shown in Figure 1a. FMC63 in the figure is a single-chain antibody targeting the CD19 antigen, and the hinge region and transmembrane region of the receptor are derived from human CD28. In the anti-CD19 E28Z CAR, CD3ε was inserted after the CD28 transmembrane domain and before the CD28 intracellular domain. In addition, the anti-CD19 E YF In the 28Z CAR, the amino acid sequence of the CD3ε intracellular domain carrying the Y / F mutation was inserted after the CD28 transmembrane domain and before the CD28 intracellular domain.

[0132] Anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 E YF Anti-CD30 in 28Z CAR-T cells 19 The membrane level of CAR receptor was measured by flow cytometry. The flow cytometry graph results are shown in Figure 1b. This shows that there was no difference in the CAR positivity rate or expression level (MFI) between the three groups.

[0133] Anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies were used to detect anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 E YF Flow cytometry was performed on 28Z CAR-T cells. The flow cytometry graph results are shown in Figure 1c. + CAR-T cells and CD8 + The percentage of CAR-T cells was shown to be similar.

[0134] 100,000 anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were each treated with CD19 +The lymphoma cell line Raji was transplanted into a round-bottom 96-well microplate at a 1:1 ratio, mixed uniformly, and then centrifuged at room temperature (400g, 1min) to promote cell-cell contact. Then, the complete medium was used to culture the cells in a 37°C incubator for 1 day before collection. In addition, 1×BFA was added 6 hours before sample collection to inhibit cytokine release, and the samples were washed once with PBS after sample collection. Then, the cells were fixed with 4% PFA at room temperature for 5 min and perforated with 0.1% Triton X-100 at room temperature for 5 min. Then, intracellular staining procedures were performed according to the usual operation to detect IL-2, IFN-γ, and TNF-α with the corresponding directly labeled antibodies. As a result, as shown in Figure 1d-1f, the anti-CD19 E28Z CAR-T cells showed a significantly higher expression of IL-2, IFN-γ, and TNF-α than the anti-CD19 E28Z CAR-T cells. YF 28Z CAR-T cells are CD19 + The cytokine levels of IL-2, IFN-γ, and TNF-α produced after specific stimulation by CD3ε cells were obviously restored and increased, implying that the ITAM motif in the CD3ε intracellular domain has an important inhibitory effect on cytokine production. However, it should be noted that anti-CD19 E YF The cytokines IFN-γ and TNF-α secreted by 28Z CAR-T cells were still significantly lower than those secreted by anti-CD19 28Z CAR-T cells (Figures 1e-1f). Thus, it was possible to reduce the production of inflammatory cytokines such as IL-1β and IL-6 due to activation of macrophages and monocytes.

[0135] 100,000 anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were each treated with CD19 +The cells were transplanted into a round-bottom 96-well microplate at a 1:1 ratio with the lymphoma cell line Raji, mixed uniformly, and then centrifuged at room temperature (400g, 1 min) to promote cell-cell contact. Next, the cells were cultured for a certain period of time in a 37°C incubator using complete medium. Samples were then collected at different times, washed once with PBS, and stained with anti-human Ki67-APC antibody according to the instructions in the Foxp3 / Transcription Factor Staining Buffer Set reagent kit, and the expression level of Ki67 was measured by flow cytometry. As a result, as shown in Figure 1g, anti-CD19 E YF 28Z CAR-T is a CD19 + The proliferation rate after stimulation with Raji cells was consistently faster than that of anti-CD19 28Z CAR-T cells and even faster than that of anti-CD19 E28Z CAR-T cells. YF 28Z CAR-T cells had the strongest proliferation ability.

[0136] 100,000 anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were each treated with CD19 + The cells were transferred to a round-bottom 96-well microplate at a 1:1 ratio with the lymphoma cell line Raji, mixed uniformly, and centrifuged at room temperature (400 g, 1 min) to remove any cell-cell contact. Next, the cells were cultured in a complete medium at 37°C in an incubator for a certain period of time. Samples were then collected at different times, washed once with PBS, stained according to the Annexin V Apoptosis Detection Kit (APC) reagent kit instructions, and then analyzed for Annexin V (Annexin V) expression in CAR-T cells. The expression levels of anti-CD19 E and V were measured by flow cytometry. As a result, as shown in Figure 1h, YF28Z CAR-T cells and anti-CD19 E28Z CAR-T cells are CD19 + For a sustained period of time following stimulation with Raji cells, the apoptosis level was significantly lower than that of anti-CD19 28Z CAR-T cells.

[0137] 100,000 anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were each treated with CD19 + The cells were transplanted into a round-bottom 96-well microplate at a 1:1 ratio with the lymphoma cell line Raji, mixed uniformly, and then centrifuged at room temperature (400g, 1 min) to promote cell-cell contact. Next, the cells were cultured for a certain period of time in a 37°C incubator using complete medium. Samples were then collected at different times, washed once with PBS, and stained with anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies to detect CAR. + The number of viable T cells was detected. As a result, as shown in Fig. 1i, CD19 + After stimulation with Raji cells, anti-CD19 E YF The 28Z CAR-T cells had the highest survival rate, followed by the anti-CD19 E28Z CAR-T cells, and the anti-CD19 28Z CAR-T cells had the lowest survival rate. YF This means that 28Z CAR-T cells had the strongest ability to survive sustainedly.

[0138] anti-CD1928Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells are CD19 + It has a toxic reaction to tumor cells. -K562 cells were resuspended in 1640 serum-free medium and pre-stained with 1x CellTraker deep red dye (37°C water bath, 30 min). Then, the cells were washed once with 1640 serum-free medium, resuspended in complete T cell medium, and stained for CD19. + The target cells were mixed with K562_CD19 (IRES mCherry) cells at a ratio of 1:1. The CAR-T cells were then mixed with the mixed target cells at a ratio of 3:1, 1:1, or 1:3, plated on a round-bottom 96-well microplate, mixed uniformly, and centrifuged at room temperature (400g, 1 min) to promote cell-cell contact. The samples were then cultured in a 37°C incubator using complete medium for one day, after which the samples were collected and placed on ice. The CD19 - K562 cells (APC + , mCherry - ) and CD19 + K562_CD19 cells (APC - , mCherry + The percentage change in CD19 in the experimental group was measured by flow cytometry. - K562 cells (APC + , mCherry - ) / CD19 + K562_CD19 cells (APC - , mCherry + The N value was calculated by dividing the total number of CD19 cells in the mixed target cell sample wells to which CAR-T cells were not added by the percentage of CD19 cells in the mixed target cell sample wells. - K562 cells (APC + , mCherry - ) / CD19 + K562_CD19 cells (APC - , mCherry + The survival rate N / N0 was calculated based on the percentage of lysis (%) of the anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 E YFThe three components of 28Z CAR-T cells are CD19 + The killing toxicity to tumor cells was equivalent.

[0139] 100,000 anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were each treated with CD19 + The cells were transferred to a 1.5 ml EP tube with lymphoma cell line Raji at a 1:1 ratio, mixed uniformly, and then centrifuged at 4°C (500 g, 2 min) to promote cell-cell contact. The cells were then stimulated with complete medium at a series of timings in a 37°C water bath. At different timings, the cells were fixed with an equal volume of 8% PFA at room temperature for 5 min, and then perforated with 0.1% Triton X-100 at room temperature for 5 min. Then, intracellular staining procedures were performed according to standard procedures. First, rabbit pErk(1 / 2) Thr202 / Tyr204 (Fig. 1k), rabbit-derived pAKT S473 (Figure 1l) and rabbit-derived primary antibody pS6 S235 / 236 (Figure 1m) were stained with primary antibodies, washed once, and then stained with Alexa647-conjugated goat anti-rabbit IgG polyclonal antibody as secondary antibodies, washed once, and then measured by flow cytometry. As a result, as shown in Figure 1k to Figure 1m, anti-CD19 E YF 28Z CAR-T cells express pErk(1 / 2) after specific stimulation with antigen. Thr202 / Tyr204 and pS6 S235 / 236 The overall levels of anti-CD19 E28Z CAR-T cells were significantly higher than those of anti-CD19 28Z CAR-T and anti-CD19 E28Z CAR-T cells. YF AKT in 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells S473 The phosphorylation levels of both CAR-T cells and anti-CD19 28Z CAR-T cells were significantly higher than those of anti-CD19 28Z CAR-T cells. Thr202 / Tyr204and the phosphorylation levels of the serine / threonine kinase AKT in the PI3K / AKT signaling pathway. S473 The phosphorylation levels of ribosomal protein S6, a downstream signaling molecule of both pathways, showed promotion of cell proliferation, viability, and anti-apoptotic activity. S235 / 236 The phosphorylation level of anti-CD19 E is indicative of the cell growth and proliferation level. YF It was revealed that 28Z CAR-T cells had the strongest proliferation and survival capabilities.

[0140] How to process experimental data: Statistical analysis of the data was performed using the software GraphPad Prism 8.0.2, and P < 0.05 (specifically, * P < 0.05, ** P < 0.01, *** P<0.001, **** Differences were considered to be statistically significant when P<0.0001. Data for each group were presented as mean ± SD.

[0141] In Figures 1d to 1f, one-way ANOVA was supplemented with Sidak's multiple comparison test.

[0142] In Figures 1g, 1h, and 1j to 1m, Sidak's multiple comparison test was added to the two-way ANOVA.

[0143] In Figure 1i, a two-way ANOVA was performed.

[0144] Test Results: (1) anti-CD19 E YF 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 28Z CAR-T cells had similar membrane levels of anti-CD19 CAR receptors and CD4 / CD8 ratios.

[0145] (2) anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 EYF Specific stimulation of 28Z CAR-T cells with Raji cells in vitro demonstrated that anti-CD19 E YF The cytokine levels of IL-2, IFN-γ, and TNF-α produced by 28Z CAR-T cells were obviously restored and increased, implying that the ITAM motif in the CD3ε intracellular domain has an important inhibitory effect on cytokine production. However, it should be noted that anti-CD19 E YF The levels of these cytokines secreted by 28Z CAR-T cells were still significantly lower than those secreted by anti-CD19 28Z CAR-T cells (Figures 1e-f). It was possible to reduce the production of proinflammatory cytokines such as IL-1β and IL-6 due to activation of phage and monocytes.

[0146] (3) anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were specifically stimulated in vitro with Raji cells and expanded for a certain period of time. Samples were taken at different times to measure the proliferation ability (i.e., Ki67 expression level). Compared with anti-CD19 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells, anti-CD19 E28Z CAR-T cells showed a significantly higher proliferation ability. YF 28Z CAR-T cells had the best proliferation ability.

[0147] (4) anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were specifically stimulated in vitro with Raji cells and expanded for a certain period of time. Samples were taken at different times to measure apoptosis levels (indicated by Annexin V). YFThe apoptosis levels of 28Z CAR-T cells were significantly lower than those of anti-CD19 28Z CAR-T cells.

[0148] (5) anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were specifically stimulated with Raji cells and amplified for a certain period of time. Samples were then taken at different times to generate amplified CAR + The number of T cells was measured and the anti-CD19 E YF The number of 28Z CAR-T cells was persistently higher than that of anti-CD19 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells.

[0149] (6) anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells and CD19 + K562:CD19 - In an in vitro killing experiment in which K562 cells were co-cultured with anti-CD19 E YF The killing ability of 28Z CAR-T cells was comparable to that of anti-CD19 28ZCAR-T cells and anti-CD19 E28Z CAR-T cells.

[0150] (7) anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells were specifically stimulated with Raji cells and expanded for a certain period of time. Samples were then taken at different times to measure the expression of the signal molecule serine / threonine protein kinase Erk(1 / 2). Thr202 / Tyr204 , serine / threonine kinase AKT S473 and ribosomal protein S6 S235 / 236 The phosphorylation level of anti-CD19 E was measured. YF28Z CAR-T cells express pErk(1 / 2) after specific stimulation with antigen. Thr202 / Tyr204 and pS6 S235 / 236 The overall level of anti-CD19 E was significantly higher than that of anti-CD19 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells. YF AKT in 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells S473 The phosphorylation levels of both CAR-T cells were significantly higher than those of anti-CD19 28Z CAR-T cells. Thr202 / Tyr204 The phosphorylation levels of AKT in the PI3K / AKT signaling pathway S473 The phosphorylation levels of ribosomal protein S6, a shared downstream signaling molecule of the two pathways, were found to promote cell proliferation, survival, and anti-apoptotic activity. S235 / 236 The phosphorylation level of anti-CD19 E is indicative of the cell growth and proliferation level. YF It was revealed that 28Z CAR-T cells had the strongest proliferation and survival capabilities.

[0151] Anti-CD19E YF The experimental design to compare the in vivo anti-subcutaneous tumor capabilities of 28Z CAR-T cells, anti-CD19 E28Z cells, and anti-CD19 28Z CAR-T cells was as follows.

[0152] Using aseptic technique, 3 x 10 7 A Raji cell-PBS resuspension was prepared at 100 μl / ml. 3 million Raji cells were then subcutaneously implanted into the left dorsal region of each 6-week-old B-NDG female mouse, which was designated as day 0. Six days later, when the Raji subcutaneous tumor had grown to a diameter of 6–7 mm, the mice were randomly divided into three groups (E YFThe mice were divided into three groups (28Z group, E28Z group, 28Z group, and vector group), and 100 ul 8 million T cells were injected into the tail vein of each mouse. After that, the tumor size was measured periodically with a caliper, and the tumor data and the survival time of the mice were recorded. The tumor area was calculated as length x width.

[0153] Experimental conclusion: anti-CD19 E YF 28Z CAR-T cells had antitumor ability. EXAMPLES

[0154] The sequence of anti-CD19 28Z CAR contains the structure of FMC63 scFv, CD28 hinge region, CD28 transmembrane domain, and CD3ζ intracellular domain (i.e., the sequence shared by Rosenberg, SA laboratory at NCBI (GenBank: HM852952.1). Anti-CD19 28Z CAR was obtained by gene synthesis, and based on this, the sequence of the CAR structure shown in Figure 2a was designed. Then, eGFP and CAR sequences were linked and encoded in the same open reading frame (ORF) via the T2A self-cleaving peptide. Therefore, the expression level of CAR could be represented by the expression amount / fluorescence intensity of eGFP. In addition, by inserting the BRS motif of the CD3ε intracellular segment after the CD28 transmembrane domain based on the overlap extension PCR technique, anti-CD19 E BRS They created a new type of CAR called the 28Z CAR.

[0155] Synthetic anti-CD19 E BRS The nucleotide sequence of 28Z CAR is as shown in SEQ ID NO: 13. ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACG

[0156] CAR was subcloned into the lentivirus expression plasmid pHAGE vector, and the CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2G in a ratio of 10:7.5:3.5, respectively, and then transfected into 293FT cells using calcium phosphate transfection to generate lentivirus particles. Next, anti-CD19 28Z CAR or anti-CD19 E were transfected into 293FT cells using ultra-high speed centrifugation. BRS The lentiviruses expressing 28Z CAR were concentrated to produce high titers of virus in small volumes (~10 8 IU / ml, and then transfected into primary T cells (MOI=10) activated with αCD3 / αCD28 Ab-beads for 1 day. Complete T cell medium (XIVO-15 + 1% PS + 10ng / ml human IL-7 + 10ng / ml human IL-15 + 5% Human AB serum, 10mM neutralizing The primary T cells were cultured using CD19 28Z NAC (anti-CD19 28Z CAR-T cells) for 1 day, then stimulated with antibodies for 4 days, after which the virus was removed. Then, on the 5th day after stimulation with antibodies, CAR-T cells with the same CAR expression levels (represented by eGFP fluorescence intensity) were sorted and the obtained T cells were incubated with anti-CD19 28Z CAR-T cells and anti-CD19 E BRS The 28Z CAR-T cells were then grown for a certain period of time, and a series of indicators were assessed for CAR-T, including CAR membrane level, CD4 and CD8 subset ratio, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro sustained viability, and in vivo antitumor ability.

[0157] Regarding the indicator detection method and results: The obtained anti-CD19 28Z CAR and anti-CD19 E BRSThe structure of 28Z CAR is shown in Figure 2a. FMC63 in the figure is a single-chain antibody targeting the CD19 antigen, and the hinge region and transmembrane region of the receptor are derived from human CD28. The motif of the human CD3ε intracellular basic rich region was inserted behind the CD28 transmembrane region and before the CD28 intracellular region.

[0158] Anti-CD19 28Z CAR-T and anti-CD19 E were detected using Alexa Fluor 647-conjugated anti-mouse FMC63 scFv antibody. BRS The CAR level on the membrane surface of 28Z CAR-T cells was measured by flow cytometry. The flow cytometry graph results are shown in Figure 2b. This shows that there was no difference in the CAR positivity rate and expression level (MFI) between the two.

[0159] Anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies were used, and anti-CD19 28Z CAR-T and anti-CD19 E BRS Flow cytometry was performed on 28Z CAR-T cells. The flow cytometry graph results are shown in Figure 2c. + CAR-T cells and CD8 + The percentage of CAR-T cells was shown to be similar.

[0160] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were each treated with CD19 +The cells were transplanted into a round-bottom 96-well microplate at a 1:1 ratio with lymphoma cell line Raji, mixed uniformly, and centrifuged at room temperature (400g, 1min) to promote cell-cell contact. Then, the complete medium was used to culture the cells in a 37°C incubator for 1 day before collection. In addition, 1×BFA was added 6 hours before sample collection to inhibit cytokine release, and the samples were washed once with PBS after sample collection. Then, the cells were fixed with 4% PFA at room temperature for 5 min and perforated with 0.1% Triton X-100 at room temperature for 5 min. Then, intracellular staining procedures were performed according to the usual operation to detect IL-2, IFN-γ, and TNF-α with the corresponding directly labeled antibodies. As a result, as shown in Figure 2d, Figure 2e, and Figure 2f, anti-CD19 E BRS 28Z CAR-T cells were treated with CD19 + After specific stimulation with the cells, cytokine levels of IL-2 and TNF-α produced were similar to those of anti-CD19 28Z CAR-T cells, whereas cytokine levels of IFN-γ were reduced compared to anti-CD19 28Z CAR-T cells.

[0161] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were each treated with CD19 + The cells were transferred to a round-bottom 96-well microplate at a 1:1 ratio with the lymphoma cell line Raji, mixed uniformly, and then centrifuged at room temperature (400g, 1 min) to promote cell-cell contact. Then, the cells were cultured in a complete medium in an incubator at 37°C for a certain period of time. The samples were then circulated at different times. After collection, the samples were washed once with PBS and stained with anti-human Ki67-APC antibody according to the instructions in the Foxp3 / Transcription Factor Staining Buffer Set reagent kit, and the expression level of Ki67 was measured by flow cytometry. As a result, as shown in Figure 2g, anti-CD19 E BRS 28Z CAR-T cells are CD19 +After stimulation with Raji cells, they proliferated faster than anti-CD19 28Z CAR-T cells for a sustained period of time.

[0162] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were each treated with CD19 + The cells were transplanted into a round-bottom 96-well microplate at a 1:1 ratio with the lymphoma cell line Raji, mixed uniformly, and then centrifuged at room temperature (400g, 1 min) to promote cell-cell contact. Then, the cells were cultured for a certain period of time in a 37°C incubator using complete medium. Samples were then collected at different times, washed once with PBS, stained according to the Annexin V Apoptosis Detection Kit (APC) reagent kit instructions, and the expression level of Annexin V in CAR-T cells was measured by flow cytometry. As a result, as shown in Figure 2h, anti-CD19 E BRS 28Z CAR-T cells are CD19 + After stimulation with Raji cells, the late apoptosis level was significantly lower than that of anti-CD19 28Z CAR-T cells.

[0163] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were each treated with CD19 + The cells were transplanted into a round-bottom 96-well microplate at a 1:1 ratio with the lymphoma cell line Raji, mixed uniformly, and then centrifuged at room temperature (400g, 1 min) to promote cell-cell contact. Next, the cells were cultured for a certain period of time in a 37°C incubator using complete medium. Samples were then collected at different times, washed once with PBS, and stained with anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies to detect CAR. + The number of surviving T cells was detected. As a result, as shown in Figure 2i, anti-CD19 E BRS 28Z CAR-T cells are CD19 +The survival rate of Raji cells after stimulation with anti-CD19 was This was clearly higher than that of 28Z CAR-T cells.

[0164] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were each treated with CD19 + The cells were transplanted into 1.5 ml EP tubes at a 1:1 ratio with lymphoma cell line Raji, mixed homogeneously, and centrifuged (500 g, 2 min) at 4°C to promote cell-cell contact. Next, the cells were stimulated at a series of timings using complete medium in a water bath at 37°C. Next, the cells were fixed at different timings by adding an equal volume of 8% PFA at room temperature for 5 min, and then perforated with 0.1% Triton X-100 at room temperature for 5 min. Then, intracellular staining procedures were performed according to the standard procedure. First, rabbit-derived pAKT S473 (Figure 2j) and rabbit-derived primary antibody pS6 S235 / 236 (Fig. 2k) was stained with a primary antibody, washed once, and then stained with an Alexa647-conjugated goat anti-rabbit IgG polyclonal antibody as a secondary antibody. After washing once, flow cytometry was performed. As a result, as shown in Fig. 2j and Fig. 2k, anti-CD19 E BRS 28Z CAR-T cells express pAKT receptors after specific stimulation with antigen. S473 and pS6 S235 / 236 The overall levels of the signaling molecule AKT were significantly higher in the anti-CD19 28Z CAR-T cells than in the anti-CD19 28Z CAR-T cells. S473 The phosphorylation level of the signaling molecule ribosomal protein S6 indicates the promotion of cell proliferation and viability. S235 / 236 The phosphorylation level of anti-CD19 E is indicative of the cell growth and proliferation level. These two indicators are important in terms of signaling pathways. BRS The proliferation and survival capacity of 28Z CAR-T cells was relatively good.

[0165] anti-CD19 28Z CAR-T cells and E BRS 28Z CAR-T cells are CD19 +It has a toxic reaction to tumor cells. - K562 cells were resuspended in 1640 serum-free medium and pre-stained with 1x CellTraker deep red dye (37°C water bath, 30 min). Then, the cells were washed once with 1640 serum-free medium, resuspended in complete T cell medium, and stained for CD19. + The target cells were mixed with K562_CD19 (IRES mCherry) cells at a ratio of 1:1. Anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were mixed with the mixed target cells at a ratio of 3:1, 1:1, or 1:3, respectively, and plated on a round-bottom 96-well microplate to mix evenly, and then centrifuged at room temperature (400g, 1min) to promote cell-cell contact. Then, the samples were cultured in a 37°C incubator with complete medium for 1 day, and then collected and placed on ice. CD19 - K562 cells (APC + , mCherry - ) and CD19 + K562_CD19 cells (APC - , mCherry + The percentage change in CD19 in the experimental group was measured by flow cytometry. - K562 cells (APC + , mCherry - ) / CD19 + K562_CD19 cells (APC - , mCherry + The percentage of patients with anti-CD19 28Z CAR-T cells or anti-CD19 E BRS 28Z Mixed target cell sample wells without CAR-T cells: N0=CD19 - K562 cells (APC + , mCherry - ) / CD19 + K562_CD19 cells (APC - , mCherry +The survival rate N / N0 was calculated based on the percentage of CD19. In other words, the formula for the killing rate was "Lysis (%) = 1-N / N0". As a result, as shown in Figure 2l, + Anti-CD19 E against tumor cells BRS The toxicity of 28Z CAR-T cells was shown to be comparable to that of anti-CD19 28Z CAR-T cells.

[0166] How to process experimental data: Statistical analysis of the data was performed using the software GraphPad Prism 8.0.2, and P < 0.05 (specifically, * P < 0.05, ** P < 0.01, *** P<0.001, **** Differences were considered to be statistically significant when P<0.0001. Data for each group were presented as Mean ± SD.

[0167] In Figures 2d to 2f, one-way ANOVA was supplemented with Sidak's multiple comparison test.

[0168] In Figures 2g, 2h, and 2j to 2l, Sidak's multiple comparison test was added to the two-way ANOVA.

[0169] In Figure 2i, a two-way ANOVA was performed.

[0170] Test Results: (1) anti-CD19 E BRS Membrane levels and CD4 / CD8 ratios of 28Z CAR-T cells and anti-CD19 28Z CAR-T cells were comparable.

[0171] (2) anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were specifically stimulated in vitro with Raji cells, and anti-CD19 E BRSThe cytokine levels of IL-2 and TNF-α produced by 28Z CAR-T cells were similar to those of anti-CD19 28Z CAR-T cells, but the cytokine levels of IFN-γ were lower than those of anti-CD19 28Z CAR-T cells.

[0172] (3) anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were specifically stimulated in vitro with Raji cells and expanded for a certain period of time. Samples were then taken at different times to assess proliferation ability (i.e., Ki67 expression level). The anti-CD19 E BRS The proliferation ability of 28Z CAR-T was obviously higher than that of anti-CD19 28Z CAR-T cells.

[0173] (4) anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were specifically stimulated in vitro with Raji cells and expanded for a certain period of time, and then samples were taken at different times to measure the apoptosis levels. BRS When 28Z CAR-T cells and anti-CD19 28Z CAR-T cells were compared, the two were comparable in the early stage, but in the later stage, anti-CD19 E BRS The apoptosis level of 28Z CAR-T cells was obviously lower than that of anti-CD19 28Z CAR-T cells.

[0174] (5) anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were specifically stimulated with Raji cells and amplified for a certain period of time. Samples were then taken at different times to generate amplified CAR + The number of T cells was measured and the anti-CD19 E BRS The number of 28Z CAR-T cells was significantly higher than that of anti-CD19 28Z CAR-T cells.

[0175] (6) anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were specifically stimulated with Raji cells and expanded for a certain period of time. Samples were then taken at different times to measure the expression of the signal molecule AKT. S473 The phosphorylation level of the signaling molecule AKT was measured. S473 The phosphorylation level of anti-CD19 E indicates the promotion of cell proliferation and viability. BRS Global pAKT in 28Z CAR-T cells S473 The levels were higher in anti-CD19 28Z CAR-T cells and anti-CD19 E BRS The proliferation and survival capacity of 28Z CAR-T cells was shown to be significantly higher than that of anti-CD19 28Z CAR-T cells.

[0176] (7) anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells were specifically stimulated with Raji cells and expanded for a certain period of time. Samples were then taken at different times and analyzed using the signal molecule S6. S235 / 236 The phosphorylation level of the signal molecule S6 was measured. S235 / 236 The phosphorylation level of anti-CD19 E indicates the growth and proliferation ability of cells. BRS Overall pS6 of 28Z CAR-T cells S235 / 236 The levels were significantly higher than those of anti-CD19 28Z CAR-T cells, and anti-CD19 E BRS The growth and proliferation ability of 28Z CAR-T cells was shown to be significantly higher than that of anti-CD19 28Z CAR-T cells.

[0177] (8) anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells and CD19 + K562:CD19 - In an in vitro killing experiment in which K562 cells were co-cultured with anti-CD19 E BRSThe killing ability of 28Z CAR-T cells was comparable to that of anti-CD19 28Z CAR-T cells.

[0178] Anti-CD19E BRS The experimental design to compare the in vivo anti-subcutaneous tumor ability of 28Z CAR-T cells and anti-CD19 28Z CAR-T cells was as follows.

[0179] Using aseptic technique, 3 x 10 7 A Raji cell-PBS resuspension was prepared at 100 μl / ml. 3 million Raji cells were then subcutaneously implanted into the left dorsal region of each 6-week-old B-NDG female mouse, which was designated as day 0. Six days later, when the Raji subcutaneous tumor had grown to a diameter of 6–7 mm, the mice were randomly divided into three groups (E BRS The mice were divided into three groups (28Z group, 28Z group, and vector group) and 100 ul 8 million T cells were injected into the tail vein of each mouse. After that, the tumor size was measured periodically with a caliper, and the tumor data and the survival time of the mice were recorded. The tumor area was calculated as length x width.

[0180] Experimental conclusion: anti-CD19 E BRS 28Z CAR-T had antitumor ability.

[0181] The above is merely a preferred embodiment of the present invention, and does not limit the present invention in any form or substance. It should be noted that those skilled in the art can make some improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be considered as the scope of protection of the present invention. Any equivalent modifications of minor modifications, alterations and variations that a technician familiar with this field can make using the technical contents disclosed above without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. In addition, any equivalent modifications, alterations and variations made to the above embodiments based on the substantial technology of the present invention are all within the scope of the technical solution of the present invention.

Claims

1. A chimeric antigen receptor comprising a CD3ε intracellular basic amino acid rich region motif, comprising an extracellular domain, a transmembrane domain and an intracellular domain, which are linked in order; the extracellular domain comprises an antigen recognition region and a hinge region; A chimeric antigen receptor having a CD3ε intracellular basic amino acid rich region motif linked to one end of the intracellular domain linked to the transmembrane domain.

2. Furthermore, (1) the amino acid sequence of the motif of the CD3ε intracellular basic amino acid-rich region is as shown in SEQ ID NO: 10; (2) the intracellular domain comprises, in order, a CD3ε intracellular basic amino acid-rich region motif, a costimulatory signaling region, and a CD3ζ intracellular segment; 2. The chimeric antigen receptor comprising the CD3ε intracellular basic amino acid rich region motif of claim 1, characterized in that it comprises one or more of the following features:

3. The chimeric antigen receptor comprising a CD3ε intracellular basic amino acid rich region motif according to claim 2, characterized in that the costimulatory signaling region is selected from one or more intracellular segments of CD27, CD28, CD134, 4-1BB, OX-40 and ICOS.

4. Furthermore, (1) the amino acid sequence of the intracellular segment of CD28 is as set forth in SEQ ID NO: 2; (2) The amino acid sequence of the CD3ζ intracellular segment is as set forth in SEQ ID NO:3; 4. The chimeric antigen receptor comprising the motif of the CD3ε intracellular basic amino acid rich region of claim 3, characterized in that it comprises one or more of the following features:

5. Furthermore, a. the antigen recognition region is selected from a single chain antibody targeting a tumor surface antigen, the tumor surface antigen being selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, fibroblast activation protein, Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, and GD2; b. the transmembrane domain is selected from one or more transmembrane regions of CD28, CD4, CD8α, OX40, or H2-Kb; c. The hinge region is selected from one or more of the following: a CD28 hinge region, a CD8α hinge region, a CD4 hinge region, an immunoglobulin IgG hinge region, or an immunoglobulin IgG hinge region and a hinge region of the CH2CH3 region; 2. The chimeric antigen receptor comprising the CD3ε intracellular basic amino acid rich region motif of claim 1, characterized in that it comprises one or more of the following features:

6. Furthermore, d. the single chain antibody is selected from FMC63; e. the amino acid sequence of the transmembrane domain of CD28 is as set forth in SEQ ID NO:4; f. The amino acid structure of the CD28 hinge region is as shown in SEQ ID NO:

7. 、 6. The chimeric antigen receptor comprising the CD3ε intracellular basic amino acid rich region motif of claim 5, characterized in that it comprises one or more of the following features:

7. Furthermore, 1) the antigen recognition region of the chimeric antigen receptor comprising the CD3ε intracellular basic amino acid-rich region motif is selected from FMC63, the hinge region is selected from the CD28 hinge region, the transmembrane domain is selected from the CD28 transmembrane domain, and the intracellular domain comprises the CD3ε intracellular basic amino acid-rich region motif, the CD28 intracellular segment, and the CD3ζ intracellular segment, which are linked in order; 2) The amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 11; 7. A chimeric antigen receptor comprising a motif of the CD3ε intracellular basic amino acid rich region according to any one of claims 1 to 6, characterized in that it comprises one or more of the following features:

8. (1) A polynucleotide sequence encoding a chimeric antigen receptor comprising the motif of the CD3ε intracellular basic amino acid rich region according to any one of claims 1 to 6, and (2) a complementary sequence of the polynucleotide sequence described in (1); A polynucleotide sequence selected from:

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

12.

10. A nucleic acid construct comprising the polynucleotide sequence of claim 8.

11. The nucleic acid construct of claim 10, wherein the nucleic acid construct is a vector.

12. The nucleic acid construct of claim 10, wherein the nucleic acid construct is a lentiviral vector and comprises an origin of replication, a 3'LTR, a 5'LTR, and the polynucleotide sequence of claim 8.

13. A lentiviral vector system comprising the nucleic acid construct of claim 10 and an auxiliary component of a lentiviral vector.

14. A genetically modified T cell comprising the polynucleotide sequence of claim 8.

15. A genetically modified T cell comprising the nucleic acid construct of claim 10.

16. A genetically modified T cell, characterized in that it is infected with the lentiviral vector system described in claim 13.

17. Use of a chimeric antigen receptor comprising a motif of the CD3ε intracellular basic amino acid rich region according to any one of claims 1 to 6 in the manufacture of a product for any one or more of the following uses: (1) generating T cells; (2) improving the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation ability of T cells; (5) improving the anti-tumor ability of T cells; and (6) suppressing the level of the cytokine IFN-γ from T cells.

18. The polynucleotide sequence of claim 8 is effective in (1) generating T cells, (2) improving the survival ability of T cells, (3) inhibiting T cell apoptosis, (4) enhancing the proliferation ability of T cells, (5) improving the anti-tumor ability of T cells, and (6) suppressing the level of cytokine IFN-γ from T cells. Use in the manufacture of products for any one or more of the above purposes.

19. Use of the nucleic acid construct according to claim 10 in the manufacture of a product having any one or more of the following uses: (1) generating T cells; (2) improving the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation ability of T cells; (5) improving the anti-tumor ability of T cells; and (6) suppressing the level of the cytokine IFN-γ from T cells.

20. Use of the lentiviral vector system described in claim 13 in the manufacture of a product having any one or more of the following uses: (1) generating T cells; (2) improving the survival ability of T cells; (3) inhibiting T cell apoptosis; (4) enhancing the proliferation ability of T cells; (5) improving the anti-tumor ability of T cells; and (6) suppressing the level of the cytokine IFN-γ in T cells.

21. Use of a chimeric antigen receptor comprising a CD3ε intracellular basic amino acid rich region motif according to any one of claims 1 to 6 in the manufacture of a tumor treatment product.

22. 10. Use of the polynucleotide sequence of claim 8 in the manufacture of a tumor treatment product.

23. 11. Use of the nucleic acid construct of claim 10 in the manufacture of a tumor treatment product.

24. 14. Use of the lentiviral vector system of claim 13 in the manufacture of a tumor treatment product.

25. 15. Use of the genetically modified T cells of claim 14 in the manufacture of a tumor treatment product.

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