Chimeric antigen receptors and their uses

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, and more specifically to chimeric antigen receptors and their uses that include a CD3ε intracellular region having a Y / F mutation or a CD3ε intracellular basic amino acid-rich region motif. [Background technology]

[0002] Chimeric antigen receptors, abbreviated as CARs, are T cells equipped with CARs that target specific tumor antigens, and these are called CAR-T cells. CAR-T therapy is widely applied in the treatment of tumors (especially in the immunotherapy of solid tumors). This therapy represents a new type of precise targeted therapy in tumor treatment, and in recent years, through optimization and improvement, it has shown promising results in clinical tumor treatment. In short, CAR-T therapy is a highly promising, accurate, rapid, efficient, and potentially curative new type of tumor immunotherapy.

[0003] However, CAR-T therapy still has many limitations. For example, it has issues such as cytokine storms encountered in the clinical treatment of tumors, poor neurotoxicity, and inferior sustained proliferative capacity. 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, when patients (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukemia or non-Hodgkin lymphoma were treated with KTE-C19 (anti-CD19 28Z) CAR-T therapy, the number of CAR-T cells in their blood was detected. The number of CAR-T cells peaked on day 14, but then showed a significant decrease on days 28 and 42, and by day 68, CAR-T cells could no longer be detected in the blood of any of the patients. 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 when 53 patients with relapsed acute lymphoblastic leukemia received anti-CD19 28Z CAR-T therapy, the median number of CAR-T cells in their blood was detected on day 14. Both of these studies indicate that 28Z CAR-T cells have relatively poor sustained proliferation and survival capabilities, and that improvements in treatment efficacy are desirable. [Overview of the project] [Problems that the invention aims to solve]

[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 enhance antitumor effects, extend the remission period of the disease, reduce the recurrence rate, and extend the patient's survival period. In view of the shortcomings of the prior art described above, the object of the present invention is to provide a chimeric antigen receptor and its applications that include a CD3ε intracellular region having a Y / F mutation or a motif of a CD3ε intracellular basic amino acid-rich region. [Means for solving the problem]

[0005] To achieve the above-mentioned objectives and other related objectives, the present invention, in a first aspect, The present invention provides a chimeric antigen receptor comprising a CD3ε intracellular domain having a Y / F mutation. The chimeric antigen receptor comprises, in order, an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises an antigen recognition region and a hinge region.

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

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

[0008] In a third embodiment, the present invention provides a nucleic acid construct comprising a polynucleotide sequence described in a second embodiment of the present invention.

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

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

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

[0012] In a fifth aspect of the present invention, the present invention provides recombinant T cells or a drug composition containing said recombinant T cells. Characterized by the fact that the cells contain the polynucleotide sequence described in the second aspect of the present invention, contain the nucleic acid construct described in the third aspect of the present invention, or are infected with the lentiviral vector system described in the fourth aspect of the present invention.

[0013] In its sixth aspect, the present invention provides applications for the production of products for any one or more of the following uses of a chimeric antigen receptor comprising a CD3ε intracellular region having the Y / F mutation described in the first aspect of the present invention, a polynucleotide sequence described in the second aspect of the present invention, a nucleic acid construct described in the third aspect of the present invention, or a lentiviral vector system described in the fourth aspect of the present invention: (1) production of T cells, (2) improvement of T cell viability, (3) inhibition of T cell apoptosis, (4) enhancement of T cell proliferation and / or viability, (5) improvement of T cell antitumor ability, and (6) suppression of cytokine IFN-γ levels and inhibition of TNF-α secretion from T cells.

[0014] In its seventh aspect, the present invention provides applications in the manufacture of tumor therapeutic products for a chimeric antigen receptor comprising a CD3ε intracellular region having the Y / F mutation described in the first aspect of the present invention, a polynucleotide sequence described in the second aspect of the present invention, a nucleic acid construct described in the third aspect of the present invention, a lentiviral vector system described in the fourth aspect of the present invention, or recombinant T cells described in the fifth aspect of the present invention.

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

[0016] T cells modified with the chimeric antigen receptor in this invention significantly improve the persistence of T cells and dramatically enhance their antitumor ability by improving their viability, reducing apoptosis levels, and improving their proliferative capacity. This advantage makes it possible to further improve tumor treatment efficacy, extend disease remission, reduce recurrence rates, and extend patient survival in clinical applications. Furthermore, these modified T cells can downregulate the expression levels of cytokines IFN-γ and TNF-α, thereby reducing the production of inflammatory cytokines such as IL-1β and IL-6 by activating macrophages and monocytes.

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

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

[0019] One end of the intracellular domain that is linked to the transmembrane domain is ligated to a motif of the CD3ε intracellular basic amino acid-rich region.

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

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

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

[0023] More preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3'LTR, a 5'LTR, and a polynucleotide sequence as described in the ninth aspect of the present invention.

[0024] In an eleventh aspect of the present invention, a lentiviral vector system is provided, comprising a nucleic acid construct and an auxiliary component of a lentiviral vector as described in a tenth aspect of the present invention.

[0025] In a twelfth aspect of the present invention, the present invention provides recombinant T cells or a drug composition containing said recombinant T cells. Characterized by the fact that the cells contain a polynucleotide sequence as described in the ninth aspect of the present invention, contain a nucleic acid construct as described in the tenth aspect of the present invention, or are infected with a lentiviral vector system as described in the eleventh aspect of the present invention.

[0026] In its thirteenth aspect, the present invention provides applications for the production of products for any one or more of the following uses of a chimeric antigen receptor containing the CD3ε intracellular basic amino acid-rich region motif described in aspect eight of the present invention, a polynucleotide sequence described in aspect nin of the present invention, a nucleic acid construct described in aspect ten of the present invention, or a lentiviral vector system described in aspect eleven of the present invention: (1) T cell production, (2) improvement of T cell viability, (3) inhibition of T cell apoptosis, (4) enhancement of T cell proliferative capacity, (5) improvement of T cell antitumor capacity, and (6) suppression of cytokine IFN-γ levels from T cells.

[0027] In its fourteenth aspect, the present invention relates to the intracellular basicity of CD3ε described in the eighth aspect of the present invention. The present invention provides applications for chimeric antigen receptors containing amino acid-rich region motifs, polynucleotide sequences described in the 9th aspect of the present invention, nucleic acid constructs described in the 10th aspect of the present invention, lentiviral vector systems described in the 11th aspect of the present invention, or recombinant T cells described in the 12th aspect of the present invention in the manufacture of tumor therapeutic products.

[0028] As described above, the chimeric antigen receptor containing the CD3ε intracellular basic amino acid-rich region motif and its applications in the present invention have the following beneficial effects.

[0029] T cells modified with the chimeric antigen receptor containing the CD3ε intracellular basic amino acid-rich region motif in this invention can significantly improve the persistence of T cells and dramatically enhance their antitumor ability by improving viability, reducing apoptosis levels, and improving proliferative capacity. This advantage makes it possible to further improve tumor treatment efficacy, extend disease remission, reduce recurrence rates, and extend patient survival in clinical applications. Furthermore, these modified T cells can downregulate the expression level of the cytokine IFNγ, thereby reducing the production of inflammatory cytokines such as IL-1β and IL-6 by activating macrophages and monocytes. [Brief explanation of the drawing]

[0030] [Figure 1a] Figure 1a shows schematic structural diagrams of anti-CD19 28Z CAR, anti-CD19 E28Z CAR, and anti-CD19 EYF28Z CAR, as well as the amino acid sequences of the CD3ε intracellular region and the CD3ε intracellular region with the Y / F mutation. FMC63 in the figure is a single-chain antibody targeting the CD19 antigen, and the receptor's hinge region and transmembrane region are derived from human CD28. In anti-CD19 E28Z CAR, CD3ε is inserted posterior to the CD28 transmembrane region and anterior to the CD28 intracellular region. In anti-CD19 EYF28Z CAR, the amino acid sequence of the CD3ε intracellular region with the Y / F mutation is inserted posterior to the CD28 transmembrane region and anterior to the CD28 intracellular region. [Figure 1b] Figure 1b shows the flow cytometry graphs at the membrane level after anti-CD19 28Z CAR, anti-CD19 E28Z CAR, and anti-CD19 EYF28Z CAR are expressed in T cells. [Figure 1c] Figure 1c is a flow cytometry graph of the proportion of CD4+ and CD8+ cells 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 shows 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 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 1h] Figure 1h shows the apoptotic 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 number of surviving 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 response of anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 EYF28Z CAR-T cells to CD19+ tumor cells. [Figure 1k] Figure 1k shows the phosphorylation levels (pErk(1 / 2)Thr202 / Tyr204) of the serine / threonine protein kinase Erk(1 / 2)Thr202 / Tyr204, a signaling molecule that 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 serine / threonine kinase AKTS473, a signaling molecule that 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 ribosomal protein S6S235 / 236, a signaling molecule that 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 schematic structural diagrams of the anti-CD19 28Z CAR and anti-CD19 EBRS28Z CAR. FMC63 in the figure is a single-chain antibody targeting the CD19 antigen, and its receptor hinge region and transmembrane region are derived from human CD28. The motif of the CD3ε intracellular basicity-rich region is inserted posterior to the CD28 transmembrane region and anterior to the CD28 intracellular region. [Figure 2b] Figure 2b is a flow cytometry graph at the membrane level after anti-CD19 28Z CAR and anti-CD19 EBRS28Z CAR are expressed in T cells. [Figure 2c] Figure 2c is a flow cytometry graph of the proportion of CD4+ and CD8+ cells in anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells. [Figure 2d] Figure 2d shows 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 shows 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 status 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 apoptotic 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 number of surviving 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 levels of serine / threonine kinase AKTS473, a signaling molecule that 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 levels of the ribosomal protein S6S235 / 236, a signaling molecule that marks cell growth and proliferation, after stimulation of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells with Raji cells (CD19 antigen). [Figure 2l] Figure 2l shows the toxic response of anti-CD19 28Z CAR-T cells and anti-CD19 EBRS28Z CAR-T cells to CD19+ tumor cells. [Modes for carrying out the invention]

[0031] The chimeric antigen receptor containing the CD3ε intracellular domain having the Y / F mutation described in the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain that are sequentially linked.

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

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

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

[0035] The intracellular domain includes, in order, a CD3ε intracellular region having a Y / F mutation (in which two tyrosines in the ITAM motif of the intracellular region are mutated to phenylalanine), an intracellular region of the co-stimulatory signaling domain, and a CD3ζ intracellular segment.

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

[0037] In a more preferred embodiment, the co-stimulatory signaling region is selected from the CD28 intracellular segment. In this case, the lethality is enhanced.

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

[0039] In one embodiment, the amino acid sequence of the CD3ζ intracellular segment is SEQ ID N As shown in O:3, specifically, the following applies: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0040] In one embodiment, the antigen recognition region is selected from single-chain antibodies that target tumor surface antigens. The tumor surface antigen is selected from one or more of the following: 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, CD19 or mesothelin, which have good target specificity, are selected.

[0042] The aforementioned transmembrane domain is selected from one or more transmembrane regions among 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 a hinge region formed by linking an immunoglobulin IgG hinge region with a CH2CH3 region. That is, it can be an IgG1 hinge or an IgG1 hinge-CH2CH3.

[0045] Selectively, the sequence of the hinge region is the CD28 hinge region. The amino acid structure is as shown in SEQ ID NO:7. Specifically, it is IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0046] In one embodiment, the single-chain antibody includes 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 array.

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

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

[0050] In one embodiment, the single-chain antibody includes a light chain variable region and a heavy chain variable region of a monoclonal antibody FMC63, wherein the light chain variable region and the heavy chain variable region are selectively linked by a linker sequence.

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

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

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

[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 This is the nucleotide sequence of a 28Z CAR. SEQ ID NO:6 is as follows (5'→3'). gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagt .

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

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

[0057] One end of the intracellular domain that is linked to the transmembrane domain is linked to a motif of CD3ε intracellular basic amino acid-rich region (Basic residue-rich sequence), abbreviated as the BRS motif.

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

[0059] The intracellular domain comprises, in order, a motif of a CD3ε intracellular basic amino acid-rich region, a co-stimulatory signaling region, and a CD3ζ intracellular segment.

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

[0061] In a more preferred embodiment, the co-stimulatory signaling region is selected from an intracellular segment of CD28. In this case, the lethality is enhanced.

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

[0063] In one embodiment, the amino acid sequence of the CD3ζ intracellular segment is as shown in SEQ ID NO:3. Specifically, it is as follows: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0064] In one embodiment, the antigen recognition region is selected from single-chain antibodies that target tumor surface antigens. The tumor surface antigen is selected from one or more of the following: 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] Furthermore, the transmembrane domain is selected from one or more transmembrane regions among 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 a hinge region formed by linking an immunoglobulin IgG hinge region with a CH2CH3 region. That is, it can be an IgG1 hinge or an IgG1 hinge-CH2CH3.

[0069] Selectively, the sequence of the hinge region is the CD28 hinge region. The amino acid structure is as shown in SEQ ID NO:7. Specifically, it is IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0070] In one embodiment, the single-chain antibody includes 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 array.

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

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

[0074] In one embodiment, the single-chain antibody includes a light chain variable region and a heavy chain variable region of a monoclonal antibody FMC63, wherein the light chain variable region and the heavy chain variable region are selectively linked by a linker sequence.

[0075] The antigen recognition region of the chimeric antigen receptor, which includes the motif of the intracellular basic amino acid-rich region of CD3ε, is selected from FMC63, and the hinge region is selected from the CD28 hinge region. The transmembrane domain is also selected from the CD28 transmembrane region. The intracellular domain includes, in order, the motif of the intracellular basic amino acid-rich region of CD3ε, the intracellular segment of CD28, and the intracellular segment of CD3ζ.

[0076] In one embodiment, a chimeric antigen receptor (Anti-CD19 E) containing a motif of the intracellular basic amino acid-rich region of CD3ε is used. BRS 28Z CAR) consists of FMC63 scFv, CD28 hinge region, CD28 transmembrane region, CD3εBRS motif and CD3ζ fine Includes the intravesicular region.

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

[0078] SEQ ID NO:11 (Anti-CD19 E BRS Amino acid sequence of 28Z CAR): Starting from FMC63 scFv, it is linked in sequence to the CD28 hinge region, the transmembrane region of CD28, the CD3ε BRS motif, and the CD3ζ intracellular region. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKNRKAKAKRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[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 Nucleotide sequence of 28Z CAR). SEQ ID NO:12 is as follows (5’→3’). gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaagaatagaaaggccaag gccaagaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa。

[0080] Each of the above-mentioned parts forming the chimeric antigen receptor of the present invention may be directly linked to one another or linked via a linker sequence. The linker sequence may be a linker sequence applied to antibodies known in the art, for example, a linker sequence containing G and S. Typically, the linker contains one or more repeating motifs. For example, the motifs may 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 consist of one, two, three, four, or five repeating 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, 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 also 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] One point to understand is that gene cloning often requires the design of appropriate enzymatic cleavage sites. In such cases, one or more unrelated residues are inevitably introduced at the end of the expressed amino acid sequence, but this does not affect the activity of the target sequence. Furthermore, for the creation of fusion proteins, promotion of recombinant protein expression, acquisition of recombinant proteins automatically secreted outside the host cell, or advantageous purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other appropriate region within the recombinant protein (including, but not limited to, appropriate linker peptides, signal peptides, leader peptides, terminal elongation, etc.). Regarding signal peptides, let's consider the following: The first thing synthesized during the translation of mRNA encoding a secreted protein is a signal peptide with a hydrophobic amino acid residue at the N-terminus. The signal peptide is recognized and bound to a receptor on the endoplasmic reticulum membrane. Once the signal peptide reaches the lumen of the endoplasmic reticulum via a tunnel formed by membrane proteins, it is hydrolyzed by signal peptidases located on the lumen surface. Induction of signal peptides allows nascent polypeptides to pass through the endoplasmic reticulum membrane and enter the lumen, ultimately leading to extracellular secretion. CAR signal peptides have diverse sources, primarily CD8 and CD28, and GM-CSF receptor signal peptides. The N-terminus or C-terminus of the fusion protein of the present invention (i.e., the CAR described above) may further contain one or more polypeptide fragments as a protein tag. Any suitable tag may be used in this text. For example, the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

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

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

[0084] Typically, the polynucleotide sequences described in this text can be obtained by PCR amplification. Specifically, primers are designed based on the nucleotide sequences disclosed in this text (especially 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 perform two or more PCR amplifications and then concatenate the fragments amplified in the correct order.

[0085] The nucleic acid construct provided in the present invention contains the above-mentioned polynucleotide sequence.

[0086] The nucleic acid construct further includes one or more regulatory sequences that are operationally linked to the polynucleotide sequence. The coding sequence of the CAR described in this invention can be manipulated in various ways to ensure the expression of the protein. The nucleic acid construct may be manipulated before insertion into the vector, depending on the differences and requirements of the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are known in the art.

[0087] The regulatory sequence can be an appropriate promoter sequence. Typically, the promoter sequence is operationally ligated to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, and may include mutated, cleaved, and hybrid promoters. Furthermore, it can be obtained from a gene encoding an extracellular or intracellular polypeptide of the same or different species as the host cell.

[0088] The regulatory sequence may be an appropriate transcriptional terminator sequence, i.e., a sequence that terminates transcription upon recognition by the host cell. The terminator sequence is operationally ligated to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that functions in a selected host cell can be used in this invention.

[0089] The regulatory sequence may be a suitable leader sequence, which is an untranslated region of mRNA important for translation in the host cell. The leader sequence is manipulably ligated to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that functions in a selected host cell can be used in this invention.

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

[0091] Typically, the expression of a CAR-encoding polynucleotide sequence is achieved by manipulating a promoter and incorporating the construct into an expression vector. The vector should be suitable for eukaryotic cell replication and integration. Typical cloning vectors include transcriptional and translational terminators, start sequences, and promoters that can regulate the expression of a desired nucleic acid sequence.

[0092] The polynucleotide sequence encoding the CAR of the present invention can be cloned into numerous types of vectors. For example, it can be cloned into plasmids, bacteriophages, phage derivatives, animal viruses, and cosmids. Furthermore, the vector is an expression vector. The expression vector can be supplied to cells in viral vector form. Viral vector technology is well known in the art. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector includes an origin of replication that plays a role in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers.

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

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

[0095] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may contain either or both of a selectable marker gene and / or reporter gene. This is convenient for identifying and selecting expressing cells from a population of cells attempted to be transfected or infected with a viral vector. On the other hand, the selectable marker can be retained on a single DNA fragment and used in cotransfection procedures. The flanking of both the selectable marker and reporter gene may both have appropriate regulatory sequences to enable expression in host cells. Effective selectable markers include, for example, antibiotic resistance genes such as neo.

[0096] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After DNA is introduced into receptor cells, the expression of the reporter gene is measured at an appropriate time. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Furthermore, suitable expression systems can be constructed using publicly known and recognized technologies or are commercially available.

[0097] Methods for introducing genes into cells and methods for expressing genes in cells are already established in this field. It is knowledge. Vectors can be easily introduced into host cells (e.g., mammalian, bacterial, yeast, or insect cells) by any method in the field. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0098] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Biological methods for introducing polynucleotides of interest into host cells include methods using DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0099] Biological methods for introducing polynucleotides into host cells include the use of viral vectors (particularly lentiviral vectors). This is 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 1, adenoviruses, and adeno-associated viruses, among others. Numerous virus-based systems for introducing genes into mammalian cells have already been developed, and lentiviruses, for example, provide a convenient platform for use in gene transfer systems. Using techniques known in the field, it is possible to introduce selected genes into vectors and package them into lentiviral particles. The recombinant viruses can then be isolated and introduced into target cells in vivo or in vitro. Numerous retroviral systems are known in this field. In addition, some implementations use adenoviral vectors. Numerous adenoviral vectors are known in this field. In addition, some implementations use lentiviral vectors.

[0100] The present invention provides a lentiviral vector system. The lentiviral vector system includes the nucleic acid construct and auxiliary components of the lentiviral vector described above.

[0101] The auxiliary components of the lentivirus include a lentiviral packaging plasmid and a cell line.

[0102] The lentiviral vector system is formed by virally packaging the nucleic acid construct with the assistance of a lentiviral packaging plasmid and a cell line. The method for constructing the lentiviral vector system is a method commonly used in this field.

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

[0104] Compared to other drug therapies, CAR-T cells can be maintained in the body for extended periods due to their self-renewal and survival capabilities. Furthermore, upon stimulation with a specific target antigen, they rapidly amplify and can kill tumor cells containing the target. Subsequently, they can form specific memory cells, such as effector memory T cells and central memory T cells.

[0105] The present invention further includes cell therapy. In this cell therapy, T cells are genetically modified to express CARs as described herein. The CAR-T cells are then injected into the patient who needs them. The injected cells are capable of killing the patient's tumor cells. Unlike antibody therapy, CAR-T cells are reproducible in the body and can sustainably control tumors. It exhibits sustained effects.

[0106] Antitumor immune responses induced by CAR-T cells can be active or passive. Furthermore, CAR-induced immune responses can be part of adoptive immunotherapy. CAR-T cells induce specific immune responses to the antigen-binding sites of CARs.

[0107] The cancers that can be treated are non-solid tumors such as hematological malignancies (e.g., leukemia and lymphoma). In particular, the diseases that can be treated using the CAR, its coding sequence, nucleic acid construct, expression vector, virus and CAR-T cells of the present invention are preferably CD19-induced diseases, and especially CD19-induced hematological malignancies.

[0108] Specifically, the term "CD19-induced diseases" in this text includes, but is not limited to, leukemias and lymphomas such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0109] The present invention provides recombinant T cells or a drug composition containing such recombinant T cells. The cells contain the polynucleotide sequence, the nucleic acid construct, or are infected with the lentiviral vector system.

[0110] The CAR-modified T cells of the present invention may be administered alone or as part of a drug composition in combination with diluents and / or other components (e.g., relevant cytokines or cell populations). Briefly speaking, the drug compositions of the present invention may include a combination of the CAR-T cells described herein and one or more pharmaceutically 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 drug composition of the present invention is administered in a manner suitable for the disease being treated (or prevented). The quantity and frequency of administration are determined, for example, by factors such as the patient's symptoms, the type of disease, and its severity.

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

[0113] The target composition can be administered by any convenient method, including spraying, injection, oral administration, intravenous infusion, implantation, or transplantation. The compositions described herein can be administered to patients by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous, or intraperitoneal injection. In one implementation, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another implementation, the T cell composition of the present invention is preferably administered by intravenous injection. The T cell composition can also be injected directly into tumors, lymph nodes, or infected sites.

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

[0115] In this text, "antitumor capacity" refers to a biological effect, which can be demonstrated by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an extension of estimated lifespan, or an improvement in various physiological symptoms related to cancer.

[0116] The terms "patient," "subject," and "individual" are interchangeable within this text and refer to any living organism capable of generating an immune response (e.g., mammals). This includes, but is not limited to, humans, dogs, cats, mice, rats, and their genetically modified species.

[0117] The chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, and the lentiviral vector system are applied to the manufacture of products for any one or more of the following uses: (1) production of T cells, (2) improvement of T cell viability, (3) inhibition of T cell apoptosis, (4) enhancement of T cell proliferation and / or viability, (5) improvement of T cell antitumor ability, and (6) suppression of cytokine IFN-γ levels and inhibition of TNF-α secretion from T cells.

[0118] By improving viability, reducing apoptosis levels, and enhancing proliferative capacity, it becomes possible to significantly improve the persistence of T cells and dramatically enhance their antitumor capabilities.

[0119] The chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, and the lentiviral vector system can also be applied to the manufacture of products for one or more of the following uses: enhancing the proliferation and survival ability, growth and proliferation ability, or sustained proliferation ability 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 recombinant T cells are used in the manufacture of tumor treatment products.

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

[0122] Selectively, the tumor is chosen from B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0123] Embodiments of the present invention will be described below through specific examples. Those skilled in the art will readily understand other advantages and effects of the present invention from the disclosures herein. Furthermore, the present invention can be implemented or applied by other different specific embodiments. Also, each detail herein can be modified or altered in various ways, based on different perspectives and applications, without departing from the spirit of the invention.

[0124] Before further describing 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 implementations described later. Furthermore, it should be understood that the present invention The terminology used in the embodiments is for the purpose of describing specific implementations and is not intended to limit the scope of protection of the present invention. Furthermore, in the specification and claims of the present invention, unless otherwise explicitly stated in the text, singular forms such as "one," "one," and "this" are included in the plural form.

[0125] Where numerical ranges are indicated in the examples, unless otherwise described in the present invention, it should be interpreted that any two endpoints of each numerical range and any number between those endpoints can be selected. Furthermore, unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, the present invention may also be implemented using any prior art methods, devices, and materials similar to or equivalent to those 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 described, the experimental methods, detection methods, and preparation methods disclosed in this invention all utilize general 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 anti-CD1928Z CAR sequence contains the FMC63 scFv, CD28 hinge region, CD28 transmembrane region, and CD28 and CD3ζ intracellular regions (i.e., the sequence shared by the Rosenberg, SA laboratory at NCBI (GenBank: HM852952.1)). The anti-CD1928Z CAR was obtained by gene synthesis, and the sequence of the CAR structure shown in Figure 1a was designed based on this. The eGFP and CAR sequences were then encoded by concatenating them into the same open reading frame (ORF) via a T2A self-cleaving peptide. Therefore, the CAR expression level could be expressed as eGFP expression level / fluorescence intensity. Furthermore, gene splicing by overlap extension PCR was used. Using PCR-based site-directed mutagenesis and recombination ligation techniques, a novel CAR was created by inserting a CD3ε intracellular region containing a Y / F mutation (in which the two tyrosines in the ITAM motif of the intracellular region have been mutated to phenylalanine) behind the CD28 transmembrane region, and anti-CD19 E YF It was named the 28Z CAR.

[0128] anti-CD19 E YF The nucleotide sequence of the 28Z CAR is as shown in SEQ ID NO:8. Specifically, it is as follows: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC

[0129] E YF Anti-CD19 E28Z C differs from 28Z only in that the intracellular region of CD3ε containing the Y / F mutation is replaced with a sequence that matches the intracellular region of CD3ε. An AR was created. The nucleotide sequence of the anti-CD19 E28Z CAR is as shown in SEQ ID NO:9, and is specifically as follows. .

[0130] The CAR was subcloned into the lentiviral expression plasmid pHAGE vector. The CAR expression plasmid was then mixed with the packaging plasmids pSPAX2 and pMD2G in a ratio of 10:7.5:3.5, respectively, and introduced into 293FT cells using calcium phosphate transfection to generate lentiviral particles. Next, using ultrafast centrifugation, anti-CD19 28Z CAR, anti-CD19 E28Z CAR, or anti-CD19 E YF Lentiviruses expressing 28Z CAR are concentrated to produce small-volume, high-titer viruses (~10 8After adjusting the concentration to IU / ml, primary T cells (MOI=10) activated with αCD3 / αCD28 Ab-beads for 1 day were transfected accordingly. Primary T cells were cultured in complete T cell medium (XIVO-15 + 1% PS + 10 ng / ml human IL-7 + 10 ng / ml human IL-15 + 5% Human AB serum, 10 mM neutralized NAC) by removing the virus after 1 day of transfection, stimulating with the antibody for 4 days, and then removing the antibody as well. On the 5th day after antibody stimulation, CAR-T cells with the same CAR expression level (indicated by eGFP fluorescence intensity) were separated, and the obtained T-cells were each treated with anti-CD19. 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF The cells were designated as 28Z CAR-T cells. After amplification for a certain period, a series of indicators were assessed for the CAR-T cells, including the CAR membrane level, the proportion of CD4 and CD8 subsets, cytokine secretion in vitro, in vitro killing ability, in vitro proliferative ability, in vitro apoptosis level, in vitro sustained survival ability, and in vivo antitumor ability.

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

[0132] Using Alexa Fluor647-conjugated anti-mouse FMC63 scFv antibody, anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells, and anti-CD19 E YF anti-CD in 28Z CAR-T cells 19. The membrane levels of CAR receptors were measured by flow cytometry. The flow cytometry graph is shown in Figure 1b. This shows that there was no difference in CAR positivity rate and expression level (MFI) among the three groups.

[0133] Using anti-humanCD4-APC and anti-human CD8-PE-Cy7 antibodies, 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 is shown in Figure 1c. From this, the CD4 of the three cells was analyzed. + CAR-T cells and CD8 + The proportion 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 are each CD19 +Lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottom 96-well microplate, homogeneously mixed, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for 1 day before sample 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 collection. Subsequently, the cells were fixed with 4% PFA at room temperature for 5min and perforated with 0.1% Triton X-100 at room temperature for 5min. Then, intracellular staining procedures were performed according to standard procedures, and IL-2, IFN-γ, and TNF-α were detected with the corresponding directly labeled antibodies. As shown in Figures 1d to 1f, anti-CD19 E28Z CAR-T cells were compared to anti-CD19 E28Z cells. YF 28Z CAR-T cells are CD19 + The cytokine levels of IL-2, IFN-γ, and TNF-α produced after specific cell stimulation were clearly restored and elevated. This indicates that the ITAM motif in the intracellular domain of CD3ε has a significant 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). Therefore, it was possible to reduce the production of inflammatory cytokines such as IL-1β and IL-6 by activating 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 are each CD19 +Lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottom 96-well microplate, homogeneously mixed, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for a set period of time. Subsequently, samples were collected at different time points, washed once with PBS, stained with anti-human Ki67-APC antibody according to the instructions of the Foxp3 / Transcription Factor Staining Buffer Set reagent kit, and then Ki67 expression levels were measured by flow cytometry. As a result, as shown in Figure 1g, anti-CD19 E YF 28Z CAR-T is CD19 + The proliferation rate of Raji cells after stimulation was consistently faster than that of anti-CD19 28Z CAR-T cells for a certain period of time, and consequently faster than that of anti-CD19 E28Z CAR-T cells. That is, anti-CD19 E YF 28Z CAR-T cells exhibited the strongest proliferative capacity.

[0136] 100,000 anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells are each CD19 + The lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottomed 96-well microplate, and after homogeneous mixing, the cells were centrifuged at room temperature (400g, 1min) to check for intercellular contact. Next, complete medium was used and the cells were cultured in a 37°C incubator for a set period of time. Subsequently, after collecting the samples at different time points, the samples were washed once with PBS, stained according to the instructions of the Annexin V Apoptosis Detection Kit (APC reagent kit), and then the annexin V (Annexin V) in CAR-T cells was analyzed. The expression level of V) was measured by flow cytometry. As a result, as shown in Figure 1h, anti-CD19 E YF28Z CAR-T cells and anti-CD19 E28Z CAR-T cells are CD19 + For a sustained period following stimulation by Raji cells, apoptosis levels were significantly lower than those 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 are each CD19 + Lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottom 96-well microplate, homogeneously mixed, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for a set period of time. Subsequently, samples were collected at different time points, washed once with PBS, and stained with anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies to perform CAR + The number of viable T cells was detected. As a result, as shown in Figure 1i, CD19 + After stimulation by Raji cells, anti-CD19 E YF The number of surviving 28Z CAR-T cells was highest, followed by anti-CD19 E28Z CAR-T cells, and the number of surviving anti-CD19 28Z CAR-T cells was lowest. This is because E YF This means that 28Z CAR-T cells have the strongest sustained survival ability.

[0138] anti-CD1928Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 E YF 28Z CAR-T cells are CD19 + It exhibits a toxic reaction against tumor cells. Therefore, CD19 -K562 cells were resuspended in serum-free 1640 medium and pre-stained with 1× CellTraker deep red dye (37°C water bath, 30 min). Subsequently, the 1640 serum-free medium was washed once, and the cells were resuspended in T cell complete medium, followed by CD19 staining. + K562_CD19 (IRES mCherry) cells were mixed in a 1:1 ratio with CAR-T cells to create target cells. Then, CAR-T cells were mixed with these mixed target cells in 3:1, 1:1, and 1:3 ratios, plated into a round-bottom 96-well microplate for uniform mixing, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for 1 day before the samples were collected and placed on ice. Finally, CD19 - K562 cells (APC + , mCherry - ) Percentage and CD19 + K562_CD19 cells (APC - , mCherry + The percentage change in ) was measured by flow cytometry, and the "CD19" of the experimental group was analyzed. - K562 cells (APC + , mCherry - ) Percentage / CD19 + K562_CD19 cells (APC - , mCherry + N was defined as "the percentage of CAR-T cells added to the mixed target cell sample wells". The N value is "N0 = CD19 - K562 cells (APC + , mCherry - ) Percentage / CD19 + K562_CD19 cells (APC - , mCherry + After using the percentage of ) as a baseline, the survival rate N / N0 was calculated. That is, the formula for the mortality rate was "Lysis(%)=1-N / N0". As a result, as shown in Figure 1j, 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 toxicity against 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 are each CD19 + The lymphoma cell line Raji was transplanted into 1.5 ml EP tubes in a 1:1 ratio, and after homogeneous mixing, the mixture was centrifuged at 4°C (500 g, 2 min) to promote cell-to-cell contact. Next, the cells were stimulated in a 37°C water bath using complete medium at a series of timings. Subsequently, equivolumes of 8% PFA were added at different timings and fixed at room temperature for 5 minutes, after which perforations were made at room temperature for 5 minutes using 0.1% Triton X-100. Then, intracellular staining procedures were performed according to the usual procedure. First, rabbit-derived pErk(1 / 2) Thr202 / Tyr204 (Figure 1k), rabbit-derived pAKT S473 (Figure 1l) and rabbit-derived primary antibody pS6 S235 / 236 Each sample (Figure 1m) was stained with primary antibodies, washed once, then stained with Alexa647-conjugated goat anti-rabbit IgG polyclonal antibody as a secondary antibody, washed once, and then flow cytometry was performed. As a result, as shown in Figures 1k to 1m, anti-CD19 E YF 28Z CAR-T cells undergo pErk(1 / 2) after specific stimulation by the antigen. Thr202 / Tyr204 and pS6 S235 / 236 The overall level was significantly higher in anti-CD19 E28Z CAR-T and anti-CD19 E28Z CAR-T cells. YF AKT of 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells S473 Phosphorylation levels were significantly higher in all cells than in anti-CD19 28Z CAR-T cells. Furthermore, among the signaling pathways following antigen activation of the chimeric antigen receptor CAR, the signaling molecule of the Raf / MAPK signaling pathway, the serine / threonine protein kinase Erk(1 / 2), was also observed. Thr202 / Tyr204Phosphorylation levels and the serine / threonine kinase AKT of the PI3K / AKT signaling pathway S473 The phosphorylation levels of both ribosomal protein S6, which is a downstream signaling molecule in the two pathways, indicate the promotion of cell proliferation, survival ability, and anti-apoptotic ability. S235 / 236 The phosphorylation level of this molecule indicates the level of cell growth and proliferation. By using the phenotypes of these three signaling molecules and examining them from the perspective of the signaling pathway, anti-CD19 E YF It was revealed that 28Z CAR-T cells have the strongest proliferative and survival capabilities.

[0140] Method for processing experimental data: Using the GraphPad Prism 8.0.2 software, statistical analysis of the data was performed, and P<0.05 (specifically, * P<0.05, ** P<0.01, *** P<0.001, **** A difference was considered statistically significant if P < 0.0001. Data for each group are presented as Mean ± SD.

[0141] Figures 1d to 1f show a one-way ANOVA with Sidak's multiple comparison test added.

[0142] Figures 1g, 1h, and 1j-1m show a two-way ANOVA with Sidak's multiple comparison test added.

[0143] Figure 1i shows the results of a two-way ANOVA.

[0144] Experimental 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 levels of anti-CD19 CAR receptors on the membrane and CD4 / CD8 ratios.

[0145] (2) anti-CD19 28Z CAR-T cells, anti-CD19 E28Z CAR-T cells and anti-CD19 EYF When the 28Z CAR-T cells were specifically stimulated in vitro with Raji cells, compared with the anti-CD19 28Z CAR-T cells, the anti-CD19 E YF The cytokine levels of IL-2, IFN-γ and TNF-α produced by the 28Z CAR-T cells were clearly restored and increased. This means that the ITAM motif in the intracellular region of CD3ε has an important inhibitory effect on cytokine production. However, it should be noted that these cytokines secreted by the anti-CD19 E YF 28Z CAR-T cells were still clearly slightly less than those of the anti-CD19 28Z CAR-T cells (Figures 1e - 1f). Therefore, it was possible to reduce the production of inflammatory cytokines such as IL-1β and IL-6 by the activation of macrophages and monocytes.

[0146] (3) The 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 amplified for a certain period of time. Then, samples were collected at different time points to measure the proliferation ability (i.e., the expression level of Ki67). As a result, compared with the anti-CD19 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells, the anti-CD19 E YF 28Z CAR-T cells had the best proliferation ability.

[0147] (4) The 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 amplified for a certain period of time. Then, samples were collected at different time points to measure the apoptosis level (indicated by annexin V). As a result, the anti-CD19 E28Z CAR-T cells and anti-CD19 E YF ​The apoptosis levels of 28Z CAR-T cells were all 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. Then, samples were collected at different time points, and the number of amplified CAR + T cells was measured. As time passed, the number of anti-CD19 E YF 28Z CAR-T cells was clearly higher than that of anti-CD19 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells continuously.

[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 where K562 was co-cultured with anti-CD19 E YF 28Z CAR-T cells, the killing ability of anti-CD19 E

[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 amplified for a certain period of time. Then, samples were collected at different time points, and the phosphorylation levels of the signal molecules serine / threonine protein kinase Erk(1 / 2) Thr202 / Tyr204 serine / threonine kinase AKT S473 and ribosomal protein S6 S235 / 236 were measured. Then, anti-CD19 E YF28Z CAR-T cells undergo pErk(1 / 2) after specific stimulation by the antigen. Thr202 / Tyr204 and pS6 S235 / 236 The overall level was significantly higher in anti-CD19 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells. YF AKT of 28Z CAR-T cells and anti-CD19 E28Z CAR-T cells S473 Phosphorylation levels were significantly higher in all cells than in anti-CD19 28Z CAR-T cells. Within the overall signaling pathway after antigen activation of the chimeric antigen receptor CAR, the signaling molecule Erk(1 / 2) of the Raf / MAPK signaling pathway was also elevated. Thr202 / Tyr204 Phosphorylation level and AKT in the PI3K / AKT signaling pathway S473 The phosphorylation levels of both ribosomal protein S6, a shared signaling molecule downstream of the two pathways, both indicate the promotion of cell proliferation, survival, and anti-apoptotic capacity. S235 / 236 The phosphorylation level of this molecule indicates the level of cell growth and proliferation. By using the phenotypes of these three signaling molecules and examining them from the perspective of the signaling pathway, anti-CD19 E YF It was revealed that 28Z CAR-T cells have the strongest proliferative and survival capabilities.

[0151] anti-CD19E YF The experimental design to compare the in vivo anti-subcutaneous tumor capacity of 28Z CAR-T cells with that of anti-CD19 E28Z cells and anti-CD19 28Z CAR-T cells was as follows:

[0152] Using aseptic techniques, 3 × 10 7 A Raji cell-PBS resuspension of 1 / ml was prepared. Then, 100 μl of 3 million Raji cells was subcutaneously transplanted into the left dorsal region of each 6-week-old B-NDG female mouse, and this point was designated as day 0. Six days later, when the Raji subcutaneous tumors had grown to a diameter of 6-7 mm, the mice were randomly divided into three groups (E YFThe mice were divided into two groups (28Z group, E28Z group, 28Z group, and vector group), and 100 µl of 8 million T cells were injected into the tail vein of each mouse. Subsequently, the size of the tumor was measured periodically using calipers, and the tumor data and the survival period of the mice were recorded. The tumor area was calculated as length × width.

[0153] Experimental conclusion: anti-CD19 E YF 28Z CAR-T cells possessed antitumor capabilities. [Examples]

[0154] The anti-CD1928Z CAR sequence contains the FMC63 scFv, CD28 hinge region, CD28 transmembrane region, and CD3ζ intracellular region (i.e., the sequence shared by the Rosenberg,SA laboratory at NCBI (GenBank: HM852952.1)). The 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. The eGFP and CAR sequences were then encoded by linking them to the same open reading frame (ORF) via a T2A self-cleaving peptide. Therefore, the CAR expression level could be indicated by the eGFP expression level / fluorescence intensity. Furthermore, by inserting the BRS motif of the CD3ε intracellular segment behind the CD28 transmembrane region based on the overlapping extension PCR technique, anti-CD19 E BRS We created a new car called the 28Z CAR.

[0155] anti-CD19 E obtained by synthesis BRS The nucleotide sequence of the 28Z CAR is as shown in SEQ ID NO:13. Specifically, it is as follows: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACG

[0156] The CAR was subcloned into the lentiviral expression plasmid pHAGE vector. The CAR expression plasmid was then mixed with packaging plasmids pSPAX2 and pMD2G in a ratio of 10:7.5:3.5, respectively, and introduced into 293FT cells using calcium phosphate transfection to generate lentiviral particles. Next, anti-CD19 28Z CAR or anti-CD19 E was obtained using ultrafast centrifugation. BRS Lentiviruses expressing 28Z CAR are concentrated to produce small-volume, high-titer viruses (~10 8 After adjusting the concentration to IU / ml, primary T cells (MOI=10) activated with αCD3 / αCD28 Ab-beads for 1 day were transfected accordingly. Complete T cell medium (XIVO-15 + 1% PS + 10 ng / ml human IL-7 + 10 ng / ml human IL-15 + 5% Human AB serum, 10 mM neutrali The culture period for primary T cells using zed NAC was as follows: after transfection with the virus for 1 day, the cells were removed, and then stimulated with the antibody for 4 days, after which the cells were also removed. On the 5th day after antibody stimulation, CAR-T cells with the same CAR expression level (indicated by eGFP fluorescence intensity) were separated, and the obtained T cells were designated as anti-CD19 28Z CAR-T cells and anti-CD19 E BRS The cells were designated as 28Z CAR-T cells. After amplification for a certain period, a series of indicators were assessed for the CAR-T cells, including the CAR membrane level, the proportion of CD4 and CD8 subsets, cytokine secretion in vitro, in vitro killing ability, in vitro proliferative ability, in vitro apoptosis level, in vitro sustained survival ability, and in vivo antitumor ability.

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

[0158] Using the Alexa Fluor647-conjugated anti-mouse FMC63 scFv antibody, anti-CD19 28Z CAR-T and anti-CD19 E BRS CAR levels on the membrane surface of 28Z CAR-T cells were measured by flow cytometry. The flow cytometry graph is shown in Figure 2b. This indicates that there was no difference in CAR positivity rate or expression level (MFI) between the two groups.

[0159] Using anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies, anti-CD19 28Z CAR-T and anti-CD19 E BRS Flow cytometry was performed on 28Z CAR-T cells. The flow cytometry graph is shown in Figure 2c. From this, the CD4 of both cells was analyzed. + CAR-T cells and CD8 + The proportion 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 are each CD19 +Lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottom 96-well microplate, homogeneously mixed, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for 1 day before sample 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 collection. Subsequently, the cells were fixed with 4% PFA at room temperature for 5 minutes and perforated with 0.1% Triton X-100 at room temperature for 5 minutes. Then, intracellular staining procedures were performed according to standard procedures, and IL-2, IFN-γ, and TNF-α were detected with the corresponding directly labeled antibodies. As a result, as shown in Figures 2d, 2e, and 2f, anti-CD19 E BRS 28Z CAR-T cells CD19 + The cytokine levels of IL-2 and TNF-α produced after specific stimulation of cells were similar to those of anti-CD19 28Z CAR-T cells, but the cytokine level of IFN-γ was lower than that of anti-CD19 28Z CAR-T cells.

[0161] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells are each CD19 + Lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottom 96-well microplate, homogeneously mixed, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for a set period of time. Subsequently, samples were rotated at different time points. After collection, the sample was washed once with PBS, and then stained with anti-human Ki67-APC antibody according to the instructions of the Foxp3 / Transcription Factor Staining Buffer Set reagent kit. Ki67 expression levels were then measured by flow cytometry. As shown in Figure 2g, anti-CD19 E BRS 28Z CAR-T cells are CD19 +After stimulation with Raji cells, the proliferation rate remained consistently faster than that of anti-CD19 28Z CAR-T cells for a certain period of time.

[0162] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells are each CD19 + Lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottom 96-well microplate, homogeneously mixed, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for a set period of time. Subsequently, samples were collected at different time points, washed once with PBS, stained according to the instructions of the Annexin V Apoptosis Detection Kit (APC reagent kit), and then 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-stage 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 are each CD19 + Lymphoma cell line Raji was transplanted in a 1:1 ratio into a round-bottom 96-well microplate, homogeneously mixed, and then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for a set period of time. Subsequently, samples were collected at different time points, washed once with PBS, and stained with anti-human CD4-APC and anti-human CD8-PE-Cy7 antibodies to perform CAR + The number of viable T cells was detected. As a result, as shown in Figure 2i, anti-CD19 E BRS 28Z CAR-T cells are CD19 +The number of Raji cells that survived after stimulation was determined by anti-CD19 There were significantly more of them than 28Z CAR-T cells.

[0164] 100,000 anti-CD19 28Z CAR-T cells and anti-CD19 E BRS 28Z CAR-T cells are each CD19 + Lymphoma cell line Raji was transplanted in a 1:1 ratio into 1.5 ml EP tubes and homogeneously mixed. Centrifugation (500 g, 2 min) at 4°C was then performed to promote cell-to-cell contact. Next, the cells were stimulated in a 37°C water bath using complete medium at a series of timings. Subsequently, equivolumes of 8% PFA were added at different timings and fixed at room temperature for 5 min. Then, perforations were performed at room temperature for 5 min using 0.1% Triton X-100. Intracellular staining procedures were then carried out according to standard procedures. First, rabbit-derived pAKT... S473 (Figure 2j) and rabbit-derived primary antibody pS6 S235 / 236 (Figure 2k) was stained with primary antibody, washed once, then stained with Alexa647-conjugated goat anti-rabbit IgG polyclonal antibody as a secondary antibody, washed once, and then flow cytometry was performed. As a result, as shown in Figures 2j and 2k, anti-CD19 E BRS 28Z CAR-T cells undergo pAKT after specific stimulation by antigens. S473 and pS6 S235 / 236 The overall level was significantly higher than in anti-CD19 28Z CAR-T cells. Furthermore, the signaling molecule AKT S473 The phosphorylation level of the signaling molecule ribosomal protein S6 indicates the promotion of cell proliferation and survival ability. S235 / 236 The phosphorylation level indicates the level of cell growth and proliferation. These two indicators, in terms of signaling pathways, are anti-CD19 E BRS The study showed that 28Z CAR-T cells had relatively good proliferative and viable capabilities.

[0165] anti-CD19 28Z CAR-T cells and E BRS 28Z CAR-T cells are CD19 +It exhibits a toxic reaction against tumor cells. Therefore, CD19 - K562 cells were resuspended in serum-free 1640 medium and pre-stained with 1× CellTraker deep red dye (37°C water bath, 30 min). Subsequently, the 1640 serum-free medium was washed once, and the cells were resuspended in T cell complete medium, followed by CD19 staining. + K562_CD19(IRES mCherry) cells were mixed in a 1:1 ratio with anti-CD19 28Z CAR-T cells and anti-CD19 E cells to create target cells. BRS 28Z CAR-T cells were mixed with the target cells in a ratio of 3:1, 1:1, and 1:3, respectively, and plated into a round-bottom 96-well microplate for uniform mixing. The mixture was then centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact. Next, the cells were cultured in complete medium in a 37°C incubator for 1 day before the samples were collected and placed on ice. Finally, CD19 - K562 cells (APC + , mCherry - ) Percentage and CD19 + K562_CD19 cells (APC - , mCherry + The percentage change in ) was measured by flow cytometry, and the "CD19" of the experimental group was analyzed. - K562 cells (APC + , mCherry - ) Percentage / CD19 + K562_CD19 cells (APC - , mCherry + The percentage of ) was defined as N. The N value is defined as anti-CD19 28Z CAR-T cells or anti-CD19 E BRS "N0=CD19" in the mixed target cell sample well without added 28Z CAR-T cells - K562 cells (APC + , mCherry - ) Percentage / CD19 + K562_CD19 cells (APC - , mCherry +After using the percentage of ) as a baseline, the survival rate N / N0 was calculated. That is, the formula for the lethality rate was "Lysis(%)=1-N / N0". As a result, as shown in Figure 2l, CD19 + anti-CD19 E against tumor cells BRS The toxicity of 28Z CAR-T cells was shown to be equivalent to that of anti-CD19 28Z CAR-T cells.

[0166] Method for processing experimental data: Using the GraphPad Prism 8.0.2 software, statistical analysis of the data was performed, and P<0.05 (specifically, * P<0.05, ** P<0.01, *** P<0.001, **** A difference was considered statistically significant if P < 0.0001. Data for each group are presented as Mean ± SD.

[0167] Figures 2d to 2f show a one-way ANOVA with Sidak's multiple comparison test added.

[0168] Figures 2g, 2h, and 2j-2l show a two-way ANOVA with Sidak's multiple comparison test added.

[0169] Figure 2i shows the results of a two-way ANOVA.

[0170] Experimental results: (1) anti-CD19 E BRS The 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 When 28Z CAR-T cells were specifically stimulated in vitro with Raji cells, 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 level of IFN-γ was lower than that 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, amplified for a certain period of time, and then samples were collected at different time points to assess their proliferative capacity (i.e., Ki67 expression level). When the bell was measured, anti-CD19 E BRS The proliferative capacity of 28Z CAR-T cells was clearly 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, amplified for a certain period of time, and then samples were collected at different time points to measure apoptosis levels. Furthermore, anti-CD19 E BRS When 28Z CAR-T cells and anti-CD19 28Z CAR-T cells were compared, they were equivalent in the early stage, but in the later stage, anti-CD19 E BRS The apoptosis level of 28Z CAR-T cells was significantly 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 by Raji cells and amplified for a certain period of time. Then, samples were collected at different time points, and the amplified CAR-T cells were analyzed. + When the number of T cells was measured, it was found that the number of anti-CD19 E cells increased over time. 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 by Raji cells, amplified for a certain period of time, and then samples were collected at different time points to analyze the signaling molecule AKT. S473 The phosphorylation level of the signal molecule AKT was measured. S473 The phosphorylation level of indicates the promotion of cell proliferation and survival ability. Then, anti-CD19 E BRS Overall pAKT of 28Z CAR-T cells S473 The level is higher than that of 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 by Raji cells, amplified for a certain period of time, and then samples were collected at different time points to detect 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 cell's growth and proliferation capacity. BRS Overall pS6 of 28Z CAR-T cells S235 / 236 The levels were clearly higher than those of anti-CD19 28Z CAR-T cells, and anti-CD19 E BRS The growth and proliferation capacity 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 in vitro toxicity experiments co-culturing K562, anti-CD19 E BRSThe killing ability of 28Z CAR-T cells was equivalent to that of anti-CD19 28Z CAR-T cells.

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

[0179] Using aseptic techniques, 3 × 10 7 A Raji cell-PBS resuspension of 1 / ml was prepared. Then, 100 μl of 3 million Raji cells was subcutaneously transplanted into the left dorsal region of each 6-week-old B-NDG female mouse, and this point was designated as day 0. Six days later, when the Raji subcutaneous tumors had grown to a diameter of 6-7 mm, the mice were randomly divided into three groups (E BRS The mice were divided into two groups (28Z group, 28Z group, and vector group), and 100 µl of 8 million T cells were injected into the tail vein of each mouse. Subsequently, the size of the tumor was measured periodically using calipers, and the tumor data and the survival period of the mice were recorded. The tumor area was calculated as length × width.

[0180] Experimental conclusion: anti-CD19 E BRS 28Z CAR-T cells possessed antitumor activity.

[0181] The above are merely preferred embodiments of the present invention and do not formally or substantially limit the present invention in any way. It should be noted that those skilled in the art can make some improvements and additions, provided they do not depart from the methods of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any equivalent modifications, alterations, and changes that can be implemented by an engineer familiar with this art using the technical content disclosed above without departing from the spirit and scope of the present invention are all equivalent embodiments of the present invention. Furthermore, any equivalent modifications, alterations, and changes implemented in the above embodiments based on the substantial art of the present invention are all within the scope of the technical solutions of the present invention.

Claims

1. A chimeric antigen receptor containing a motif of an intracellular basic amino acid-rich region of CD3ε, It includes an extracellular domain, a transmembrane domain, and an intracellular domain that are linked in order, The extracellular domain includes an antigen recognition region and a hinge region. One end of the intracellular domain that is linked to the transmembrane domain is linked to a motif of a CD3ε intracellular basic amino acid-rich region. The amino acid sequence of the motif in the intracellular basic amino acid-rich region of the aforementioned CD3ε is the sequence shown in SEQ ID NO:

10. The intracellular domain includes, in order, a motif of a CD3ε intracellular basic amino acid-rich region, a co-stimulatory signaling region, and a CD3ζ intracellular segment. Chimeric antigen receptor.

2. The co-stimulatory signaling region is selected from one or more intracellular segments among CD27, CD28, CD134, 4-1BB, OX-40, and ICOS, resulting in a chimeric antigen receptor containing a motif of an intracellular basic amino acid-rich region of CD3ε according to claim 1.

3. Furthermore, (1) The amino acid sequence of the intracellular segment of CD28 is the sequence shown in SEQ ID NO: 2, (2) The amino acid sequence of the CD3ζ intracellular segment is the sequence shown in SEQ ID NO:

3. A chimeric antigen receptor containing a motif of an intracellular basic amino acid-rich region of CD3ε according to claim 2, characterized by including one or more of the features of the above.

4. Furthermore, a. The antigen recognition region is selected from single-chain antibodies that target tumor surface antigens, and the tumor surface antigens are CD19, mesothelin, CD20, CD22, CD123, CD30, C Selected from one or more of D33, 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 a hinge region formed by linking an immunoglobulin IgG hinge region with a CH2CH3 region. A chimeric antigen receptor containing a motif of an intracellular basic amino acid-rich region of CD3ε according to claim 1, characterized by including one or more of the features of the above.

5. Furthermore, d. The single-chain antibody is selected from FMC63, e. The amino acid sequence of the transmembrane region of CD28 is the sequence shown in SEQ ID NO:

4. f. The amino acid structure of the CD28 hinge region is the sequence shown in SEQ ID NO:

7. A chimeric antigen receptor containing a motif of an intracellular basic amino acid-rich region of CD3ε according to claim 4, characterized by including one or more of the features of the above.

6. Furthermore, 1) The antigen recognition region of the chimeric antigen receptor containing the motif of the intracellular basic amino acid-rich region of CD3ε is selected from FMC63, the hinge region is selected from the CD28 hinge region, the transmembrane domain is selected from the transmembrane region of CD28, and the intracellular domain includes, in order, the motif of the intracellular basic amino acid-rich region of CD3ε, the intracellular segment of CD28, and the intracellular segment of CD3ζ. 2) The amino acid sequence of the chimeric antigen receptor is the sequence shown in SEQ ID NO:

11. A chimeric antigen receptor comprising a motif of an intracellular basic amino acid-rich region of CD3ε according to any one of claims 1 to 5, characterized by including one or more of the features of the above.

7. An isolated polynucleotide, (1) A polynucleotide encoding a chimeric antigen receptor containing a motif of an intracellular basic amino acid-rich region of CD3ε as described in any one of claims 1 to 5, and (2) A polynucleotide complementary to the polynucleotide of (1), A polynucleotide selected from the following.

8. The polynucleotide according to claim 7, characterized in that the polynucleotide has the base sequence shown in SEQ ID NO:

12.

9. A nucleic acid construct comprising the polynucleotide described in claim 7.

10. The nucleic acid construct according to claim 9, characterized in that the nucleic acid construct is a vector.

11. The nucleic acid construct according to claim 9 is a lentiviral vector comprising a replication origin, a 3'LTR, a 5'LTR, and the polynucleotide described in claim 7.

12. The nucleic acid construct and the lentiviral vector auxiliary component described in claim 9 Lusvector system.

13. Recombinant T cells characterized by containing the polynucleotide described in claim 7.

14. Recombinant T cells characterized by comprising the nucleic acid construct described in claim 9.

15. Recombinant T cells characterized by being infected with the lentiviral vector system described in claim 12.

16. Use of a chimeric antigen receptor comprising a CD3ε intracellular basic amino acid-rich region motif according to any one of claims 1 to 5 in the manufacture of a product for any one or more of the following uses: (1) production of T cells, (2) improvement of T cell viability, (3) inhibition of T cell apoptosis, (4) enhancement of T cell proliferative capacity, (5) improvement of T cell antitumor capacity, and (6) suppression of cytokine IFN-γ levels from T cells.

17. Use of the polynucleotide according to claim 7 in the manufacture of a product for any one or more of the following uses: (1) production of T cells, (2) improvement of T cell viability, (3) inhibition of T cell apoptosis, (4) enhancement of T cell proliferative capacity, (5) improvement of T cell antitumor capacity, and (6) suppression of cytokine IFN-γ levels from T cells.

18. Use of the nucleic acid construct according to claim 9 in the manufacture of a product for any one or more of the following uses: (1) production of T cells, (2) improvement of T cell viability, (3) inhibition of T cell apoptosis, (4) enhancement of T cell proliferative capacity, (5) improvement of T cell antitumor capacity, and (6) suppression of cytokine IFN-γ levels from T cells.

19. Use of the lentiviral vector system according to claim 12 in the manufacture of a product for any one or more of the following uses: (1) production of T cells, (2) improvement of T cell viability, (3) inhibition of T cell apoptosis, (4) enhancement of T cell proliferative capacity, (5) improvement of T cell antitumor capacity, and (6) suppression of T cell cytokine IFN-γ levels.

20. Use in the manufacture of a tumor therapeutic product of a chimeric antigen receptor comprising a motif of an intracellular basic amino acid-rich region of CD3ε as described in any one of claims 1 to 5.

21. Use of the polynucleotide according to claim 7 in the manufacture of a tumor treatment product.

22. Use of the nucleic acid construct according to claim 9 in the manufacture of a tumor treatment product.

23. Use of the lentiviral vector system according to claim 12 in the manufacture of an oncological therapeutic product.

24. Use of genetically modified T cells according to claim 13 in the manufacture of a tumor treatment product.

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