Chimeric antigen receptor immune cell with modified CD3 zeta region and application of chimeric antigen receptor immune cell

Abstract

The invention provides a chimeric antigen receptor immune cell with a modified CD3 zeta region and application of the chimeric antigen receptor immune cell. Specifically, a cytokine receptor fragment is used for replacing an ITAM part fragment in a CD3 zeta region in the chimeric antigen receptor, so that synergistic activation of various downstream signals is realized, and solid tumor response higher than that of traditional second-generation CAR is obtained. The chimeric antigen receptor immune cell provided by the invention has the advantages of self-activation, reduced cell depletion level and improved cell dryness. The chimeric antigen receptor immune cell disclosed by the invention has an application prospect in the anti-tumor field.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to a chimeric antigen receptor immune cell with a modified CD3ζ region and its applications. Background Technology

[0002] CAR-T cell therapy has achieved significant results and cures in some indications of hematological malignancies, with several products currently on the market. However, in the treatment of solid tumors, which have a higher incidence rate, CAR-T cell therapy products based on second-generation CAR structures have performed far below expectations. This is mainly due to a combination of limiting factors, including CAR-induced initiation activation being inferior to TCR; insufficient stemness and limited proliferation of CAR T cells, as well as a lack of memory response; poor infiltration within solid tumors; and an immunosuppressive microenvironment.

[0003] Therefore, there is a need in this field to develop a chimeric antigen receptor immune cell that is well activated, capable of proliferating in large quantities, and reduces cell exhaustion. Summary of the Invention

[0004] The purpose of this invention is to provide a chimeric antigen receptor immune cell that is well activated, capable of proliferating in large quantities, and reduces cell exhaustion.

[0005] In a first aspect of the invention, a chimeric antigen receptor (CAR) is provided, the chimeric antigen receptor comprising a substituted intracellular signal transduction domain, the intracellular signal transduction domain comprising one or more immune receptor tyrosine activation domains (ITAMs), wherein the one or more ITAMs comprise one or more substituted fragments derived from an exogenous polypeptide.

[0006] In another preferred embodiment, the intracellular signal transduction domain is an intracellular signal transduction domain derived from immune cell activation-related receptors.

[0007] In another preferred embodiment, the immune cell activation-related receptor is selected from the group consisting of CD3ζ, CD3γ, CD3δ, and CD3ε.

[0008] In another preferred embodiment, the exogenous polypeptide is a receptor associated with immune cell activation signaling pathways.

[0009] In another preferred embodiment, the exogenous polypeptide is a cytokine receptor.

[0010] In another preferred embodiment, the exogenous polypeptide is an interleukin receptor.

[0011] In another preferred embodiment, the exogenous polypeptide is selected from the group consisting of: IL-1β receptor, IL-2 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-21 receptor, IL-23 receptor, IFN-γ receptor, TNF-α receptor, TGF-β receptor, GM-CSF receptor, DAP10, DAP12, FcRγ, or combinations thereof.

[0012] In another preferred embodiment, the exogenous polypeptide is selected from the group consisting of IL2RB, IL21R, IL7R, IL10R, or combinations thereof.

[0013] In another preferred embodiment, the intracellular signal transduction domain is derived from CD3γ, CD3δ, or CD3ε.

[0014] In another preferred embodiment, the intracellular signal transduction domain includes an immune receptor tyrosine activation domain (ITAM).

[0015] In another preferred embodiment, the length of the substituted sequence fragment in the ITAM is 1-20 aa, more preferably 5-15 aa, and even more preferably 11-13 aa.

[0016] In another preferred embodiment, the length of each of the one or more substituted segments is independently 1-20 aa, more preferably 5-15 aa, and even more preferably 11-13 aa.

[0017] In another preferred embodiment, the length of the substituted fragment is the same as the length of the sequence fragment being substituted.

[0018] In another preferred embodiment, the substituted fragment contains tyrosine (Y).

[0019] In another preferred embodiment, the substituted fragment contains a tyrosine (Y) mutated to a phenylalanine (F).

[0020] In another preferred embodiment, the amino acid sequence of the substituted fragment is as shown in SEQ ID NO:1, 2, 3, 4 or 5, or has at least 90% amino acid sequence identity with it.

[0021] In another preferred embodiment, the intracellular signal transduction domain is derived from the intracellular signal transduction domain (CD3ζ region) of CD3ζ.

[0022] In another preferred embodiment, the intracellular signal transduction domain comprises three immune receptor tyrosine activation domains ITAM1, ITAM2, and ITAM3, sequentially from the N-terminus to the C-terminus.

[0023] In another preferred embodiment, the ITAM3 includes a substitution fragment R1 that replaces tyrosine at position 12 and its neighboring amino acid fragment, or tyrosine at position 23 and its neighboring amino acid fragment, or a combination thereof, in the wild-type ITAM3 amino acid sequence (SEQ ID NO:30).

[0024] In another preferred embodiment, the R1 fragment is derived from IL2RB or IL21R.

[0025] In another preferred embodiment, the amino acid sequence of R1 is as shown in SEQ ID NO:1, 2, 3, 4 or 5.

[0026] In another preferred embodiment, the ITAM3 includes substitution fragments R1 and R2, which respectively replace tyrosine at position 12 and its neighboring amino acid fragments and tyrosine at position 23 and its neighboring amino acid fragments in the wild-type ITAM3 amino acid sequence (SEQ ID NO:30).

[0027] In another preferred embodiment, the R1 and R2 segments are each independently derived from IL2RB or IL21R.

[0028] In another preferred embodiment, fragment R1 originates from IL2RB and fragment R2 originates from IL21R; or

[0029] The R1 fragment originates from IL21R and the R2 fragment originates from IL2RB; or

[0030] Both fragments R1 and R2 originate from IL2RB.

[0031] In another preferred embodiment, the amino acid sequences of R1 and R2 are each independently shown as SEQ ID NO:1, 2, 3, 4 or 5.

[0032] In another preferred embodiment, the R2 fragment is derived from IL-12, and the sequence of the R2 fragment is shown in SEQ ID NO:46.

[0033] In another preferred embodiment, the tyrosine residue at position 12 of ITAM3 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 3-13, 4-14, 5-15, 6-16, or 7-17 of SEQ ID NO:30.

[0034] In another preferred embodiment, the tyrosine residue at position 23 of ITAM3 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 17-27, 18-28, or 19-29 of SEQ ID NO:30.

[0035] In another preferred embodiment, the ITAM3 includes substitution fragments R1 and R2, wherein:

[0036] R1 replaces the sequence segment shown at positions 7-17 in the wild-type ITAM3 amino acid sequence;

[0037] R2 replaces the sequence segment shown at positions 18-28 in the wild-type ITAM3 amino acid sequence.

[0038] In another preferred embodiment, the amino acid sequence of R1 is as shown in SEQ ID NO:1 or 3; and the amino acid sequence of R2 is as shown in SEQ ID NO:2 or 4.

[0039] In another preferred embodiment, the amino acid sequence of R1 is as shown in SEQ ID NO:1, 2, 3 or 4; and the amino acid sequence of R2 is as shown in SEQ ID NO:5.

[0040] In another preferred embodiment, the amino acid sequence of R1 is shown in SEQ ID NO:5; and the amino acid sequence of R2 is shown in SEQ ID NO:1, 2, 3 or 4.

[0041] In another preferred embodiment, the amino acid sequence of R1 is shown in SEQ ID NO:1; and the amino acid sequence of R2 is shown in SEQ ID NO:5.

[0042] In another preferred embodiment, the amino acid sequence of R1 is shown in SEQ ID NO:1; and the amino acid sequence of R2 is shown in SEQ ID NO:46.

[0043] In another preferred embodiment, the ITAM2 includes a substitution fragment R3 that replaces tyrosine at position 12 and its neighboring amino acid fragment, or tyrosine at position 24 and its neighboring amino acid fragment, or a combination thereof, in the wild-type ITAM2 amino acid sequence (SEQ ID NO:29).

[0044] In another preferred embodiment, the R3 fragment is derived from IL2RB or IL21R.

[0045] In another preferred embodiment, the amino acid sequence of R3 is as shown in SEQ ID NO:1, 2, 3, 4 or 5.

[0046] In another preferred embodiment, the ITAM2 includes substitution fragments R3 and R4, which respectively replace tyrosine at position 12 and its neighboring amino acid fragments and tyrosine at position 24 and its neighboring amino acid fragments in the wild-type ITAM2 amino acid sequence (SEQ ID NO:29).

[0047] In another preferred embodiment, the R3 and R4 fragments are each independently derived from IL2RB or IL21R.

[0048] In another preferred embodiment, fragment R3 originates from IL2RB and fragment R4 originates from IL21R; or

[0049] The R3 fragment originates from IL21R and the R4 fragment originates from IL2RB; or

[0050] Both R3 and R4 fragments originate from IL2RB.

[0051] In another preferred embodiment, the amino acid sequences of R3 and R4 are each independently shown as SEQ ID NO:1, 2, 3, 4 or 5.

[0052] In another preferred embodiment, the R4 fragment is derived from IL-12, and the sequence of the R4 fragment is shown in SEQ ID NO:46.

[0053] In another preferred embodiment, the tyrosine residue at position 12 of ITAM2 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 3-13, 4-14, 5-15, 6-16, or 7-17 of SEQ ID NO:29.

[0054] In another preferred embodiment, the tyrosine residue at position 24 of ITAM2 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 17-27, 18-28, or 19-29 of SEQ ID NO:29.

[0055] In another preferred embodiment, the ITAM2 includes substitution fragments R3 and R4, wherein:

[0056] R3 replaces the sequence segment shown at positions 7-17 in the wild-type ITAM2 amino acid sequence;

[0057] R4 replaces the sequence segment shown at positions 19-29 in the wild-type ITAM2 amino acid sequence.

[0058] In another preferred embodiment, the amino acid sequence of R3 is as shown in SEQ ID NO:1 or 3; and the amino acid sequence of R4 is as shown in SEQ ID NO:2 or 4.

[0059] In another preferred embodiment, the amino acid sequence of R3 is as shown in SEQ ID NO:1, 2, 3 or 4; and the amino acid sequence of R4 is as shown in SEQ ID NO:5.

[0060] In another preferred embodiment, the amino acid sequence of R3 is shown in SEQ ID NO:5; and the amino acid sequence of R4 is shown in SEQ ID NO:1, 2, 3 or 4.

[0061] In another preferred embodiment, the amino acid sequence of R3 is shown in SEQ ID NO:2; and the amino acid sequence of R4 is shown in SEQ ID NO:46.

[0062] In another preferred embodiment, the amino acid sequence of R3 is shown in SEQ ID NO:2; and the amino acid sequence of R4 is shown in SEQ ID NO:5.

[0063] In another preferred embodiment, the ITAM1 includes a substitution fragment R5 that replaces tyrosine at position 14 and its neighboring amino acid fragment, or tyrosine at position 25 and its neighboring amino acid fragment, or a combination thereof, in the wild-type ITAM1 amino acid sequence (SEQ ID NO:28).

[0064] In another preferred embodiment, the R5 fragment is derived from IL2RB or IL21R.

[0065] In another preferred embodiment, the amino acid sequence of R5 is as shown in SEQ ID NO:1, 2, 3, 4 or 5.

[0066] In another preferred embodiment, the ITAM1 includes substitution fragments R5 and R6, which respectively replace tyrosine at position 14 and its neighboring amino acid fragments and tyrosine at position 25 and its neighboring amino acid fragments in the wild-type ITAM1 amino acid sequence (SEQ ID NO:28).

[0067] In another preferred embodiment, the R5 and R6 fragments are each independently derived from IL2RB or IL21R.

[0068] In another preferred embodiment, the R5 fragment originates from IL2RB and the R6 fragment originates from IL21R; or

[0069] The R5 fragment originates from IL21R and the R6 fragment originates from IL2RB; or

[0070] Both R5 and R6 fragments originate from IL2RB.

[0071] In another preferred embodiment, the amino acid sequences of R5 and R6 are each independently shown as SEQ ID NO:1, 2, 3, 4 or 5.

[0072] In another preferred embodiment, the tyrosine residue at position 14 of ITAM1 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 5-15, 6-16, 7-17, or 8-18 of SEQ ID NO:28.

[0073] In another preferred embodiment, the tyrosine residue at position 25 of ITAM1 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 19-29, 20-30, or 21-31 of SEQ ID NO:28.

[0074] In another preferred embodiment, the intracellular signal transduction domain has the same sequence length as the wild-type CD3ζ region.

[0075] In another preferred embodiment, the intracellular signal transduction domain has one or more substitutions selected from the group consisting of the wild-type CD3ζ region (SEQ ID NO:20):

[0076] (I) The amino acids at positions 20-30 of ITAM1 are replaced by the sequence shown in SEQ ID NO:5;

[0077] (II) The amino acids at positions 7-17 of ITAM2 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4 or 5;

[0078] (III) The amino acids at positions 19-29 of ITAM2 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4 or 5;

[0079] (IV) The amino acids at positions 7-17 of ITAM3 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4 or 5;

[0080] (V) The amino acids at positions 18-28 of ITAM3 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4 or 5.

[0081] In another preferred embodiment, the intracellular signal transduction domain further comprises one or more substitutions selected from the group consisting of the wild-type CD3ζ region (SEQ ID NO:20):

[0082] (VI) The amino acids at positions 105-108 are mutated to the sequence shown in YRHQ (SEQ ID NO:6);

[0083] (VII) The amino acids at positions 6-9 of ITAM1 (SEQ ID NO:28) are mutated to the sequence shown in YRHQ (SEQ ID NO:6);

[0084] (VIII) The amino acid sequence at positions 24-27 of ITAM2 (SEQ ID NO:29) is mutated to the sequence shown in YRHQ (SEQ ID NO:6);

[0085] The amino acid sequence (IX)ITAM3 (SEQ ID NO:30) is mutated from position 12 to 15 to the sequence shown in YRHQ (SEQ ID NO:6).

[0086] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 86-96 substituted with a sequence as shown in SEQ ID NO:1 or 5.

[0087] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 86-96 substituted with a sequence as shown in SEQ ID NO:1 or 3; and amino acids 55-65 substituted with a sequence as shown in SEQ ID NO:2 or 4.

[0088] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 86-96 substituted with a sequence as shown in SEQ ID NO:1 or 3; and amino acids 51-61 substituted with a sequence as shown in SEQ ID NO:2 or 4.

[0089] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 81-91 substituted with a sequence as shown in SEQ ID NO:1 or 3; and amino acids 55-65 substituted with a sequence as shown in SEQ ID NO:2 or 4.

[0090] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 81-91 substituted with a sequence as shown in SEQ ID NO:1 or 3; and amino acids 51-61 substituted with a sequence as shown in SEQ ID NO:2 or 4.

[0091] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 86-96 substituted with the sequence shown in SEQ ID NO:1 or 3; amino acids 55-65 substituted with the sequence shown in SEQ ID NO:2 or 4; and amino acids 27-37, 67-77, or 97-107 substituted with the sequence shown in SEQ ID NO:5.

[0092] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 86-96 substituted with a sequence as shown in SEQ ID NO:1 or 3; and amino acids 55-65 substituted with a sequence as shown in SEQ ID NO:5.

[0093] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 86-96 substituted with the sequence shown in SEQ ID NO:5; and amino acids 55-65 substituted with the sequence shown in SEQ ID NO:1 or 3.

[0094] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 55-65 substituted with the sequence shown in SEQ ID NO:2, amino acids 67-77 substituted with the sequence shown in SEQ ID NO:46, amino acids 86-96 substituted with the sequence shown in SEQ ID NO:1, and amino acids 97-107 substituted with the sequence shown in SEQ ID NO:5.

[0095] In another preferred embodiment, the substituted intracellular signal transduction domain, relative to the wild-type CD3ζ region as shown in SEQ ID NO:20, has amino acids 55-65 substituted with the sequence shown in SEQ ID NO:2, amino acids 67-77 substituted with the sequence shown in SEQ ID NO:5, amino acids 86-96 substituted with the sequence shown in SEQ ID NO:1, and amino acids 97-107 substituted with the sequence shown in SEQ ID NO:46.

[0096] In another preferred embodiment, the intracellular signal transduction domain is mutated at amino acid positions 105-108 relative to the wild-type CD3ζ region to the sequence shown in YRHQ (SEQ ID NO:6).

[0097] In another preferred embodiment, the amino acid sequence of the intracellular signal transduction domain is shown in any one of SEQ ID NO:7-19, 31-39 and 47-48.

[0098] In another preferred embodiment, the amino acid sequence of the intracellular signal transduction domain is shown in any one of SEQ ID NO:7-19 and 31-39.

[0099] In another preferred embodiment, the amino acid sequence of the intracellular signal transduction domain is shown in SEQ ID NO:9.

[0100] In another preferred embodiment, the amino acid sequence of the intracellular signal transduction domain is as shown in SEQ ID NO:47 or 48.

[0101] In another preferred embodiment, the CAR further comprises an extracellular domain and a transmembrane domain.

[0102] In another preferred embodiment, the extracellular domain and transmembrane domain are derived from the NKG2D molecule.

[0103] In another preferred embodiment, the CAR further comprises one or more intracellular co-stimulatory domains.

[0104] In another preferred embodiment, one or more intracellular co-stimulatory domains are connected to the N-terminus, C-terminus, or both sides of the intracellular signal transduction structural domain.

[0105] In another preferred embodiment, the CAR has a structure shown in any one of the following formulas Ia-Ie:

[0106] L-EB-H-TM-C-ICS(Ia)

[0107] L-EB-H-TM-CC-ICS(Ib);

[0108] L-EB-H-TM-ICS-C(Ic);

[0109] L-EB-H-TM-C-ICS-C(Id);

[0110] L-EB-H-TM-ICS-CC(Ie);

[0111] In the formula,

[0112] Each "-" independently represents a linking peptide or peptide bond;

[0113] L is either a signal peptide or a non-signal peptide;

[0114] EB is an extracellular binding domain;

[0115] H represents the region with no hinge or no hinge;

[0116] TM is a transmembrane domain;

[0117] C is the domain with no or no intracellular co-stimulation.

[0118] ICS is the intracellular signal transduction domain that contains the immune receptor tyrosine activation domain (ITAM).

[0119] In another preferred embodiment, the extracellular binding domain is an antigen-binding fragment that specifically binds to tumor-associated antigens.

[0120] In another preferred embodiment, the tumor-associated antigen is selected from the group consisting of: BCMA, CD28, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD 51. CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD12 5. CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEACAM6(NC A-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1. Jagged 2. KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / KATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR 7. TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, Claudin18.2, or combinations thereof.

[0121] In another preferred embodiment, the tumor-associated antigen is selected from the group consisting of EDB, Claudin18.2, CD19, or combinations thereof.

[0122] In another preferred embodiment, the extracellular binding domain is an antibody, an antigen-binding fragment (Fab), a single-chain antibody (scFv), a single-domain antibody (nanobody), a natural ligand, a bispecific T-cell binder (BiTE), a dual-affinity retargeting protein (DART), a designed ankyrin repeat protein (DARPins), an affinity multimer (avimer), or a combination thereof.

[0123] In another preferred embodiment, the extracellular binding domain is scFv.

[0124] In another preferred embodiment, the amino acid sequence of the extracellular binding domain is shown in SEQ ID NO:23.

[0125] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or combinations thereof.

[0126] In another preferred embodiment, H is a hinge region derived from CD8.

[0127] In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO:24.

[0128] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0129] In another preferred embodiment, the TM is a transmembrane region derived from CD28.

[0130] In another preferred embodiment, the TM amino acid sequence is shown in SEQ ID NO:25.

[0131] In another preferred embodiment, C is an intracellular co-stimulatory domain of a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0132] In another preferred embodiment, C is an intracellular co-stimulatory domain derived from 4-1BB or CD28, or a combination thereof.

[0133] In another preferred embodiment, the C amino acid sequence is as shown in SEQ ID NO:26.

[0134] In a first aspect of the invention, a chimeric antigen receptor is also provided, the chimeric antigen receptor comprising a substituted intracellular signal transduction domain, the intracellular signal transduction domain having one or more substitutions selected from the group consisting of the wild-type CD3ζ region (SEQ ID NO:20):

[0135] (I) The amino acids at positions 6-9 of ITAM1 (SEQ ID NO:28) are mutated to the sequence shown in YRHQ (SEQ ID NO:6);

[0136] (II) The amino acids 24-27 of ITAM2 (SEQ ID NO:29) are mutated to the sequence shown in YRHQ (SEQ ID NO:6);

[0137] (III) The amino acid sequence at positions 12-15 of ITAM3 (SEQ ID NO:30) is mutated to the sequence shown in YRHQ (SEQ ID NO:6).

[0138] In a second aspect of the invention, a nucleic acid molecule is provided that encodes a chimeric antigen receptor as described in the first aspect of the invention.

[0139] In another preferred embodiment, the nucleic acid molecule is DNA, RNA, or a combination thereof.

[0140] In a third aspect of the invention, a carrier is provided, said carrier containing nucleic acid molecules as described in the second aspect of the invention.

[0141] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0142] In another preferred embodiment, the vector is a lentiviral vector.

[0143] In another preferred embodiment, the vector further includes a selection from the group consisting of: promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), etc.

[0144] In a fourth aspect of the invention, a host cell is provided, said host cell containing a vector or chromosome as described in the third aspect of the invention, or having an exogenous nucleic acid molecule as described in the second aspect of the invention integrated into it, or expressing a chimeric antigen receptor as described in the first aspect of the invention.

[0145] In a fifth aspect of the invention, an engineered immune cell is provided, said immune cell containing a vector as described in the third aspect of the invention or a chromosome in which an exogenous nucleic acid molecule as described in the second aspect of the invention is integrated or which expresses a chimeric antigen receptor as described in the first aspect of the invention.

[0146] In another preferred embodiment, the engineered immune cells are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, or combinations thereof.

[0147] In another preferred embodiment, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

[0148] In another preferred embodiment, the engineered immune cell is a CAR-T cell.

[0149] In a sixth aspect of the present invention, a method for preparing engineered immune cells as described in the fifth aspect of the present invention is provided, comprising the steps of: transducing a nucleic acid molecule as described in the second aspect of the present invention or a vector as described in the third aspect of the present invention into immune cells, thereby obtaining the engineered immune cells.

[0150] In another preferred embodiment, the method further includes a step of testing the function and effectiveness of the obtained engineered immune cells.

[0151] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a nucleic acid molecule as described in the second aspect of the invention, a carrier as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, and / or engineered immune cells as described in the fifth aspect of the invention, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0152] In another preferred embodiment, the formulation is a liquid formulation.

[0153] In another preferred embodiment, the dosage form of the preparation is an injection.

[0154] In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1 × 10⁻⁶. 3- 1×10 8 Cells / ml, preferably 1×10⁻⁶ 4 -1×10 7 Cells / ml

[0155] In an eighth aspect of the invention, there is provided the use of a nucleic acid molecule as described in the second aspect of the invention, a carrier as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, an engineered immune cell as described in the fifth aspect of the invention, and / or a pharmaceutical composition as described in the seventh aspect of the invention, for the preparation of a medicament or formulation for the prevention and / or treatment of a disease.

[0156] In another preferred embodiment, the disease is a tumor.

[0157] In another preferred embodiment, the tumor high expression is selected from the group consisting of tumor-associated antigens: EDB, Claudin18.2, CD19, or combinations thereof.

[0158] In another preferred embodiment, the tumor is selected from the group consisting of solid tumors, hematomas, or combinations thereof.

[0159] In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or combinations thereof.

[0160] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, or combinations thereof.

[0161] In an eighth aspect of the invention, a method for treating a disease is provided, comprising administering to a subject requiring treatment an effective amount of engineered immune cells as described in the fifth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention.

[0162] In another preferred embodiment, the disease is a tumor.

[0163] In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells).

[0164] In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).

[0165] In another preferred embodiment, the method may be used in combination with other treatment methods.

[0166] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0167] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0168] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0169] Figure 1 A schematic diagram of the construction of the anti-EDB-Fn chimeric antigen receptor is shown, including wild-type or mutant EDB-28ζ (EDB-28z) CAR.

[0170] Figure 2 The study demonstrated the in vitro antigen-dependent proliferation and killing effects of different mutants.

[0171] Figure 3 The positive rate of CAR expression in CAR-T cells targeting EDB was shown in each group.

[0172] Figure 4 The in vitro activity of freshly prepared EDB-targeted CAR-T cells was demonstrated.

[0173] Figure 5 The in vitro activity of each group of EDB-targeted CAR-T cells after two rounds of stimulation with U87MG tumor cells at an effector-target ratio of 2:1 within 7 days was demonstrated.

[0174] Figure 6 The study showcased the residual U87MG-luc tumor cells at the bottom of the plate after two rounds of tumor challenge within 7 days using CAR-T cells targeting EDB in each group.

[0175] Figure 7 The results of live cell counts after co-incubation of EDB-targeted CAR-T cells with U87MG tumor cells are presented.

[0176] Figure 8 The changes in the proportion of CD4 / CD8 subtypes in CAR-T cells after co-incubation of EDB target CAR-T cells with U87MG tumor cells were shown.

[0177] Figure 9 The study demonstrated the flow cytometry analysis of CAR-T cell exhaustion, stemness, and apoptosis after co-incubation of EDB-targeted CAR-T cells with U87MG tumor cells.

[0178] Figure 10 The in vivo tumor-suppressing activity of CAR-T cells targeting EDB was demonstrated.

[0179] Figure 11 The in vitro proliferation activity of EDB-28Z and EDB-S71 was demonstrated.

[0180] Figure 12 The in vivo activity comparison between EDB-28Z and EDB-S71 was presented.

[0181] Figure 13 The study demonstrated the detection of CAR-T cells in the peripheral blood of NCG mice 7 days after CAR-T infusion.

[0182] Figure 14 The study demonstrated the concentrations of certain cytokines in the peripheral blood of NCG-bearing mice 7 days after CAR-T infusion.

[0183] Figure 15 The study demonstrated the pathological examination of various tissues and organs and the weight monitoring of NCG mice 7 days after CAR-T infusion.

[0184] Figure 16 The abundance of various CAR-T cells in the tumors of NCG-bearing mice was shown 7 days after CAR-T infusion.

[0185] Figure 17 This study demonstrates the function of CAR-T cells within the tumor of NCG-bearing mice 7 days after CAR-T infusion.

[0186] Figure 18 The mitochondrial activity of EDB-S71 and EDB-28Z after tumor stimulation was demonstrated.

[0187] Figure 19 and 20 This study demonstrates the population and function of CAR-T cells within the tumor of NCG-bearing mice 14 days after CAR-T infusion. The definitions of each cluster and markers are as follows: Figure 19 As shown.

[0188] Figure 21 The CAR positivity rate of EDB-S71 reinfused cells in each group was shown in the dose-effect activity assay.

[0189] Figure 22 The results of dose-effect activity assays and reimplantation challenge of EDB-S71 were presented.

[0190] Figure 23 This study demonstrates the memory response of four NCG mice with CR in the EDB-S71 M and H groups to tumors after contralateral reimplantation.

[0191] Figure 24 The sustained killing capability of Claudin18.2 target at an effective-to-target ratio of 1:1 is demonstrated, and the figure shows the real-time dynamic detection by RTCA.

[0192] Figure 25 This demonstrates the in vivo tumor-suppressing effect and proliferative capacity of Claudin18.2-targeted CAR-T cells.

[0193] Figure 26 The construction of a downstream signaling mutant based on the S71 structure was demonstrated.

[0194] Figure 27 The antigen-dependent activation of signaling pathways in each mutant was demonstrated.

[0195] Figure 28 The study demonstrated the activation and apoptosis of CAR-T cells and tumor cells after multiple rounds of stimulation.

[0196] Figure 29 The experimental procedures for sustained in vivo responses in CAR-T cells of various mutants were demonstrated.

[0197] Figure 30 The study showed the changes in tumor volume in NCG-bearing mice after infusion of various groups of CAR-T cells.

[0198] Figure 31 Analysis of 4D-DIA phosphorylated proteomics was presented.

[0199] Figure 32 The KEGG pathway analysis of 4D-DIA phosphorylation proteomics is presented, and the figure shows the top 20 pathways with the most significant upregulation of S71 compared to 28Z.

[0200] Figure 33 Principal component analysis of 4D-DIA phosphorylated proteomics.

[0201] Figure 34 This is a combined analysis of 4D-DIA phosphorylated proteomics and 4D proteomics. 34-A shows the phosphorylation levels at the S71 mutation site and other related sites; 34-B shows the Vocano plot after combined analysis of phosphorylated proteomics and proteomics and normalization.

[0202] Figure 35 The results of antigen-dependent proliferation assays of EDB CAR-T cells containing S71, SX1, or SX2 sequences and U87MG tumor cells were presented. Detailed Implementation

[0203] Through extensive and in-depth research, the inventors have developed for the first time a chimeric antigen receptor immune cell with a modified CD3ζ region and its application. To improve the efficacy of CAR-T therapy for solid tumors, this invention modifies and reshapes the signaling pathway within the second-generation CAR molecule using site-directed mutagenesis and other techniques. By antigen-dependent synergistic activation of the Jak / STAT signaling pathway, the sustained surveillance and killing capabilities of the second-generation CAR are significantly enhanced, the proliferative potential of immune cells is stimulated, and excessive exhaustion of CAR-T cells is significantly reduced through modification of redundant signals. These findings have been validated on various types of solid tumor targets. Based on these findings, this invention was completed.

[0204] the term

[0205] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0206] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0207] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0208] "Transduction," "transfection," "conversion," or the terms used herein refer to the process of delivering exogenous polynucleotides to host cells, where they are transcribed and translated to produce polypeptide products, including the introduction of exogenous polynucleotides into host cells (e.g., E. coli) using plasmid molecules.

[0209] "Gene expression" or "expression" refers to the process by which genes are transcribed, translated, and modified post-translationally to produce their RNA or protein products.

[0210] "Polynucleotide" refers to a polymer of nucleotides of any length, including deoxyribonucleotides (DNA), ribonucleotides (RNA), their hybrid sequences, and analogs. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. As used herein, the term polynucleotide refers to interchangeable single-stranded and double-stranded molecules. Unless otherwise stated, polynucleotides in any embodiment described herein include double-stranded forms and two complementary single strands that constitute a double-stranded form, as known or predictable.

[0211] Substitution of conserved amino acids is known in the art. In some embodiments, potential substituted amino acids are in one or more of the following group: glycine, alanine; and valine, isoleucine, leucine, and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine, lysine, arginine, and histidine; and / or phenylalanine, tryptophan, and tyrosine; methionine and cysteine. Furthermore, the present invention also provides non-conserved amino acid substitutions that allow for substitution of amino acids from different groups.

[0212] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0213] The term “giving” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0214] This invention relates to the modification of chimeric antigen receptor (CAR) and intracellular signal transduction domain.

[0215] A chimeric antigen receptor (CAR) consists of an extracellular antigen recognition domain, a transmembrane domain, and an intracellular domain. Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.

[0216] The extracellular domain includes a target-specific binding element (extracellular binding domain). This extracellular domain can be an antibody, antigen-binding fragment (Fab), single-chain antibody (scFv), or single-domain antibody (nanobody) based on antigen-antibody specific binding, or a natural sequence or derivative thereof based on ligand-receptor specific binding. The CD3ζ region optimized in this invention is versatile and can be used for any antigen that can be targeted by CAR immune cells. In a preferred embodiment, the target antigen of the CAR of this invention includes, but is not limited to: EDB, CLDN18.2, CD19, or combinations thereof.

[0217] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker (or hinge region)" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2-100 amino acids, and most preferably 3-50 amino acids.

[0218] The CAR of this invention, when expressed in T cells, is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, resulting in a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with one or more intracellular domains derived from the co-stimulatory molecule and the ζ chain.

[0219] Intracellular domains include the costimulatory signaling region and the ζ-chain portion. The costimulatory signaling region refers to a portion of the intracellular domain that includes costimulatory molecules. Costimulatory molecules are cell surface molecules required for an effective lymphocyte response to antigens, not antigen receptors or their ligands.

[0220] The intracellular domains of the CAR of this invention include the CD3ζ signal transduction domain, referred to as the CD3ζ region or CD3z region. The three immune receptor tyrosine activation domains of the CD3ζ region are named ITAM1, ITAM2, and ITAM3 sequentially from the proximal to the distal membrane. This invention achieves synergistic activation of multiple downstream signals by replacing a portion of the ITAM fragment with a cytokine receptor fragment, thereby obtaining a higher response rate in solid tumors than traditional second-generation CARs. As used herein, the term "replacement fragment" refers to the cytokine receptor fragment or its derivative used to replace the ITAM fragment, which can be derived from any cytokine receptor. In one embodiment, the cytokine receptor is an interleukin receptor.

[0221] In one embodiment, the substituted fragment is derived from interleukin-2 receptor β (IL2RB). In one embodiment, the substituted fragment is a cytokine receptor fragment containing tyrosine (Y) and its neighboring amino acids. In another embodiment, the substituted fragment is a derived sequence of a cytokine receptor fragment containing tyrosine (Y) and its neighboring amino acids. In yet another embodiment, the tyrosine (Y) in the substituted fragment is point-mutated to phenylalanine (F). For example, the substituted fragment may have the amino acid sequence shown in any one of SEQ ID NO: 1-4. The tyrosine (Y) and its neighboring amino acids may be tyrosine (Y) and its N-terminal 3-5 amino acids and C-terminal 3-5 amino acids.

[0222] In one embodiment, the substituted fragment is derived from the interleukin-21 receptor (IL21R). For example, the substituted fragment may have the amino acid sequence shown in SEQ ID NO:5.

[0223] In one embodiment, the substitution fragment is used to replace tyrosine (Y) and its neighboring amino acids in ITAM3. In one embodiment, the substitution fragment is used to replace tyrosine (Y) and its neighboring amino acids in ITAM2 and ITAM3. In one embodiment, the substitution fragment is used to replace tyrosine (Y) and its neighboring amino acids in ITAM1, ITAM2, and ITAM3.

[0224] Each ITAM naturally contains two tyrosine residues. In one embodiment, a substitution fragment may replace one or two tyrosine residues and their neighboring amino acids in ITAM. In one embodiment, a substitution fragment from IL2RB or IL21R or its YF variant (e.g., SEQ ID NO: 1, 2, 3, 4, or 5) may replace one or more of the following in ITAM3: the first tyrosine residue (position 12) and its neighboring amino acid; the second tyrosine residue (position 23) and its neighboring amino acid; the first tyrosine residue (position 12) and its neighboring amino acid; and the second tyrosine residue (position 24) and its neighboring amino acid.

[0225] Each ITM can contain substitution fragments from the same or different sources. For example, in ITM3, the first tyrosine (position 12) and its neighboring amino acid can be replaced by a substitution fragment from IL2RB, while the second tyrosine (position 23) and its neighboring amino acid can be replaced by a substitution fragment from IL21R. Alternatively, the first and second tyrosines and their neighboring amino acids in ITM3 can both be replaced by substitution fragments from IL2RB.

[0226] In one embodiment, the position of the tyrosine residue in the ITAM sequence after substitution can be the same as the position of the tyrosine residue in the wild-type ITAM, or shifted by 1 or 2 amino acids. After substitution, the first tyrosine residue in ITAM3 can be located at position 12 (the same as in the wild type), or it can be shifted towards the N-terminus or C-terminus (e.g., at positions 10, 11, 13, or 14). Exemplary sequences with tyrosine shifts are shown in SEQ ID NO: 11-13, but are not limited thereto. This shift does not attenuate or substantially does not attenuate the signal transduction function of CD3ζ.

[0227] In one embodiment, the CD3ζ region of the CAR of the present invention has the following mutation relative to the wild-type CD3ζ region as shown in SEQ ID NO:20:

[0228] (I) Amino acids at positions 86-96 are mutated to the sequence shown in SEQ ID NO: 1 or 3; and

[0229] (II) The amino acids at positions 55-65 are mutated to the sequence shown in SEQ ID NO:2 or 4.

[0230] In one embodiment, the CD3ζ region of the CAR of the present invention is identical to the wild-type CD3ζ region shown in SEQ ID NO:20, except for the mutations (I) and (II) described above. In another embodiment, the CD3ζ region of the CAR of the present invention is further mutated to amino acids 105-108 as shown in YRHQ (SEQ ID NO:6) relative to the wild-type CD3ζ region.

[0231] The present invention also provides a CAR having a YRHQ-substituted signal transduction domain. In some embodiments, YRHQ may substitute one or more amino acid segments of ITAM1, ITAM2, and ITAM3 in the CD3ζ region. For example, it may substitute amino acids 6-9 of ITAM1 (SEQ ID NO:28), amino acids 24-27 of ITAM2 (SEQ ID NO:29), and / or amino acids 12-15 of ITAM3 (SEQ ID NO:30).

[0232] In one embodiment, the CD3ζ region of the CAR of the present invention has the following mutation relative to the wild-type CD3ζ region as shown in SEQ ID NO:20:

[0233] (1) The amino acids at positions 55-65 are replaced by the sequence shown in SEQ ID NO:2;

[0234] (2) The amino acids at positions 67-77 are replaced by the sequence shown in SEQ ID NO:46;

[0235] (3) The amino acids at positions 86-96 are replaced by the sequence shown in SEQ ID NO:1;

[0236] (4) The amino acids at positions 97-107 are replaced by the sequence shown in SEQ ID NO:5.

[0237] In one embodiment, the CD3ζ region of the CAR of the present invention has the following mutation relative to the wild-type CD3ζ region as shown in SEQ ID NO:20:

[0238] (1) The amino acids at positions 55-65 are replaced by the sequence shown in SEQ ID NO:2;

[0239] (2) The amino acids at positions 67-77 are replaced by the sequence shown in SEQ ID NO:5;

[0240] (3) The amino acids at positions 86-96 are replaced by the sequence shown in SEQ ID NO:1;

[0241] (4) The amino acids at positions 97-107 are replaced by the sequence shown in SEQ ID NO:46.

[0242] The present invention also provides CARs comprising other CD3ζ-based signal transduction domain variants. In some embodiments, ITAM1, ITAM2, or ITAM3 of the signal transduction domain variant is deleted. In some embodiments, at least one tyrosine (Y) and its neighboring amino acid are deleted from ITAM1, ITAM2, or ITAM3 of the signal transduction domain variant. In some embodiments, at least one tyrosine (Y) and its neighboring amino acid fragment in the signal transduction domain variant are shifted forward or backward by 1, 2, or 3 amino acids. In some embodiments, the shifted or backward fragment may be further replaced by the substitution fragments described above. In some embodiments, the substitution fragments described above are inserted into a non-ITAM region, for example, between the transmembrane region and ITAM1. Other exemplary signal transduction domain variants are shown in SEQ ID NO:34-39.

[0243] In some embodiments, a substituted fragment of the interleukin-21 receptor is inserted into the ITAM region. Other exemplary variants of the signal transduction domain are shown in SEQ ID NO:40-45.

[0244] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0245] Chimeric antigen receptor immune cells (CAR-immune cells)

[0246] In this invention, a chimeric antigen receptor immune cell is provided, which comprises the chimeric antigen receptor combination of this invention.

[0247] The chimeric antigen receptor immune cells of the present invention can be CAR-T cells, CAR-NK cells, or CAR-macrophages. Preferably, the chimeric antigen receptor immune cells of the present invention are CAR-T cells.

[0248] As used herein, the terms "CAR-T cell", "CAR-T", and "CAR-T cell of the present invention" all refer to the CAR-T cell described in the fifth aspect of the present invention.

[0249] CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action of CAR-T cells is not limited by MHC; (2) Given that many tumor cells express the same tumor markers, once the CAR gene targeting a certain tumor marker is constructed, it can be widely used; (3) CAR can utilize both tumor protein markers and glycolipid non-protein markers, thus expanding the target range of tumor markers; (4) Using the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0250] As used herein, the terms "CAR-NK cell," "CAR-NK," and "CAR-NK cell of the present invention" all refer to the CAR-NK cell described in the fifth aspect of the present invention. The CAR-NK cell of the present invention can be used to treat tumors.

[0251] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infections and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

[0252] Compared with CAR-T cells, CAR-NK cells have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzymes, without killing normal cells in the body; (2) they release very little cytokine, thus reducing the risk of cytokine storm; and (3) they are very easy to expand in vitro and develop into "ready-made" products. Otherwise, they are similar to CAR-T cell therapy.

[0253] carrier

[0254] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0255] This invention also provides vectors comprising the nucleic acid molecules of this invention. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0256] In short, the expression cassette or nucleic acid sequence of this invention is typically operatively linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0257] The expression constructs of the present invention can also be used with standard gene delivery protocols for nucleic acid immunotherapy and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0258] This nucleic acid can be cloned into many types of vectors. For example, it can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0259] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0260] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0261] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located in a 30–110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible to maintain promoter function when an element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to initiate transcription.

[0262] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0263] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain either or both of an optional marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0264] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0265] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0266] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0267] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0268] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0269] In a preferred embodiment of the present invention, the carrier is a lentiviral carrier.

[0270] preparation

[0271] This invention provides a formulation comprising the chimeric antigen receptor CAR as described in the first aspect of this invention, the nucleic acid molecule as described in the second aspect of this invention, the carrier as described in the third aspect of this invention, or the host cell as described in the fourth aspect of this invention, or the engineered immune cell as described in the fifth aspect of this invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, more optimal 1×10 4 -1×10 7 Cells / ml

[0272] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0273] Therapeutic applications

[0274] This invention includes therapeutic applications using cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of this invention. The transduced T cells can target tumor cell markers (such as EDB), synergistically activating T cells and evoking an immune response, thereby significantly enhancing their efficiency in killing tumor cells. T cells containing the modified CD3ζ region of this invention can proliferate rapidly and, upon stimulation by tumor cells, exhibit low exhaustion levels while being activated, thus increasing T cell levels and enhancing anti-tumor effects.

[0275] Therefore, the present invention also provides a method for stimulating a T-cell-mediated immune response against a target cell population or tissue of a mammal, comprising the step of administering the CAR-cells of the present invention to a mammal.

[0276] In one embodiment, the present invention includes a type of cell therapy in which the patient's own T cells (or those from a heterologous donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This method results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0277] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and sustain for an extended period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, anti-EDB CAR-T cells elicit a specific immune response against anti-EDB cells.

[0278] Although the data disclosed herein specifically discloses a combination of scFv, hinge and transmembrane regions, and CD28 co-stimulatory domains, and CD3ζ signaling domains with specific modifications, the invention should be interpreted as including any number of variations in each of the components of the construct.

[0279] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Cancers include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) and solid tumors. Types of cancers treatable with the CAR of this invention include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0280] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0281] The CAR-modified T cells of the present invention can also be used as a type of vaccine for in vitro immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0282] For in vitro immunization, at least one of the following occurs in vitro before the cells are administered into a mammal: i) cell expansion, ii) introduction of nucleic acid encoding CAR into the cells, and / or iii) cryopreservation of the cells.

[0283] In vitro procedures are well known in the art and are discussed more fully below. Simply put, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0284] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.

[0285] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need of the treatment.

[0286] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-7, IL-15, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may comprise target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0287] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined by clinical trials.

[0288] When referring to "effective dose," "immunologically effective dose," "antitumor effective dose," "tumor-suppressive effective dose," or "therapeutic dose," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that: pharmaceutical compositions comprising T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0289] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0290] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant therapeutic modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for specific tumor patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0291] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 6 One to 1×10 10 The CAR-T cells of the present invention are administered to a patient, for example, via intravenous infusion.

[0292] The main advantages of this invention include:

[0293] 1) It was discovered that the CD3z region of CAR intracellular signaling can be modularly modified, and multiple signals can be synergistically activated through fusion mutations of the CAR receptor and cytokine receptor. The mutant CAR of this invention has a higher response rate in solid tumors compared to traditional second-generation CARs.

[0294] 2) Compared with traditional second-generation CARs, the mutant CAR of the present invention has higher antigen-dependent proliferation capacity, higher sustained killing effect, and higher safety.

[0295] 3) Intracellular signal remodeling improves the exhaustion caused by self-activation in traditional second-generation CARs. The mutant CAR of this invention has lower exhaustion and apoptosis levels and improved cell stemness.

[0296] 4) The modified CD3z region of this invention has been validated in multi-target CAR and has universality.

[0297] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0298] Example 1: Construction of Chimeric Antigen Receptors

[0299] like Figure 1 As shown, all constructs contain the scfv of L19, the hinge region of CD8α, the transmembrane region of CD28, the intracellular region of CD28, and the intracellular region of wild-type or mutant CD3z. Specifically, 28z contains the intracellular region of wild-type CD3z. In the mutant CD3z structure of S69, Y142 (based on the amino acid sequence of the complete CD3z molecule) and adjacent amino acids of wild-type CD3z are replaced by Y510 and adjacent amino acids of IL2RB. The mutant CD3z structure of S70 is based on the S69 mutation, with the addition of a YXXQ motif after Y153 of CD3z. Based on the mutant CD3z of S69 and S70, S71 and S72 replace Y111 and adjacent amino acids of wild-type CD3z with Y392 and adjacent amino acids of IL2RB. The structure of S11 is derived from a sequence from Kagoya, Japan, and serves as a control group.

[0300] Example 2: CAR-T cell preparation and proliferation experiment

[0301] T cells in each group were activated with CD3 / CD28 magnetic beads for 36 hours, followed by lentiviral transduction. Cell sorting was performed using anti-Fab magnetic beads, and the CAR positivity rate was then detected. Figure 3 ).

[0302] After all cytokines were removed and the cells in each group were starved for 12 hours, live cell labeling was performed using CFSE fluorescent dye. The cells were then co-incubated with tumor cells (U87MG-ffluc) at an effector-to-target ratio of 2:1 for 3 days, followed by flow cytometry analysis. The results were compared with unincubated cells, and antigen-dependent cell proliferation was calculated. Figure 2 -A). Imaging of the remaining tumor cells at the bottom of the plate after 3 days of incubation ( Figure 2-B). S11 is the CAR structure from Kagoya, Japan, and EFL28 is the inventor's previous research result; both are control groups. After initial in vitro activity screening, S69-S72 of the EDB target exhibited strong antigen-dependent proliferation and sustained tumor killing, and were selected as the subjects for subsequent experimental research.

[0303] Example 3: In vitro activity of EDB-targeted CAR-T cells

[0304] CAR-T cells from each group were co-incubated with U87MG at an effector-to-target ratio of 2:1. After 24 hours, the secretion of TNF-α (4-A) and IFN-γ (4-B) in the cell culture supernatant was detected by ELISA. Figure 4 AB). U87MG tumor cells overexpressing luc were constructed. CAR-T cells from different groups were co-incubated with U87MG-luc tumor cells at different effector-to-target ratios. After 24 hours, the remaining tumor cells at the bottom of the plate were detected and normalized, and the in vitro killing efficiency was calculated. Figure 4 C).

[0305] The results showed that S71 and 28Z had similar in vitro killing and factor release capabilities.

[0306] Following tumor challenge, CAR-T cells from each group were co-incubated with U87MG at an effector-to-target ratio of 2:1. After 24 hours, TNF-α levels in the cell culture supernatant were detected using ELISA. Figure 5 A) and IFN-γ Figure 5 B) secretion. U87MG tumor cells overexpressing luc were constructed. CAR-T cells from different tumor-challenged groups were co-incubated with U87MG-luc tumor cells at different effector-to-target ratios. After 24 hours, the remaining tumor cells at the bottom of the plate were detected and normalized, and the in vitro killing efficiency was calculated. Figure 5 C).

[0307] The results showed that, after tumor stimulation challenge, S71 exhibited stronger cell lysis and IFN-γ release compared to 28z. Figure 6 As shown, S71 has a more durable tumor clearance capability.

[0308] Example 4: Detection of antigen-dependent proliferation of CAR-T cells targeting EDB

[0309] After removing all cytokines, the EDB target CAR-T cells in each group were co-incubated with U87MG tumor cells at the same cell concentration and an effector-target ratio of 2:1 for 5 days. The viable cells were counted and the expansion fold was calculated using trypan blue staining.

[0310] like Figure 7 As shown, S71 exhibits good antigen-dependent proliferation ability. Figure 8The study showed changes in the proportion of CD4 / CD8 subtypes in CAR-T cells, suggesting that S71 CD8+ cells had a stronger proliferative capacity compared to other groups. Figure 9 (AC) shows the flow cytometry detection of CAR-T cell exhaustion, stemness, and apoptosis.

[0311] Example 5: In vivo tumor-suppressive activity of EDB-targeted CAR-T cells

[0312] NCG mice were subcutaneously injected with 1E6 WT-U87MG tumor cells. Seven days later, tumor volume was measured and mice were randomly assigned to groups. The average tumor volume in each group was approximately 75 mm. 3 After grouping, NCG mice underwent tail vein infusion of 1.5E6 CAR+ cells / mouse from each group, or untreated T cells (MOCK). Tumor volume and body weight were measured and monitored every 3-4 days after infusion.

[0313] like Figure 10 As shown, group S71 exhibits stronger tumor suppressor activity and was selected as the subsequent mutation structure.

[0314] Example 6: Comparison of in vitro proliferation activities of EDB-28Z and EDB-S71

[0315] After removing all cytokines, EDB-28Z and EDB-S71 CAR-T cells were placed in 1% FBS medium for 12 hours before CFSE live cell labeling. The labeled CAR-T cells were then co-incubated with U87MG tumor cells at an effector-to-target ratio of 2:1 for 5 days. Flow cytometry was used to detect changes in CFSE fluorescence intensity in CD4 / CD8 subset CAR-T cells.

[0316] like Figure 11 As shown, EDB-S71 has a stronger antigen-dependent proliferation ability compared to EDB-28Z.

[0317] Example 7: Comparison of in vivo activities of EDB-28Z and EDB-S71

[0318] NCG mice were subcutaneously injected with 1E6 doses of WT-U87MG on day -8. Tumor volume was measured on day -1 and mice were randomly assigned to groups. The mean tumor volume per group was approximately 75 mm. 3 After grouping, NCG mice underwent tail vein infusion of EDB-28Z, EDB-S71, or untreated T cells (MOCK) 1.5E6 CAR+ cells / mouse on day 0. Tumor volume changes were as follows: Figure 12 As shown in the figure. The results indicate that EDB-S71 has a stronger ability to inhibit tumor growth than EDB-28Z.

[0319] Example 8: Detection of in vivo antitumor activity of CAR-T cells

[0320] NCG mice were subcutaneously injected with 1,4E6 doses of WT-U87MG on day -8. Tumor volume was measured on day -1 and mice were randomly assigned to groups. The mean tumor volume for each group was approximately 100 mm. 3 After grouping, NCG mice received tail vein infusion of EDB-28Z, EDB-S71, or untreated T cells (MOCK) 1.5E7 CAR+ cells / mouse on day 0. All NCG mice were sacrificed on day 7 for subsequent studies. Peripheral blood was collected after sacrifice on day 7. After removing erythrocytes, the percentage of CD3+CD8+ cells (13-A) and the PD1+ / CD3+ ratio (13-B) were detected and statistically analyzed by flow cytometry. Serum was separated, and various cytokines were detected using a Luminex assay kit.

[0321] like Figure 13 As shown in (AB), EDB-S71 exhibits higher cell abundance and lower depletion in peripheral blood. Figure 14 As shown, EDB-S71 exhibits stronger release of effector and chemokine factors and lower release of CRS-related inflammatory and inhibitory factors compared to EDB-28Z.

[0322] Mice were sacrificed on day 7, and their tissues and organs were separated for H&E staining. Figure 15 A) No obvious organic lesions or necrosis were found in the pathological examination of CAR-T in each group. Significant changes in body weight were observed in mice after CAR-T infusion in each group, with EDB-28Z showing a more severe weight loss. Figure 15 B).

[0323] Mice were sacrificed on day 7, and tumor tissue was isolated for CD3 staining immunohistochemical analysis. Fields of view were randomly selected from the edges and center of each tumor, and IHC-positive cells were analyzed in each field of view using ImageJ. Results are as follows: Figure 16 As shown, EDB-S71 has a higher distribution and infiltration rate within the tumor compared to EDB-28Z.

[0324] Mice were sacrificed on day 7, and tumor tissue was isolated for DAPI+CD8+GranzymB+PD1+CD31+ multicolor immunofluorescence. Figure 17 A); Halo panoramic scan analysis showed that the total number of CD8+ cells of EDB-S71 within the tumor ( Figure 17 B) and CD8+GZMB+PD1-( Figure 17 C) had a higher proportion of highly active, low-exhaustion cell subsets; while EDB-S71 had a lower proportion of PD1+ positivity in CD8+ cells. Figure 17 D).

[0325] CAR-T cells from each group (CAR+CD8+) were co-incubated with tumor cells at an effector-to-target ratio of 2:1. After 18 hours, cells from each group were collected for Seahorse assay, and their OCR data were plotted and statistically analyzed. The mitochondrial activity of EDB-S71 and EDB-28Z after tumor stimulation was as follows: Figure 18 As shown in (AB).

[0326] Example 9: Study on the population and function of CAR-T cells in vivo after reinfusion

[0327] On day -8, 1E6 units of U87MG tumor cells were subcutaneously injected. On day -1, tumor volume was measured and patients were randomly assigned to groups. The average volume of each group was approximately 70 mm². 3 On day 0, mice were infused with either EDB-28z or EDB-S71 3E6CAR-positive cells per mouse. On day 14 post-infusion, mice were sacrificed, and tumor tissue from each mouse was extracted for CyTOF analysis. The definitions and markers of each cluster are shown in the figure.

[0328] Figure 20 A-20B shows the percentage of each cluster in the EDB-28Z and EDB-S71 groups, respectively; Figure 20 C shows the distribution of T cell subsets; Figure 20 D shows the expression of cluster characteristic markers with significant differences. The results show that EDB-S71, which has been infiltrating tumors for 14 days, has stronger sustained activation and killing ability, lower exhaustion and more complete expression of co-stimulatory molecules than EDB-28Z.

[0329] Example 10: Dose-effect activity assay of EDB-S71

[0330] On day -7, 2E6 doses of WT-U87MG tumor cells were subcutaneously injected. On day -1, tumor volume was measured and patients were randomly assigned to groups, with each group having a tumor volume of approximately 135 mmHg. 3 On day 0, 15E6 EDB-S71 cells were infused per mouse, and the CAR positivity rates in each group were 0%, 30%, 60%, and 90%, respectively. On day 53, tumors were reimplanted on the contralateral side of all CR mice.

[0331] Figure 21 The CAR positivity rate of EDB-S71 reinfused cells in each group was shown. Figure 22 A shows the tumor volume change curves over time for each group; Figure 22 B shows the tumor volume changes for each mouse in each group (groups are indicated by the color of the curve, compared to...). Figure 21 (Corresponding to the middle group). In the EDB-S71 M and H groups, the tumors of 2 mice in each group completely regressed.

[0332] Figure 23 This study demonstrates the tumor memory response in four NCG mice with complete renal function (CR) in the EDB-S71 M and H groups after contralateral tumor reimplantation. The results show that the EDB-S71 M and H groups exhibited a good memory response.

[0333] Example 11: Detection of in vitro tumor-killing activity of Claudin 18.2 CAR-T

[0334] Freshly frozen human PBMCs were used to select negative human CD3+ T cells using magnetic bead cocktails. After activation with CD3 / CD28 magnetic beads for 24 hours, the activated T cells were transduced with Claudin18.2 CAR using lentivirus at an MOI of 10. On the fifth day after transduction, CAR-T cells were sorted for CAR+ using magnetic beads to achieve a CAR positivity rate of over 90%. The sorted CAR-T cells were cultured until day 14 for subsequent experiments.

[0335] CLDN18.2-U87MG tumor cells were plated into xCELLigence 16-well plates on day 0. After the tumor cells adhered stably for 24 hours, CAR-T cells from each group, which had been starved for 12 hours with serum and all cytokines removed, were co-incubated with the tumor cells at an effector-to-target ratio of 1:1. The detector detected the remaining adherent tumor cells at the bottom of the plate every 15 minutes.

[0336] Figure 24 The sustained killing capability of Claudin18.2 target at an effective-to-target ratio of 1:1 is demonstrated, and the figure shows the real-time dynamic detection by RTCA.

[0337] Example 12: In vivo tumor suppression and proliferation capacity of Claudin18.2 target CAR-T cells

[0338] The experimental procedure is as follows Figure 25 As shown in Figure A. Mice were subcutaneously injected with 1E6 RFP-CLDN-U87MG tumor cells on day -13. Tumor measurements and randomization were performed on day -1. The tumor volume in each group was approximately 166 mm². 3 On day 0, 5E6 CAR-positive cells were infused per cell, with CAR-T cells being CLDN-28Z-P2A-luc and CLDN-S71-P2A-luc.

[0339] Figure 25 BC demonstrates tumor growth and quantification. Figure 25 DE demonstrated the quantitative distribution and proliferation level of CAR-T cells co-expressing luciferase in vivo. The results showed that CLDN-S71 exhibited better anti-tumor effects and proliferation capacity than CLDN-28Z.

[0340] Example 13 Construction and Activity Detection of Downstream Signaling Mutants Based on S71 Structure

[0341] The structures of each mutant are as follows Figure 26 As shown. S71-FY is the mutant in S71 where the first Tyr mutation in the ITAM2 mutation structure is Phe, and ITAM3 remains unchanged; S71-YF is the mutant in S71 where ITAM2 remains unchanged, and the first Tyr mutation in the ITAM3 mutation structure is Phe; S71-FF is the mutant in S71 where the first Tyr mutation in both the ITAM2 and ITAM3 mutation structures is Phe.

[0342] Flow cytometry was used to detect the signal activation intensity of p-STAT5, p-STAT3, and p-ERK after co-incubation of each mutant with CLDN-U87MG tumor cells for 0 min, 30 min, and 60 min. Each mutant showed varying degrees of antigen-dependent signaling pathway activation. Figure 27 ).

[0343] Multiple rounds of tumor cell stimulation were performed on each mutant CAR-T cell group (please describe the experimental methods in detail, including the type of tumor cells, the ratio or number of cells co-incubated, etc.) to detect cell activation and apoptosis. CLDN18.2-U87MG tumor cells were plated into 24-well plates on day 0. After 24 hours of stable adhesion, CAR-T cells from each group, starved for 12 hours after removing serum and all cytokines, were co-incubated with tumor cells at a 1:1 effector-to-target ratio. Every 48 hours, an equal number of tumor cells at a 1:1 effector-to-target ratio were added, for a total of three additions. After the third addition, 48 hours later, the remaining CAR-T cells were harvested for flow cytometry analysis to detect the expression of various markers on the cell surface. The experimental procedure is as follows: Figure 28 As shown in Figure A. The expression of CD69 on the surface of CAR-T cells in each group was detected after three rounds of stimulation. Figure 28 B) and apoptosis Figure 28 C).

[0344] Example 14: Detection of sustained in vivo response of CAR-T cells from various mutants

[0345] The experimental procedure is as follows Figure 29 As shown. Figure 30 The study demonstrated the changes in tumor volume in NCG-bearing mice after infusion of CAR-T cells from various groups. Through repeated tumor implantation in mice with complete tumor regression, the study observed different sustained responses in each group.

[0346] Example 15: Phosphorylated proteomics analysis of CAR-T cells

[0347] CLDN-28Z and CLDN-S71 were prepared from T cells from three different donors. After co-incubating with tumor cells for 30 min, the CAR-T cells were lysed and analyzed by mass spectrometry. Figure 31 The results of 4D-DIA phosphorylated proteomics analysis show the number of phosphorylated proteins and the proportion of phosphorylation types that differ between S71 and 28Z. Figure 32 The study showed the top 20 pathways with the highest upregulation of S71 compared to 28Z. Figure 33 Principal component analysis of 4D-DIA phosphorylated proteomics. Figure 34 This study combined 4D-DIA phosphorylation proteomics with 4D proteomics analysis.

[0348] Example 16: Preparation and Functional Detection of CAR-T Cells Containing SX1 or SX2 Sequences

[0349] Based on EDB-S71 CAR-T cells, the S71 sequence was replaced with SX1 (SEQ ID NO:47) or SX2 (SEQ ID NO:48), and EDB-SX1 and EDB-SX2 CAR-T cells were prepared using the same method as in Example 2. The blank control group was named Tmock.

[0350] The antigen-dependent proliferation capacity of EDB-targeted CAR-T cells was tested. The experimental method was as follows: Tmock group, EDB-S71, EDB-SX1 or EDB-SX2 CAR-T cell groups were stained with CFSE dye and then incubated with U87MG tumor cells at the same cell concentration in a cytokine-free medium for 3 days at an effector-target ratio of 2:1. The fluorescence intensity of immune cells, which reflects the proliferation of immune cells, was detected by flow cytometry at 0h, 24h, 48h, 72h and 120h.

[0351] The results are as follows Figure 25 As shown in Table 1. As shown in the figure, the results of Tmock are as follows: Figure 25 As shown in A, the result of S71 is as follows: Figure 25 As shown in B, the results of SX1 and SX2 are respectively as follows: Figure 25 As shown in C and D, SX1 and SX2 exhibited long-lasting antigen-dependent proliferation ability under co-incubation conditions of 72h and 120h, with SX2 showing particularly excellent antigen-dependent proliferation.

[0352] Table 1 Results of antigen-dependent proliferation detection

[0353]

[0354] The sequence of the present invention:

[0355] IL2RB replace-1: LNTDA Y LSLQE(SEQ ID NO:1)

[0356] IL2RB replace-2: GEDDA Y CTFPS (SEQ ID NO:2)

[0357] IL2RB replace-3: LNTDA F LSLQE (SEQ ID NO:3)

[0358] IL2RB replace-4: GEDDA F CTFPS (SEQ ID NO:4)

[0359] IL21R replace-1: GPPRS Y LRQWV (SEQ ID NO:5)

[0360] YRHQ(SEQ ID NO:6)

[0361] S69 CD3ζICD (underlined part is replaced by IL2RB)

[0362] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK LNTDAYLSLQE ATKDTYDALHMQALPPR(SEQ ID NO:7)

[0363] S70 SCD3ζICD (Underlined part is IL2RB substitution, italicized part is YXXQ mutation)

[0364]

[0365] S71 CD3ζICD (underlined part represents IL2RB replacement)

[0366] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN GEDDAYCTFPS DKMAEAYSEIGMKGERRRGK LNTDAYLSLQE ATKDTYDALHMQALPPR(SEQ ID NO:9)

[0367] S72 CD3ζICD (underlined part is IL2RB substitution, italicized part is YXXQ mutation)

[0368]

[0369] S71-V1 CD3ζICD (underlined part represents IL2RB replacement)

[0370] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQ GEDDAYCTFPS ELQKDKMAEAYSEIGMKGERRRGK LNTDAYLSLQE ATKDTYDALHMQALPPR(SEQ ID NO:11)

[0371] S71-V2 CD3z ICD (underlined part is the IL2RB replaced part)

[0372] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN GEDDAYCTFPS DKMAEAYSEIGMKGER LNTDAYLSLQE GLSTATKDTYDALHMQALPPR(SEQ ID NO:12)

[0373] S71-V3 CD3z ICD (underlined part is replaced by IL2RB)

[0374] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQ GEDDAYCTFPS ELQKDKMAEAYSEIGMKGER LNTDAYLSLQE GLSTATKDTYDALHMQALPPR(SEQ ID NO:13)

[0375] S131 CD3z ICD (underlined part is IL2RB substituted part, bold underlined part is IL21R substituted part)

[0376]

[0377] S132 CD3z ICD (underlined part is IL2RB substituted part, bold underlined part is IL21R substituted part)

[0378]

[0379] S133 CD3z ICD (underlined part is IL2RB substituted part, bold underlined part is IL21R substituted part)

[0380]

[0381] S134 CD3z ICD (underlined part is IL2RB substituted part, bold underlined part is IL21R substituted part)

[0382]

[0383] S135 CD3z ICD (underlined part is IL2RB substituted part, bold underlined part is IL21R substituted part)

[0384]

[0385] S136 CD3z ICD (Bold underlined part represents IL21R replacement)

[0386]

[0387] CD3ζICD (Wild Type)

[0388] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLST ATKDTYDALHMQALPPR(SEQ ID NO:20)

[0389] CD3ζICD (ITAM 1 truncated variant)

[0390] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK(SEQ ID NO:21)

[0391] IL2 SP

[0392] MYRMQLLSCIALSLALVTNS(SEQ ID NO:22)

[0393] L19 scfv

[0394] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK(SEQ ID NO:23)

[0395] CD8a hinge

[0396] AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:24)

[0397] CD28 TM

[0398] FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:25)

[0399] CD28 ICD

[0400] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:26)

[0401] IL2RB ICD

[0402] LTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV(SEQ ID NO:27)

[0403] CD3ζICD ITAM1 domain

[0404] ADAPAYQQGQNQLYNELNLGRREEYDVLDKR(SEQ ID NO:28)

[0405] CD3ζICD ITAM2 domain

[0406] PQRRKNPQEGLYNELQKDKMAEAYSEIGM(SEQ ID NO:29)

[0407] CD3ζICD ITAM3 domain

[0408] ERRRGKGHDGLYQGLSTATKDTYDALHMQ(SEQ ID NO:30)

[0409] S71FY CD3ζICD (underlined part represents IL2RB substitution, italics indicate Tyr-Phe mutation)

[0410]

[0411] S71YF CD3ζICD (underlined part represents IL2RB substitution, italics indicate Tyr-Phe mutation)

[0412]

[0413] S71FF CD3ζICD (underlined part represents IL2RB substitution, italics indicate Tyr-Phe mutation)

[0414]

[0415] S109 CD3ζICD

[0416] RVKERRRGKGPPRSYLRQWVQALPPRFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNGEDDAYCTFPSDKMAEAYSEIGMKG(SEQ ID NO:34)

[0417] S110 CD3ζICD

[0418] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRLNTDAYLSLQERKNDKMAEAYSEIGMKGERRRGKGPPRSYLRQWVQALPPR(SEQ ID NO:35)

[0419] S111 CD3ζICD

[0420] RVKFSRSADAPAYQQGPPRSYLRQWVGQGRREEYDVLDKRRGRDPEMGGKPQRRKNGEDDAYCTFPSDKMAEAYSEIGMKGERRRGKLNTDAYLSLQEQALPPR(SEQ ID NO:36)

[0421] S113 CD3ζICD

[0422] RVKFSRSAQLGPPRSYLRQWVYNELNGEDDAYCTFPSLGKRRGRDPEMGGKPQRRKNDKMAEAYSEIGMKGERRRGKLNTDAYLSLQEQALPPR(SEQID NO:37)

[0423] S114 CD3ζICD

[0424] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNDKMAEAYSEIGMKGERRRGKGPPRSYLRQWVQALPPR(SEQ IDNO:38)

[0425] S115 CD3ζICD

[0426] RVKFSRSERRRGKGPPRSYLRQWVQRGRDPEMGGKPQRRKNGEDDAYCTFPSDKMAEAYSEIGMKGALPPR(SEQ ID NO:39)

[0427] S131 CD3ζICD

[0428] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNGEDDAYCTFPSDKMAEAYSEIGMKGERRRGKLNTDAYLSLQEGPPRSYLRQWVQALPPR(SEQ ID NO:40)

[0429] S132 CD3ζICD

[0430] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNGEDDAYCTFPSDGPPRSYLRQWVKGERRRGKLNTDAYLSLQEATKDTYDALHMQALPPR(SEQ ID NO:41)

[0431] S133 CD3ζICD

[0432] RVKFSRSADAPAYQQGQNQLYNELNLGPPRSYLRQWVRRGRDPEMGGKPQRRKNGEDDAYCTFPSDKMAEAYSEIGMKGERRRGKLNTDAYLSLQEATKDTYDALHMQALPPR(SEQ ID NO:42)

[0433] S134 CD3ζICD

[0434] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNGPPRSYLRQWVDKMAEAYSEIGMKGERRRGKLNTDAYLSLQEATKDTYDALHMQALPPR(SEQ ID NO:43)

[0435] S135 CD3ζICD

[0436] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNLNTDAYLSLQEDKMAEAYSEIGMKGERRRGKGPPRSYLRQWVATKDTYDALHMQALPPR(SEQ ID NO:44)

[0437] S136 CD3ζICD

[0438] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGPPRSYLRQWVATKDTYDALHMQALPPR(SEQ ID NO:45)

[0439] IL-12 substitution part: PTHDGYLPSNI(SEQ ID NO:46)

[0440] SX1 SCD3ζICD (underscore indicates IL-2RB substitution part; bold indicates IL-21R signaling; italic indicates IL-12 substitution part):

[0441]

[0442] SX2 SCD3ζICD (Underlined indicates IL-2RB substitution; bold indicates IL-21R substitution; italic indicates IL-12 substitution)

[0443]

[0444] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A chimeric antigen receptor (CAR), characterized in that, The chimeric antigen receptor includes a substituted intracellular signal transduction domain, the intracellular signal transduction domain including one or more immune receptor tyrosine activation domains (ITAMs), wherein the one or more ITAMs include one or more substituted fragments derived from an exogenous polypeptide.

2. The CAR as described in claim 1, characterized in that, The exogenous polypeptide is selected from the group consisting of: IL-1β receptor, IL-2 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-21 receptor, IL-23 receptor, IFN-γ receptor, TNF-α receptor, TGF-β receptor, GM-CSF receptor, DAP10, DAP12, FcRγ, or combinations thereof.

3. The CAR as described in claim 1, characterized in that, The amino acid sequence of the substituted fragment is as shown in SEQ ID NO:1, 2, 3, 4, 5 or 46, or has at least 90% amino acid sequence identity with it.

4. The CAR as described in claim 1, characterized in that, The immune cell activation-related receptor CD3ζ is described, and the intracellular signal transduction domain includes three immune receptor tyrosine activation domains ITAM1, ITAM2 and ITAM3 from the N-terminus to the C-terminus.

5. The CAR as described in claim 4, characterized in that, The ITAM3 described herein includes a substitution fragment R1, which replaces tyrosine at position 12 and its adjacent amino acid fragment, or tyrosine at position 23 and its adjacent amino acid fragment, or a combination thereof, in the wild-type ITAM3 amino acid sequence (SEQ ID NO:30). The ITAM3 includes substitution fragments R1 and R2, which respectively replace tyrosine at position 12 and its adjacent amino acid fragments and tyrosine at position 23 and its adjacent amino acid fragments in the wild-type ITAM3 amino acid sequence (SEQ ID NO: 30); wherein The tyrosine residue at position 12 of ITAM3 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 3-13, 4-14, 5-15, 6-16, or 7-17 of SEQ ID NO:

30. The tyrosine residue at position 23 of ITAM3 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 17-27, 18-28, or 19-29 of SEQ ID NO:

30.

6. The CAR as described in claim 5, characterized in that, The amino acid sequence of R1 is shown in SEQ ID NO:1 or 3, and the amino acid sequence of R2 is shown in SEQ ID NO:2 or 4. The amino acid sequence of R1 is shown in SEQ ID NO:1, 2, 3 or 4, and the amino acid sequence of R2 is shown in SEQ ID NO:5 or 46; or The amino acid sequence of R1 is shown in SEQ ID NO:5, and the amino acid sequence of R2 is shown in SEQ ID NO:1, 2, 3, 4 or 46.

7. The CAR as described in claim 4, characterized in that, The ITAM2 described herein includes a substitution fragment R3, which replaces tyrosine at position 12 and its adjacent amino acid fragment, or tyrosine at position 24 and its adjacent amino acid fragment, or a combination thereof, in the wild-type ITAM2 amino acid sequence (SEQ ID NO:29). The ITAM2 includes substitution fragments R3 and R4, which respectively replace tyrosine at position 12 and its adjacent amino acid fragments and tyrosine at position 24 and its adjacent amino acid fragments in the wild-type ITAM2 amino acid sequence (SEQ ID NO: 29); wherein The tyrosine residue at position 12 of ITAM2 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 3-13, 4-14, 5-15, 6-16, or 7-17 of SEQ ID NO:

29. The tyrosine residue at position 24 of ITAM2 and its adjacent amino acid fragments refer to the sequence fragments shown at positions 17-27, 18-28, or 19-29 of SEQ ID NO:

29.

8. The CAR as described in claim 7, characterized in that, The amino acid sequence of R3 is shown in SEQ ID NO:1 or 3, and the amino acid sequence of R4 is shown in SEQ ID NO:2 or 4. The amino acid sequence of R3 is shown in SEQ ID NO:1, 2, 3 or 4, and the amino acid sequence of R4 is shown in SEQ ID NO:5 or 46; or The amino acid sequence of R3 is shown in SEQ ID NO:5; the amino acid sequence of R4 is shown in SEQ ID NO:1, 2, 3, 4 or 46.

9. The CAR as described in claim 4, characterized in that, The intracellular signal transduction domain has one or more substitutions selected from the group consisting of the wild-type CD3ζ region (SEQ ID NO:20): (I) The amino acids at positions 20-30 of ITAM1 are replaced by the sequence shown in SEQ ID NO:5; (II) The amino acids at positions 7-17 of ITAM2 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4 or 5; (III) The amino acids at positions 19-29 of ITAM2 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4, 5 or 46; (IV) The amino acids at positions 7-17 of ITAM3 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4 or 5; (V) The amino acids at positions 18-28 of ITAM3 are replaced by sequences as shown in SEQ ID NO:1, 2, 3, 4, 5 or 46; (VI) The amino acids at positions 105-108 are mutated to the sequence shown in YRHQ (SEQ ID NO:6); (VII) The amino acids at positions 6-9 of ITAM1 (SEQ ID NO:28) are mutated to the sequence shown in YRHQ (SEQ ID NO:6); (VIII) The amino acid sequence at positions 24-27 of ITAM2 (SEQ ID NO:29) is mutated to the sequence shown in YRHQ (SEQ ID NO:6); The amino acid sequence (IX)ITAM3 (SEQ ID NO:30) is mutated from position 12 to 15 to the sequence shown in YRHQ (SEQ ID NO:6).

10. The CAR as claimed in claim 1, characterized in that, The amino acid sequence of the intracellular signal transduction domain is shown in any one of SEQ ID NO: 7-19, 31-39 and 47-48.

11. The CAR as claimed in claim 1, characterized in that, The amino acid sequence of the intracellular signal transduction domain is shown in any one of SEQ ID NO:9, 47-48.

12. A chimeric antigen receptor (CAR), characterized in that, The chimeric antigen receptor includes a substituted intracellular signal transduction domain, wherein the intracellular signal transduction domain has one or more substitutions selected from the group consisting of the wild-type CD3ζ region (SEQ ID NO:20): (I) The amino acids at positions 6-9 of ITAM1 (SEQ ID NO:28) are mutated to the sequence shown in YRHQ (SEQ ID NO:6); (II) The amino acids 24-27 of ITAM2 (SEQ ID NO:29) are mutated to the sequence shown in YRHQ (SEQ ID NO:6); (III) The amino acid sequence at positions 12-15 of ITAM3 (SEQ ID NO:30) is mutated to the sequence shown in YRHQ (SEQ ID NO:6).

13. A nucleic acid molecule encoding a chimeric antigen receptor as described in claim 1 or 12.

14. A vector comprising the nucleic acid molecule as described in claim 13.

15. A host cell containing a vector as described in claim 14 or a chromosome in which an exogenous nucleic acid molecule as described in claim 13 is integrated or which expresses a chimeric antigen receptor as described in claim 1 or 12.

16. An engineered immune cell, said immune cell containing a vector as described in claim 14 or a chromosome in which an exogenous nucleic acid molecule as described in claim 13 is integrated or which expresses a chimeric antigen receptor as described in claim 1 or 12.

17. A method for preparing engineered immune cells as described in claim 16, comprising the following steps: The engineered immune cells are obtained by transducing the nucleic acid molecule as described in claim 13 or the vector as described in claim 14 into immune cells.

18. A pharmaceutical composition comprising a nucleic acid molecule as claimed in claim 13, a carrier as claimed in claim 14, a host cell as claimed in claim 15, and / or engineered immune cells as claimed in claim 16, and a pharmaceutically acceptable carrier, diluent, or excipient.

19. Use of a nucleic acid molecule as claimed in claim 13, a carrier as claimed in claim 14, or a host cell as claimed in claim 15, an engineered immune cell as claimed in claim 16, and / or a pharmaceutical composition as claimed in claim 18, for the preparation of a medicament or formulation for the prevention and / or treatment of a disease.

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