Intracellular signaling domain of chimeric antigen receptor and use thereof

Abstract

The present invention belongs to the technical field of biomedicine, and specifically relates to an intracellular signaling domain of a chimeric antigen receptor and the use thereof. The intracellular signaling domain comprises at least two of a1)-a7): a1) FCE or a portion thereof; a2) CD3ζ or a portion thereof; a3) CD40 or a portion thereof; a4) TLR2 or a portion thereof; a5) TLR4 or a portion thereof; a6) IFNgR or a portion thereof; and a7) PI3Krecruit or a portion thereof. The chimeric antigen receptor comprising the intracellular domain can maintain a CAR-positive rate while ensuring the viability and recovery yield of immune cells, thereby better ensuring the activity of CAR-immune cells. Immune cells expressing the chimeric antigen receptor comprising the intracellular domain exhibit significantly enhanced phagocytic, cytotoxic and anti-exhaustion capacities in addition to T cell activation level, thereby achieving more potent antitumor activity and enhanced tumor infiltration.

Description

Intracellular signal transduction region of chimeric antigen receptor and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the intracellular signal transduction region of chimeric antigen receptors and its applications. Background Technology

[0002] Chimeric antigen receptor (CAR) technology has made significant progress in the treatment of hematological malignancies, particularly acute lymphoblastic leukemia (ALL), lymphoma, and plasma cell myeloma (PCM). However, the efficacy of CAR-T cell therapy in the treatment of solid tumors remains unsatisfactory.

[0003] Macrophages are the main phagocytic cells in the body, capable of engulfing and digesting pathogens such as bacteria, viruses, and cancer cells. In addition to their powerful phagocytic capacity, macrophages also possess antigen-presenting capabilities, activating adaptive immune responses. Since the first engineered carcinoembryonic antigen (CEA)-targeted CAR molecules into human monocytes in 2006, CAR-macrophage / monocyte (CAR-M) has become a promising candidate due to its strong phagocytic capacity, antigen-presenting function, and ability to enter / modulate the tumor microenvironment and stimulate / initiate adaptive responses.

[0004] CAR-M therapy is similar to CAR-T therapy, but its products are centered around macrophages. It requires extracting macrophages from the patient and introducing CAR molecules into them through genetic engineering to ultimately kill tumor cells. As key effector cells of the innate immune system, macrophages have a powerful phagocytic function. Among them, tumor-associated macrophages (TAMs) are the most important immune cells in the tumor microenvironment (TME), playing a dual role in both killing and promoting tumor growth. They play a crucial role in the occurrence, development, invasion, metastasis, immune evasion, and angiogenesis and lymphangiogenesis of malignant tumors.

[0005] CARs are artificially designed transmembrane receptors, typically comprising four parts: a single-chain variable fragment (scFv) that recognizes tumor antigens, a hinge domain, a transmembrane domain, and an intracellular signaling domain (ICD). Most CAR molecules used in CAR-T products have an ICD region composed of CD3ζ and 41-BB or CD28 regions, responsible for transducing antigen recognition signals into T cells, thereby activating the cellular immune response.

[0006] In CAR-M design, the selection of different ICD regions endows CAR-Ms with varying anti-tumor capabilities. Currently, CAR-M involves introducing CAR molecules into macrophages through genetic engineering, enabling them to specifically recognize and kill cancer cells expressing corresponding antigens. As a newcomer in the field of cell therapy, the structure of CAR-M is still in the exploration and optimization stage compared to CAR-T. Among these, the intracellular signal transduction region, as a crucial part of the functional domains related to CAR-M, is a major direction for CAR-M optimization and upgrading. Summary of the Invention

[0007] The present invention provides a chimeric antigen receptor (CAR) intracellular signal transduction region comprising at least two of FCE, CD3ζ, CD40, TLR2, TLR4, IFNgR, and PI3K, a chimeric antigen receptor containing the intracellular signal transduction region, biomaterials associated with the intracellular signal transduction region or the chimeric antigen receptor, modified immune cells containing the chimeric antigen receptor, and their use in the prevention or treatment of tumors.

[0008] According to one aspect of the invention, a chimeric antigen receptor (CAR) intracellular signal transduction region is provided, comprising at least two (i.e., two, three, four, five, six, or seven, preferably two) of a1)-a7):

[0009] a1) FCE or a part thereof; a2) CD3ζ or a part thereof; a3) CD40 or a part thereof; a4) TLR2 or a part thereof; a5) TLR4 or a part thereof; a6) IFNgR or a part thereof; a7) PI3K recruit or a part thereof.

[0010] In some embodiments, the chimeric antigen receptor (CAR) intracellular signal transduction region includes:

[0011] (i) at least one of a1)-a2); and

[0012] (ii) at least one of a3)-a7).

[0013] In some embodiments, the chimeric antigen receptor (CAR) intracellular signal transduction region includes:

[0014] (i) any one of a1)-a2); and

[0015] (ii) any one of a3)-a7).

[0016] In some embodiments, the chimeric antigen receptor (CAR) intracellular signal transduction region comprises: a1) and a3); or a1) and a4); or a1) and a5); or a1) and a6); or a1) and a7); or a2) and a3); or a2) and a5); or a2) and a6); or a2) and a7).

[0017] In some embodiments, the intracellular signal transduction region of the chimeric antigen receptor (CAR) from the N-terminus to the C-terminus includes: a3) and a1); or a1) and a4); or a1) and a5); or a1) and a6); or a1) and a7); or a3) and a2); or a2) and a5); or a2) and a6); or a2) and a7).

[0018] In some embodiments, the amino acid sequence of the FCE is as follows:

[0019] b1) The amino acid sequence as shown in SEQ ID NO.7; or

[0020] b2) An amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with and has the same function as the amino acid sequence shown in SEQ ID NO.7.

[0021] In some embodiments, the amino acid sequence of CD3ζ is as follows:

[0022] c1) The amino acid sequence as shown in SEQ ID NO. 8; or

[0023] c2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO.8 and has the same function.

[0024] In some embodiments, the amino acid sequence of CD40 is as follows:

[0025] d1) The amino acid sequence as shown in SEQ ID NO.13; or

[0026] d2) An amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with and has the same function as the amino acid sequence shown in SEQ ID NO.13.

[0027] In some embodiments, the amino acid sequence of the TLR2 is as follows:

[0028] e1) The amino acid sequence as shown in SEQ ID NO. 11; or

[0029] e2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO.11 and has the same function.

[0030] In some embodiments, the amino acid sequence of the TLR4 is as follows:

[0031] f1) The amino acid sequence as shown in SEQ ID NO.12; or

[0032] f2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO. 12 and has the same function.

[0033] In some embodiments, the amino acid sequence of the IFNgR is as follows:

[0034] g1) The amino acid sequence as shown in SEQ ID NO. 9; or

[0035] g2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO.9 and has the same function.

[0036] In some embodiments, the amino acid sequence of the PI3K recruit is:

[0037] h1) The amino acid sequence as shown in SEQ ID NO. 10; or

[0038] h2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO.10 and has the same function.

[0039] Chimeric antigen receptors containing this intracellular domain can maintain CAR positivity while ensuring cell viability and recovery, thus better guaranteeing CAR-cell activity. Cells containing this intracellular domain exhibited significantly enhanced phagocytosis, killing, anti-exhaustion capabilities, and T cell activation / activation levels, achieving stronger anti-tumor activity and greater tumor invasiveness.

[0040] According to another aspect of the invention, a chimeric antigen receptor (CAR) is provided, comprising: (a) an extracellular ligand-binding domain having binding affinity for a ligand; (b) a transmembrane domain; and (c) the intracellular signal transduction region described above in this invention.

[0041] In some embodiments, the extracellular ligand-binding domain is selected from the following ligand-binding domains: antibodies, antigen-binding fragments, antibody mimics, and receptors.

[0042] In some embodiments, the antibody or the antigen-binding fragment is selected from: single-chain antibody (scFv), monoclonal antibody, antigen-binding fragment (Fab), nanobody, biantibody, triantibody, microantibody, F(ab')2 fragment, F(ab)v fragment, single-domain antibody (sdAb), V H Structural domain, V L Domain, Fv fragment, VNAR domain and V HH Structural domain; further, it is a single-chain antibody (scFv).

[0043] In some embodiments, the antibody mimicry is selected from: affinity molecules, Afflilin, Affimer, α-antibodies, high-affinity polymers, DARPin, Fynomer, Kunitz domain peptides, nano-CLAMPs, and their bioactive fragments.

[0044] In some embodiments, the ligands are selected from: CD1a, CD1b, CD1c, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16 (FcγRIII), CD17, CD18, CD19, CD20, CD21, CD22, CD 23. CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32(FcγRII), CD33, CD34, CD35, CD36, CD37 , CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD 52. CD53, CD54, CD56 (NCAM), CD57, CD61 (integrin β3), CD62L, CD63, CD64, CD66b, CD68, CD69, CD70, CD 73. CD74, CD79a (Igα), CD79b (Igβ), CD80, CD83, CD85k (ILT3), CD86, CD88, CD93 (C1Rqp), CD94, CD 95, CD99, CD103, CD105 (endothelial glycoprotein), CD107a, CD107b, CD114 (G-CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (cohesin-1), CD141, CD146, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1).1), CD163, CD183, CD191, CD193(CCR3), CD194(CCR4), CD195(CCR5), CD197(CCR7), CD203c, CD205(DEC-205), CD207 (langesin), CD209(DC-SIGN), CD223, CD235, CD244(2B4), CD252(OX40L), CD267, CD268(BAFF-R), CD273(B7-DC, PD-L2), CD276 (B7-H3), CD279 (PD1), CD282 (TLR2), CD284 (TLR4), CD294, CD304 (neuroflavin-1), CD305, CD314 (NKG2D), CD319 (CRACC), CD326, CD328 (Siglec-7), CD335 (NKp46), fetal acetylcholine receptor (AChR), ADGRE2, alpha-fetoprotein (AFP), ALK, BCMA, BDCA3, C3AR, Lewis A (CA19.9), carbonic anhydrase IX (CA1X), calciretinoin, cancer antigen-125 (CA-125), CCR1, CCR4, CDS, carcinoembryonic antigen (CEA), chromogranin, CLEC12A, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), CS-1, CSPG4, cytokeratin, desmin, DLK1, DLL3, EGFRvIII (epidermal growth factor variant III), EGFR and its allotypes, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP) 2) Epithelial glycoprotein 40 (EGP-40), epithelial membrane protein (EMA), ERBB, epithelial tumor antigen (ETA), FAP, folate-binding protein (FBP), FcγR1, FcεRIα, FITC, FLT3, FOLR1, FOLR3, prolactin, gangliosides, giant cystic disease fluid protein (GCDFP-15), GD2 (ganglioside G2), GD3, GM2, GM3, glial fibrillary acidic protein (GFAP), gpA33, glycopeptides, phosphatidylinositol proteoglycan 2 (GPC2), phosphatidylinositol proteoglycan 3 (GPC3), carcinoembryonic antigen (h5T4), influenza hemagglutinin (HA), human epidermal growth factor receptor 2 (HER2), HLA-DR, HM1.24. HMB-45 antigen, HPV E6, HPV E7, ICAM-1, IgG, IgD, IgE, IgM, IL-13 receptor α1, integrin, integrin B7, interleukin-13 receptor subunit α-2 (IL-13Rα2), κ light chain, kinase insertion domain receptor (KDR), λ light chain, LILRB2, Lewis Y (LeY), LGR5, Ly49, Ly108, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE), melanoma antigen family A1 (MAGE-A1), protein melanin A (melanoma antigen recognized by T lymphocytes; MART-1), MCSP, c-Met, MICA / B, mesothelin, muscle-specific actin (MSA), mesothelin (MSLN), pyruvate kinase isoenzyme M2 dimer form (tumor M2-PK). Mucin 1 (Muc-1), Mucin 16 (Muc-16), myo-D1, Necl-2, neurofilaments, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO, cancer-testis antigen NY-ESO-1, abnormal p53 protein, PAP (prostatic acid phosphatase), PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, prostaglandins, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSM) A) Ral-B, K-Ras (V-Ki-ras2 Kirston rat sarcoma virus oncogene), abnormal ras protein, ROR1, SLAMF7 / CS1, receptor tyrosine protein kinase erb-B2, erb-B3, erb-B4, spermin 17 (Sp17), STEAP1 (prostate six-transmembrane epithelial antigen 1), synaptophysin, tumor-associated glycoprotein 72 (TAG-72), TALLA-1, TARP (T cell receptor γ variable reading frame protein), T EM-8, human telomerase reverse transcriptase (hTERT), TIM-3, TLR4, TRBC1, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, TYRP1, tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), Vα24, nephroblastoma protein (WT-1), or any combination thereof; further comprising phosphatidylinositol proteoglycan 3 (GPC3), human epidermal growth factor receptor 2 (HER2), or prostate-specific membrane antigen (PSMA).

[0045] In some embodiments, the amino acid sequence of the extracellular ligand binding domain is as follows:

[0046] i1) An amino acid sequence as shown in SEQ ID NO. 2, 3, or 4; or

[0047] The amino acid sequences shown in i2) and i1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and have the same function.

[0048] In some embodiments, the transmembrane domain is selected from the following transmembrane domains: CD4, CD8, CD28, PD-1, OX40, 4-1BB, CTLA-4, CD2, CD3D, CD3E, CD3G, CD3ζ, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150 (SLAMF1), CD166, CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD300, CD3 57(GITR), A2aR, ICAM-1, 2B4, BTLA, DAP10, FcRα, FcRβ, Fyn, GAL9, IL7, IL12, IL15, KIR, KIR2DL4, KIR2DS1, LAG-3, Lck, LAT, LPA5, LRP, NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, SLP-76, SIRPα, pTα, T cell receptor peptides (e.g., TCRα and TCRβ), TIM3, TRIM, ZAP70, or any combination thereof; further comprising a CD8 transmembrane domain.

[0049] In some embodiments, the amino acid sequence of the transmembrane domain is as follows:

[0050] j1) The amino acid sequence as shown in SEQ ID NO. 6; or

[0051] The amino acid sequences shown in j2) and j1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and have the same function.

[0052] In some embodiments, the chimeric antigen receptor further includes an extracellular hinge domain and / or an extracellular leader domain.

[0053] In some embodiments, the extracellular hinge domain is selected from the following hinge domains: CD8, CD28, CD4, IgG, PD-1, CTLA-4, CD2, LFA-1 (CD11a / CD18), CD5, CD27 (TNFRSF7), CD70, 4-1BB, OX40 (CD134), ICOS (CD278), IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgE Fc region, IgM Fc region, IgA Fc region, or any combination thereof; further, the CD8 extracellular hinge domain.

[0054] In some embodiments, the amino acid sequence of the extracellular hinge domain is as follows:

[0055] k1) The amino acid sequence as shown in SEQ ID NO.5; or

[0056] The amino acid sequences shown in k2) and k1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and the same function.

[0057] In some embodiments, the extracellular leader domain includes the CD8 extracellular leader domain.

[0058] In some embodiments, the amino acid sequence of the extracellular leader domain is as follows:

[0059] l1) The amino acid sequence as shown in SEQ ID NO.1; or

[0060] The amino acid sequences shown in l2) and l1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and have the same function.

[0061] In some embodiments, the chimeric antigen receptor includes, from the N-terminus to the C-terminus, an extracellular leader domain, an extracellular ligand-binding domain having binding affinity for ligands, an extracellular hinge domain, a transmembrane domain, and the intracellular signal transduction region described above in this invention.

[0062] In some embodiments, the amino acid sequence of the chimeric antigen receptor is:

[0063] m1) an amino acid sequence as shown in any of SEQ ID NO. 15-19, 21-24, 31-35, 37-40, 47-51, 53-56; or

[0064] The amino acid sequences shown in m2) and m1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and have the same function.

[0065] According to another aspect of the present invention, biomaterials related to the intracellular signal transduction region of the chimeric antigen receptor or the chimeric antigen receptor described above are provided, said biomaterials comprising any one of n1)-n9):

[0066] n1) The intracellular signal transduction region of the chimeric antigen receptor or the nucleic acid molecule encoding the chimeric antigen receptor of the present invention;

[0067] n2) contains an expression cassette containing the nucleic acid molecule described in n1);

[0068] n3) A carrier containing the nucleic acid molecule described in n1);

[0069] n4) A carrier containing the expression box described in n2);

[0070] n5) A cell containing the nucleic acid molecules described in n1);

[0071] n6) Cells containing the expression cassette described in n2);

[0072] n7) Cells containing the carrier described in n3);

[0073] n8) contains cells containing the carrier described in n4);

[0074] n9) A cell containing the intracellular signal transduction region of the chimeric antigen receptor described above, or a chimeric antigen receptor of the present invention.

[0075] In some embodiments, the nucleic acid molecule may be an RNA molecule or a DNA molecule.

[0076] In some embodiments, any of the vectors n3)-n4) comprises a viral vector or a non-viral vector: wherein the viral vector is, for example, but not limited to, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector (e.g., a lentiviral vector or a gamma retroviral vector); and the non-viral vector is, for example, a plasmid, bacteriophage, lipid nanoparticle, transfection reagent, and exosome. In some embodiments, the adeno-associated virus vector comprises an Ad5 vector (e.g., an Adf35 viral vector).

[0077] In some embodiments, any of the cells described in n5)-n9) does not contain reproductive material.

[0078] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor of the present invention is as follows:

[0079] o1) nucleotide sequences as shown in any of SEQ ID NO. 76-80, 82-85, 92-96, 98-101, 86-90, 42-45; or

[0080] The nucleotide sequences shown in o2) and o1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and the same function.

[0081] According to another aspect of the present invention, a modified immune cell is provided, comprising the chimeric antigen receptor intracellular signal transduction region, chimeric antigen receptor, nucleic acid molecule in biomaterial, expression cassette in biomaterial, or carrier in biomaterial as described above.

[0082] In some embodiments, the immune cells comprise macrophages, monocytes, dendritic cells, T cells (e.g., induced pluripotent stem cell (iPSC)-derived T cells), stem cells, regulatory T cells (Tregs), natural killer (NK) cells (e.g., induced pluripotent stem cell (iPSC)-derived NK cells), γδ T cells, or natural killer T (NKT) cells. In some embodiments, the immune cells comprise macrophages, monocytes, dendritic cells, or stem cells. In a preferred embodiment, the immune cells comprise macrophages and / or monocytes. In a preferred embodiment, the immune cells comprise monocytes. In a preferred embodiment, the immune cells comprise macrophages.

[0083] In some embodiments, the modified immune cells exhibit enhanced effector activity compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit enhanced tumor-killing and / or phagocytic activity compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit enhanced resistance to exhaustion compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit enhanced tumor infiltration ability compared to unmodified immune cells.

[0084] In some embodiments, the modified immune cells exhibit increased polarization of the M1 phenotype compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit increased expression of one or more markers of the M1 phenotype compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit decreased expression of one or more markers of the M2 phenotype compared to unmodified immune cells.

[0085] In some implementations, one or more markers of the M1 phenotype include one or more of CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, CD68, CD69, MHC I, CD64, CD32, CD16, IL1R, IFIT family members, or ISG family members.

[0086] In some embodiments, one or more markers of the M2 phenotype include one or more of CD206, CD163, CD209, FIZZ1, and Ym1 / 2.

[0087] According to another aspect of the present invention, a method for preparing the modified immune cells described above is provided, the method comprising the step of delivering a nucleic acid molecule, expression cassette, or vector encoding the above-described biological material of the present invention into the immune cells.

[0088] In some embodiments, the nucleic acid molecule may be an RNA molecule or a DNA molecule.

[0089] According to another aspect of the present invention, a pharmaceutical composition is provided comprising the chimeric antigen receptor intracellular signal transduction region, chimeric antigen receptor, biomaterial, or modified immune cells described above. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0090] According to another aspect of the present invention, a method for preventing or treating a disease or ailment of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0091] In some embodiments, the subject may include mammals, such as humans or non-human mammals. In some embodiments, the non-human mammals may include, but are not limited to, non-human primates (e.g., monkeys, orangutans), mice, rats, hamsters, gerbils, cats, dogs, guinea pigs, rabbits, horses, sheep, cattle, pigs, etc.

[0092] According to another aspect of the present invention, the use of the above-described chimeric antigen receptor intracellular signal transduction region, chimeric antigen receptor, biomaterial, or modified immune cell of the present invention in the preparation of a medicament for the prevention or treatment of a disease or ailment is provided.

[0093] In some implementations, the disease or condition includes a tumor.

[0094] In some embodiments, the tumor comprises at least one of a solid tumor and a hematoma; preferably a solid tumor.

[0095] In some embodiments, the solid tumors include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, adrenocortical carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdomyosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germinal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial endodermal tumor, gonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, and medrogenic carcinoma. At least one of the following: mesothelioma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal and sinus carcinoma, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal carcinoma, retinoblastoma, salivary gland tumor, sarcoma, Cézare syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and single myeloma; further, breast cancer, gastric cancer, ovarian cancer, colon cancer, lung cancer, bladder cancer, prostate cancer, pancreatic cancer, or liver cancer.

[0096] In some embodiments, the hematologic malignancy is selected from at least one of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphoblastic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, and preleukemia. Attached Figure Description

[0097] Figure 1 shows a schematic diagram of part of the HER2 CAR molecular structure.

[0098] Figure 2 shows a schematic diagram of part of the PSMA CAR molecular structure.

[0099] Figure 3 shows a schematic diagram of part of the GPC3 CAR molecular structure.

[0100] Figure 4 shows the recovery rate of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0101] Figure 5 shows the recovery rate of macrophages transfected with IVT RNA of each PSMA CAR molecule.

[0102] Figure 6 shows the recovery rate of macrophages transfected with IVT RNA of each GPC3 CAR molecule.

[0103] Figure 7 shows the viability of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0104] Figure 8 shows the viability of macrophages transfected with IVT RNA of each PSMA CAR molecule.

[0105] Figure 9 shows the viability of macrophages transfected with IVT RNA of each GPC3 CAR molecule.

[0106] Figure 10 shows the percentage of CAR molecules expressed on the surface of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0107] Figure 11 shows the percentage of CAR molecules expressed on the surface of macrophages transfected with IVT RNA of each PSMA CAR molecule.

[0108] Figure 12 shows the percentage of CAR molecules expressed on the surface of macrophages transfected with IVT RNA of each GPC3 CAR molecule.

[0109] Figure 13 shows the percentage of CD80 molecules expressed on the surface of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0110] Figure 14 shows the percentage of CD80 molecules expressed on the surface of macrophages transfected with IVT RNA of each PSMA CAR molecule.

[0111] Figure 15 shows the percentage of CD80 molecules expressed on the surface of macrophages transfected with IVT RNA of each GPC3 CAR molecule.

[0112] Figure 16 shows the percentage of CD163 molecules expressed on the surface of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0113] Figure 17 shows the percentage of CD163 molecules expressed on the surface of macrophages transfected with IVT RNA of each PSMA CAR molecule.

[0114] Figure 18 shows the percentage of CD163 molecules expressed on the surface of macrophages transfected with IVT RNA of each GPC3 CAR molecule.

[0115] Figure 19 shows the phagocytic results of OE19 cells by macrophages transfected with IVT RNA of each HER2 CAR molecule after 2 hours.

[0116] Figure 20 shows the phagocytic results of LnCap cells by macrophages transfected with IVT RNA of various PSMA CAR molecules over 2 hours.

[0117] Figure 21 shows the phagocytic results of HepG2 cells by macrophages transfected with IVT RNA of each GPC3 CAR molecule after 2 hours.

[0118] Figure 22 shows the 48-hour killing effect of macrophages transfected with IVT RNA of each HER2 CAR molecule on OE19-luci cells.

[0119] Figure 23 shows the 48-hour killing effect of macrophages transfected with IVT RNA of various PSMA CAR molecules on LnCap-luci cells.

[0120] Figure 24 shows the 48-hour killing effect of macrophages transfected with IVT RNA of each GPC3 CAR molecule on HepG2-luci cells.

[0121] Figure 25 shows the viability of monocytes transfected with adenoviruses expressing each HER2 CAR molecule.

[0122] Figure 26 shows the viability of monocytes transfected with adenovirus expressing each PSMA CAR molecule.

[0123] Figure 27 shows the viability of monocytes transfected with adenovirus expressing each GPC3 CAR molecule.

[0124] Figure 28 shows the recovery rate of monocytes transfected with adenoviruses expressing each HER2 CAR molecule.

[0125] Figure 29 shows the recovery rate of monocytes transfected with adenoviruses expressing each PSMA CAR molecule.

[0126] Figure 30 shows the recovery rate of monocytes transfected with adenoviruses expressing each GPC3 CAR molecule.

[0127] Figure 31 shows the percentage of CAR molecules expressed in monocytes transfected with adenoviruses expressing each HER2 CAR molecule.

[0128] Figure 32 shows the percentage of CAR molecules expressed in monocytes transfected with adenoviruses expressing each PSMA CAR molecule.

[0129] Figure 33 shows the percentage of CAR molecules expressed in monocytes transfected with adenovirus expressing each GPC3 CAR molecule.

[0130] Figure 34 shows the expression of CD80 molecules on the surface of monocytes transfected with adenoviruses expressing various HER2 CAR molecules.

[0131] Figure 35 shows the expression of CD80 molecules on the surface of monocytes transfected with adenoviruses expressing various PSMA CAR molecules.

[0132] Figure 36 shows the expression of CD80 molecules on the surface of monocytes transfected with adenoviruses expressing each GPC3 CAR molecule.

[0133] Figure 37 shows the MFI of CD163 molecule expression on the surface of monocytes transfected with adenoviruses expressing various HER2 CAR molecules.

[0134] Figure 38 shows the expression of CD163 molecules on the surface of monocytes transfected with adenoviruses expressing various PSMA CAR molecules.

[0135] Figure 39 shows the MFI of CD163 molecule expression on the surface of monocytes transfected with adenovirus expressing each GPC3 CAR molecule.

[0136] Figure 40 shows the phagocytic effect of OE19 cells on monocytes transfected with adenoviruses expressing each HER2 CAR molecule after 2 hours.

[0137] Figure 41 shows the phagocytic results of LnCap cells by monocytes transfected with adenoviruses expressing various PSMA CAR molecules after 2 hours.

[0138] Figure 42 shows the phagocytic effect of HepG2 cells on monocytes transfected with adenoviruses expressing each GPC3 CAR molecule after 2 hours.

[0139] Figure 43 shows the 48-hour killing effect of OE19-luci cells on monocytes transfected with adenoviruses expressing various HER2 CAR molecules.

[0140] Figure 44 shows the 48-hour killing effect of LnCap-luci cells on monocytes transfected with adenoviruses expressing various PSMA CAR molecules.

[0141] Figure 45 shows the 48-hour killing effect of HepG2-luci cells on monocytes transfected with adenoviruses expressing each GPC3 CAR molecule.

[0142] Figure 46 shows the expression levels of CAR molecules on monocytes and macrophages after transfection with adenoviruses expressing each HER2 CAR molecule.

[0143] Figure 47 shows the expression levels of CAR molecules on monocytes and macrophages after transfection with adenoviruses expressing various PSMA CAR molecules.

[0144] Figure 48 shows the expression levels of CAR molecules on monocytes and macrophages after transfection with adenoviruses expressing each GPC3 CAR molecule.

[0145] Figure 49 shows the in vitro phagocytic levels of OE19 cells by monocytes and macrophages after transfection with adenoviruses expressing various HER2 CAR molecules, 2 hours later.

[0146] Figure 50 shows the in vitro phagocytic levels of LnCap cells by monocytes and macrophages after transfection with adenoviruses expressing various PSMA CAR molecules, 2 h later.

[0147] Figure 51 shows the in vitro phagocytic levels of HepG2 cells by monocytes and macrophages after transfection with adenoviruses expressing each GPC3 CAR molecule 2 h.

[0148] Figure 52 shows the in vitro killing levels of monocytes and macrophages after transfection with adenoviruses expressing various HER2 CARs at different effector-to-target ratios (E:T = 5:1 and E:T = 1:1) for 48 hours.

[0149] Figure 53 shows the in vitro killing levels of monocytes and macrophages after transfection with adenoviruses expressing various PSMA CARs at different effector-to-target ratios (E:T = 5:1 and E:T = 1:1) for 48 hours.

[0150] Figure 54 shows the in vitro killing levels of monocytes and macrophages after transfection with adenoviruses expressing various GPC3 CARs at different effector-to-target ratios (E:T = 5:1 and E:T = 1:1) for 48 hours.

[0151] Figure 55 shows the exhaustion level of monocytes transfected with adenoviruses expressing each HER2 CAR after 4 days of co-incubation with OE19-luci target cells.

[0152] Figure 56 shows the exhaustion level of monocytes transfected with adenoviruses expressing various PSMA CARs after 4 days of co-incubation with LnCap-luci target cells.

[0153] Figure 57 shows the exhaustion level of monocytes transfected with adenoviruses expressing each GPC3 CAR after 4 days of co-incubation with HepG2-luci target cells.

[0154] Figure 58 shows the CAR expression of monocytes transfected with adenoviruses expressing each HER2 CAR after co-incubation with OE19-luci target cells for 4 days.

[0155] Figure 59 shows the CAR expression of monocytes transfected with adenoviruses expressing various PSMA CARs after co-incubation with LnCap-luci target cells for 4 days.

[0156] Figure 60 shows the CAR expression of monocytes transfected with adenoviruses expressing each GPC3 CAR after 4 days of co-incubation with HepG2-luci target cells.

[0157] Figure 61 shows the changes in M1 phenotype levels after monocytes transfected with adenoviruses expressing each HER2 CAR were co-incubated with OE19-luci target cells for 4 days.

[0158] Figure 62 shows the changes in M1 phenotype levels after monocytes transfected with adenoviruses expressing various PSMA CARs were co-incubated with LnCap-luci target cells for 4 days.

[0159] Figure 63 shows the changes in M1 phenotype levels after monocytes transfected with adenoviruses expressing each GPC3 CAR were co-incubated with HepG2-luci target cells for 4 days.

[0160] Figure 64 shows the long-term killing effect of monocytes transfected with adenoviruses expressing each HER2 CAR at an E:T ratio of 5:1 against OE19-luci target cells.

[0161] Figure 65 shows the long-term killing effect of adenovirus-transfected monocytes expressing each HER2 CAR molecule and OE19-luci target cells at an E:T ratio of 1:1.

[0162] Figure 66 shows the long-term killing effect of monocytes transfected with adenoviruses expressing each HER2 CAR at an E:T ratio of 1:5 against OE19-luci target cells.

[0163] Figure 67 shows the long-term killing effect of adenoviruses transfected with each PSMA CAR and LnCap-luci target cells at an E:T ratio of 5:1.

[0164] Figure 68 shows the long-term killing effect of adenovirus-transfected monocytes expressing various PSMA CAR molecules against LnCap-luci target cells at an E:T ratio of 1:1.

[0165] Figure 69 shows the long-term killing effect of adenoviruses transfected with each PSMA CAR and LnCap-luci target cells at an E:T ratio of 1:5.

[0166] Figure 70 shows the long-term killing effect of monocytes transfected with adenoviruses expressing each GPC3 CAR at an E:T ratio of 5:1 against HepG2-luci target cells.

[0167] Figure 71 shows the long-term killing effect of adenovirus-transfected monocytes expressing each GPC3 CAR molecule and HepG2-luci target cells at an E:T ratio of 1:1.

[0168] Figure 72 shows the long-term killing effect of monocytes transfected with adenoviruses expressing each GPC3 CAR and HepG2-luci target cells at E:T = 1:5.

[0169] Figure 73 shows the infiltration and killing results of monocytes transfected with adenoviruses expressing each HER2 CAR against OE19-NLS mCherry monoclonal 3D tumor spheres at E:T = 5:1 and 1:1.

[0170] Figure 74 shows the infiltration and killing results of LnCap-NLS mCherry monoclonal 3D tumor spheres by monocytes transfected with adenoviruses expressing various PSMA CARs at E:T = 5:1 and 1:1.

[0171] Figure 75 shows the infiltration and killing results of monocytes transfected with adenoviruses expressing each GPC3 CAR against HepG2-NLS mCherry monoclonal 3D tumor spheres at E:T = 5:1 and 1:1.

[0172] Figure 76 shows the plasmid map of pVAX1.

[0173] Figure 77 shows the 48-h killing effect of macrophages expressing various combinations of GPC3 CAR molecules and a single ICD CAR molecule on HepG2-luci cells under E:T = 5:1 and 1:1 conditions.

[0174] When the above figure includes a legend, the processing of the horizontal axis from left to right corresponds to the processing from top to bottom in the legend. Taking Figure 43 as an example: when E:T = 5:1, 1:1, 1:5, or 1:10, the processing of the horizontal axis from left to right is UTD, HER2-FCE, HER2-FCE+IFNR, HER2-FCE+PI3K, HER2-CD3ζ, and HER2-CD3ζ+PI3K, respectively. Detailed Implementation

[0175] Macrophages are immune cells specifically designed to detect, engulf, and destroy target cells, such as pathogens or tumor cells. Macrophages are effective effectors of the innate immune system, capable of performing at least three distinct anti-tumor functions: engulfing dead and dying cells, microorganisms, cancer cells, cell debris, or other foreign substances; cytotoxicity against tumor cells; and tumor antigen presentation to coordinate adaptive anti-tumor immune responses.

[0176] Although CAR-M therapy has shown promise in cancer treatment, its application in solid tumors still faces many challenges. The dense tissue structure and complex tumor microenvironment of solid tumors limit the infiltration and killing effects of CAR-M cells. Moreover, macrophages are easily induced into M2-type macrophages that promote tumor growth in the tumor microenvironment, thus weakening their anti-tumor function.

[0177] This disclosure, through design and screening, reveals that compared to first-generation CARs containing CD3ζ, chimeric antigen receptors containing the intracellular domain of this invention, while essentially maintaining (or even improving) the CAR positivity rate, can ensure the viability and yield of immune cells (e.g., monocytes / macrophages), thus better guaranteeing the activity of CAR-immune cells (e.g., monocytes / macrophages). Immune cells (e.g., monocytes / macrophages) containing the chimeric antigen receptor containing the intracellular domain of this invention exhibit significantly enhanced phagocytosis, killing, and anti-exhaustion capabilities, as well as T cell activation / activation levels, achieving stronger anti-tumor activity and greater tumor invasiveness. Furthermore, the extracellular ligand-binding domain in the chimeric antigen receptor can be adjusted based on different targets, all of which can achieve effective killing and inhibition of target cells, demonstrating the powerful anti-tumor ability of the new generation of CAR-immune cells (e.g., monocytes / macrophages).

[0178] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0179] definition

[0180] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0181] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0182] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0183] Monocytes are a subset of leukocytes that play a crucial role in maintaining homeostasis, pathogen recognition and clearance, and inflammation. Following the generation and development of bone marrow progenitor cells, monocytes circulate in the vascular system, bone marrow, and spleen; they do not proliferate under homeostasis. Whether during normal development or due to pathogen attack, once monocytes migrate to peripheral tissues, they differentiate into dendritic cells or macrophages.

[0184] Macrophages are long-lived, phagocytic cells specialized in the innate immune system. Along with neutrophils, they are first responders to infection. Macrophages participate in the recognition, phagocytosis, and degradation of cellular debris and pathogens. They also play a role in initiating adaptive immune responses by presenting antigens to T cells and inducing other antigen-presenting cells to express co-stimulatory molecules. Furthermore, in the early stages of inflammation, macrophages play a crucial role by releasing cytokines and chemokines, which in turn recruit other immune cells to the site of inflammation. Macrophages are present in most tissues and therefore have diverse functions. Besides initiating immune and inflammatory responses to pathogens, macrophages also play a role in maintaining tissue homeostasis and tissue repair and remodeling. Macrophages also appear to play a key role in the tumor microenvironment, particularly in stromal remodeling, angiogenesis, metastasis, and tumor progression. Macrophages originate from many sources. Macrophages residing in tissues can differentiate from circulating monocytes or be generated in embryonic regions near the fetal liver, yolk sac, or dorsal aorta during embryonic development, and thus exist independently of monocytes in adulthood.

[0185] Different factors can lead to different phenotypes and activation states of macrophages. Generally, macrophages can be classified into two subtypes based on their function and activation: classically activated M1 macrophages and alternatively activated M2 macrophages. M1 macrophages mainly play a role in Th1 cell recruitment, pathogen resistance, and tumor control through innate and adaptive immune responses. M1 macrophages have strong antigen-presenting activity and secrete a large number of pro-inflammatory cytokines, such as interleukin-1 (IL-1), IL-6, TNF-α, nitric oxide (NO), and reactive oxygen species (ROS). M1 macrophages express high levels of major histocompatibility complex II (MHC II), CD68, CD80, CD86, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, CD69, MHC I, CD64, CD32, CD16, IL1R, IFIT family members or ISG family members, as well as chemokines targeting Th1 cells, such as CXCL9 and CXCL12. M2 macrophages can be activated by parasitic or fungal infection, immune complexes, apoptotic cells, macrophage colony-stimulating factor (M-CSF), IL-13, TGF-β, and type 2 helper T cell (Th2) IL-4, IL-33, and IL-25 via Th2 cell replacement. M2 macrophages express high levels of CD206, CD163, CD209, FIZZ1, Ym1 / 2, etc.

[0186] As used herein, the terms “effective amount” and “therapeutic effective amount” are interchangeable and refer to the amount of a compound, formulation, material, or composition that effectively achieves a particular biological outcome or provides a therapeutic or preventative benefit, as described herein. Such outcomes may include, but are not limited to, antitumor activity determined by any suitable method in the art.

[0187] As used in this article, the terms "effective function" or "effective activity" refer to specific activities performed by immune cells in response to stimuli. For example, the effector function of macrophages involves phagocytosis, which engulfs and digests cell debris, foreign substances, microorganisms, cancer cells, and other unhealthy cells.

[0188] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0189] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa. The phrase "at least 85% identity" compared to a reference sequence, as mentioned herein, can include at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0190] As used in this article, the term "single-chain antibody" or "scFv" refers to antibodies formed using recombinant DNA technology, in which immunoglobulin heavy and light chain fragments are linked to the Fv region via amino acid modifications. Various methods for generating single-chain antibodies are known.

[0191] The term “treatment” as used in this article can refer to treatment and / or prevention, achieving therapeutic effects by suppressing, alleviating or eradicating a disease state.

[0192] In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from the subject. The immune cells can be autologous or derived from an allogeneic or universal donor. Cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumor, and / or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In some embodiments, cells can be obtained from blood collected from the subject using any number of isolation techniques known to those skilled in the art. In some embodiments, cells from the subject's circulating blood are obtained via apheresis or leukoablation. In some embodiments, differentiation of the immune cells (e.g., monocytes) after enrichment includes stimulation with GM-CSF. In some embodiments, the cells will contain blood cells (e.g., monocytes, lymphocytes, etc.). Combinations of leukocytes, platelets, plasma, and / or erythrocytes, such as apheresis compositions, are used for enrichment. In some embodiments, selection of immune cells (e.g., monocytes) includes selection based on CD14 positivity. Immune cell precursors can differentiate into immune cells in vivo or in vitro. Non-limiting examples of precursor immune cells include hematopoietic stem cells, common myeloid progenitor cells, promyelocytes, promonocytes, premonocytes, or intermediates thereof. For example, induced pluripotent stem cells (iPSCs) can be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells can be derived from normal human tissues, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs can differentiate into bone marrow lineages (e.g., monocytes, macrophages, dendritic cells, or precursors thereof).

[0193] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0194] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0195] Example

[0196] Example 1. Construction of CAR-ICD in vitro transcription (IVT) plasmids with different combinations and preparation of IVT RNA

[0197] Different CAR-ICD IVT vector plasmids were constructed, with pVAX1 (purchased from Nanjing GenScript) selected as the plasmid. Based on the schematic diagrams of some CAR-ICD vector backbones shown in Figures 1-3, where the amino acid sequences of each component are shown in Table 1 and the nucleotide sequences are shown in Table 3, the encoding nucleic acid sequences were synthesized (the amino acid sequences of the CAR are shown in Table 2, and the nucleotide sequences are shown in Table 3). These sequences were then inserted into the pVAX1 plasmid (plasmid map shown in Figure 76) via the EcoR V site to prepare the IVT plasmid.

[0198] Table 1. Amino acid sequences of each component

[0199] Table 2. Amino acid sequence of CAR

[0200] Table 3. Nucleotide sequences of each component and CAR

[0201] The IVT plasmid was digested with BspQI restriction endonuclease (purchased from Takara), and the linearized DNA was purified and recovered using DNA magnetic beads (purchased from Shenji Biotechnology). Using the cap3011 RNA preparation kit (purchased from Shenji Biotechnology), linearized DNA was transcribed into mRNA in vitro. The IVT RNA was then purified and recovered using RNA magnetic beads (purchased from Shenji Biotechnology), yielding HER2 CAR-ICD molecules (HER2 CAR-FCE, HER2 CAR-FCE+IFNgR, HER2 CAR-FCE+PI3K, HER2 CAR-FCE+TLR2, HER2 CAR-FCE+TLR4, HER2 CAR-CD40+FCE, HER2 CAR-CD3ζ, HER2 CAR-CD3ζ+IFNgR, HER2 CAR-CD3ζ+PI3K, HER2 CAR-CD3ζ+TLR4, HER2 CAR-CD40+CD3ζ) and PSMA CAR-ICD molecules (PSMA CAR-FCE, PSMA CAR-FCE+IFNgR, PSMA CAR-FCE+PI3K, PSMA...). CAR-FCE+TLR2, PSMACAR-FCE+TLR4, PSMA CAR-CD40+FCE, PSMA CAR-CD3ζ, PSMA CAR-CD3ζ+IFNgR, PSMA CAR-CD3ζ+PI3K, PSMA CAR-CD3ζ+TLR4, PSMA CAR-CD40+CD3ζ), GPC3 CAR-ICD molecule (GPC3 CAR-FCE, GPC3 CAR-FCE+IFNgR, GPC3 CAR-FCE+PI3K, GPC3 CAR-FCE+TLR2, GPC3 CAR-FCE+TLR4, GPC3 CAR-CD40+FCE, GPC3CAR-CD3ζ, GPC3 CAR-CD3ζ+IFNgR, GPC3 CAR-CD3ζ+PI3K, GPC3 CAR-CD3ζ+TLR4, GPC3 IVT RNA of CAR-CD40+CD3ζ, GPC3 CAR-IFNR, GPC3 CAR-PI3K, GPC3 CAR-TLR2, GPC3 CAR-TLR4, GPC3 CAR-CD40).

[0202] Example 2. Detection of HER2, PSMA and GPC3 CAR molecular expression under IVT RNA transfection

[0203] Blood samples obtained from a commercially available company (with relevant ethical approvals) were centrifuged at 200g for 10 minutes to remove platelets and some red blood cells. The supernatant was discarded, and the cell pellet was mixed with PBS, then added to Ficoll (lymphocyte separation medium, purchased from Sigma), and centrifuged at 1200 rpm for 30 minutes. After centrifugation, mononuclear cells (PBMCs) were recovered and washed once with phosphate-buffered saline (PBS) by centrifugation. PBMCs were incubated with CD14+ magnetic beads (purchased from Miltenyi) for 15 min. CD14+ cells were separated using a CD14 sorting column (purchased from Miltenyi). CD14+ monocytes were harvested and monocytes were induced to differentiate into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) (refer to the induction method in the reference "MAS, et al. An Immune Atlas of Clear Cell Renal Cell Carcinoma. Cell. 2017 May 4; 169(4):736-749.e18.").

[0204] 1) After monocytes were induced to differentiate into macrophages, IVT RNA containing different HER2 CAR-ICD molecules from Example 1 was added, along with Lipofectamine. TM MessengerMAX TM Cells were harvested 48 hours after transfection using a 2:1 plasmid ratio mixture (pre-mixed and incubated at room temperature for 10 min) of the transfection reagent (Thermo). One negative control group (UTD group) consisted of untransfected macrophages. Cells were harvested 48 hours after transfection, with at least 1 × 10⁶ cells per group. 6 After mixing the macrophages with PBS, centrifuge at 400g for 5 minutes, add 2μL of FCR Blocking (Mittennis, 130-059-901), incubate at room temperature in the dark for 10 minutes, then add the antibody mixture from Table 4, incubate at 4℃ in the dark for 30 minutes, centrifuge at 400g for 5 minutes, wash once with PBS, and perform flow cytometry analysis. The amount of macrophages recovered (Figure 4) and viability (viability was obtained by dividing the number of recovered viable macrophages by the total number of recovered cells) were analyzed by flow cytometry (Figure 7), as well as the percentage of CAR molecule expression (Figure 10), CD80 molecule expression (Figure 13), and CD163 molecule expression (Figure 16) on the macrophage membrane surface were detected by flow cytometry.

[0205] Table 4. Catalog of Antibodies for Flow Cytometry Detection

[0206] The results showed that the combined ICD-CAR was superior to the single ICD-CAR in terms of recovery and viability; all combinations also showed good positive expression levels of CAR molecules; phenotypic information showed that the groups transfected with IVT RNA all tended towards M1 (CD80) + ) polarization, while simultaneously exhibiting M2 (CD163) polarization. + ) resistance.

[0207] 2) After monocytes were induced to differentiate into macrophages, IVT RNA containing different PSMA CAR-ICD molecules from Example 1 was added, along with Lipofectamine. TM MessengerMAX TM Cells were harvested 48 hours after transfection using a 2:1 plasmid ratio mixture (pre-mixed and incubated at room temperature for 10 min) of the transfection reagent (Thermo). One negative control group (UTD group) consisted of untransfected macrophages. Cells were harvested 48 hours after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were collected, mixed with PBS, and centrifuged at 400g for 5 minutes. The cell pellet was mixed with PBS, and PSMA protein (purchased from Acro) (4 μl / test) was added and incubated at room temperature for 20 minutes. Unbound PSMA protein was removed by centrifugation at 400g for 5 minutes. After washing, cells were subjected to flow cytometry analysis of PSMA-targeting CAR molecules, and the recovery amount of macrophages (Figure 5) and viability (Figure 8) of each group were analyzed. The percentage of CAR molecules expressed on the macrophage membrane surface (Figure 11), the percentage of CD80 molecules expressed (Figure 14), and the percentage of CD163 molecules expressed (Figure 17) were also analyzed by flow cytometry.

[0208] The results showed that the combined ICD-CARs were superior to the individual ICD-CARs in terms of recovery and viability; all combinations also exhibited good positive expression levels of CAR molecules; phenotypic information showed that the groups transfected with IVT RNA tended towards M1 (CD80) + ) polarization, while simultaneously exhibiting M2 (CD163) polarization. + ) resistance.

[0209] 3) After monocytes were induced to differentiate into macrophages, IVT RNA containing different GPC3 CAR-ICD molecules from Example 1 was added, along with Lipofectamine. TM MessengerMAX TMCells were harvested 48 hours after transfection using a 2:1 plasmid ratio mixture (pre-mixed and incubated at room temperature for 10 min) of the transfection reagent (Thermo). One negative control group (UTD group) consisted of untransfected macrophages. Cells were harvested 48 hours after transfection, with at least 1 × 10⁶ cells per group. 6 Cells were collected, mixed with PBS, and centrifuged at 400g for 5 minutes. The cell pellet was mixed with PBS, and GPC3 protein (purchased from Acro) (4 μl / test) was added and incubated at room temperature for 20 minutes. Unbound GPC3 protein was removed by centrifugation at 400g for 5 minutes. After washing, the cells were subjected to flow cytometry detection of GPC3-targeting CAR molecules, and the macrophage recovery amount (Figure 6) and viability (Figure 9) of each group were analyzed. The percentage of CAR molecules expressed on the macrophage membrane surface (Figure 12), the percentage of CD80 molecules expressed (Figure 15), and the percentage of CD163 molecules expressed (Figure 18) were also detected by flow cytometry.

[0210] The results showed that the combined ICD-CARs were superior to the individual ICD-CARs in terms of recovery rate and viability; all combinations also exhibited good positive expression levels of CAR molecules; phenotypic information showed that the groups transfected with IVT RNA tended towards M1 (CD80) + ) polarization, while simultaneously exhibiting M2 (CD163) polarization. + ) resistance.

[0211] Example 3. In vitro phagocytosis assay of HER2, PSMA, and GPC3 CAR-M transfected with IVT RNA.

[0212] OE19 cells (human esophageal cancer cells, purchased from COBIOER BIOSCIENCES) / LnCap cells (purchased from COBIOER Biotechnology) / HepG2 cells (purchased from Shanghai Anwei Biotechnology) were suspended in PBS. DiR dye (final concentration 5 μM) was added to the cell suspension and incubated at 37°C for 20 minutes. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed X-VIVO15 medium (purchased from Lonza, hereinafter referred to as "complete medium") containing 10% FBS (purchased from Gibco) to a density of 1×10⁶ cells / mL. 6 Cells / mL.

[0213] 1) The IVT RNAs of HER2 CAR-FCE, HER2 CAR-FCE+IFNgR, HER2 CAR-FCE+PI3K, HER2 CAR-FCE+TLR2, HER2 CAR-FCE+TLR4, HER2 CAR-CD40+FCE, HER2 CAR-CD3ζ, HER2 CAR-CD3ζ+IFNgR, HER2 CAR-CD3ζ+PI3K, HER2 CAR-CD3ζ+TLR4, and HER2 CAR-CD40+CD3ζ prepared in Example 1 were transfected into macrophages obtained according to the method in Example 2 (the transfection method was the same as in Example 2). Two days later, the cells were resuspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0214] The negative macrophage group (UTD group) consisted of untransfected RNA macrophages suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until the density reached 1×10⁶ cells / mL. 6 Cells / mL.

[0215] Transfected macrophages and negative macrophages are collectively referred to as effector cells, while OE19 cells are referred to as target cells. Effector cells and target cells are added to culture dishes at an effector-to-target ratio of 1:1 (cell number ratio). After culturing for 2 hours, cells are collected, stained with anti-CD11b-FITC (purchased from eBioscience), and analyzed by flow cytometry for cells containing the DiR label in the CD11b+ population; these cells are macrophages that have engulfed OE19 cells.

[0216] The results showed that the HER2 CAR combination with ICD-CAR was superior to the ICD-CAR alone in terms of phagocytosis ability, especially the phagocytosis ability of the PI3K-containing ICD was significantly improved (see Figure 19).

[0217] 2) The IVT RNAs prepared in Example 1 for PSMA CAR-FCE, PSMA CAR-FCE+IFNgR, PSMA CAR-FCE+PI3K, PSMA CAR-FCE+TLR2, PSMA CAR-FCE+TLR4, PSMA CAR-CD40+FCE, PSMA CAR-CD3ζ, PSMA CAR-CD3ζ+IFNgR, PSMA CAR-CD3ζ+PI3K, PSMA CAR-CD3ζ+TLR4, and PSMA CAR-CD40+CD3ζ were transfected into macrophages obtained according to the method in Example 2 (the transfection method was the same as in Example 2). Two days later, the cells were resuspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium until the density was 1×10⁻⁶. 6 Cells / mL.

[0218] The negative macrophage group (UTD group) consisted of untransfected RNA macrophages suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until the density reached 1×10⁶ cells / mL. 6 Cells / mL.

[0219] Transfected macrophages and negative macrophages are collectively referred to as effector cells, while LnCap cells (purchased from eBioscience) are referred to as target cells. Effector cells and target cells are added to culture dishes at an effector-to-target ratio of 1:1 (cell number ratio). After culturing for 2 hours, cells are collected, stained with anti-CD11b-FITC (purchased from eBioscience), and analyzed by flow cytometry for cells containing DiR markers in the CD11b+ population; these cells are macrophages that have phagocytosed target cells.

[0220] The results showed that the PSMA CAR combination with ICD-CAR was superior to the ICD-CAR alone in terms of phagocytosis capacity, especially the phagocytosis capacity of the ICD containing PI3K (see Figure 20).

[0221] 3) The IVT RNAs of GPC3 CAR-FCE, GPC3 CAR-FCE+IFNgR, GPC3 CAR-FCE+PI3K, GPC3 CAR-FCE+TLR2, GPC3 CAR-FCE+TLR4, GPC3 CAR-CD40+FCE, GPC3 CAR-CD3ζ, GPC3 CAR-CD3ζ+IFNgR, GPC3 CAR-CD3ζ+PI3K, GPC3 CAR-CD3ζ+TLR4, and GPC3 CAR-CD40+CD3ζ prepared in Example 1 were transfected into macrophages obtained according to the method in Example 2 (the transfection method was the same as in Example 2). Two days later, the cells were resuspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶. 6 Cells / mL.

[0222] The negative macrophage group (UTD group) consisted of untransfected RNA macrophages suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until the density reached 1×10⁶ cells / mL. 6 Cells / mL.

[0223] Transfected macrophages and negative macrophages were collectively referred to as effector cells, while HepG2 cells (purchased from Shanghai Anwei Biotechnology) were referred to as target cells. Effector cells and target cells were added to culture dishes at an effector-to-target ratio of 1:1 (cell number ratio). After culturing for 2 hours, cells were collected, stained with anti-CD11b-FITC (purchased from eBioscience), and analyzed by flow cytometry for DiR-labeled cells in the CD11b+ population; these cells were macrophages that phagocytosed target cells.

[0224] The results showed that the GPC3 CAR combination ICD-CAR was superior to the ICD-CAR alone in terms of phagocytosis capacity, especially the phagocytosis capacity of the ICD containing PI3K (see Figure 21).

[0225] Example 4. In vitro killing assay of HER2, PSMA and GPC3 CAR-M transfected with IVT RNA

[0226] The target cells for cytotoxicity included OE19-luciferase, LnCap-luciferase, and HepG2-luciferase. OE19, LnCap, and HepG2 cells were seeded in 6-well plates and cultured to a confluence of 60–80%. Commercially available luciferase lentivirus (purchased from Genomics) was used to modify the target cells at an MOI of 10. Puromycin was used to screen the modified cells, resulting in stable luciferase-expressing OE19, LnCap, and HepG2 cell lines for in vitro cytotoxicity assays.

[0227] OE19-luciferase cells (OE19-luciferase) were harvested and resuspended in preheated complete culture medium until the cell density was approximately 1 × 10⁻⁶. 5 Cells / mL. 100 μL per well (1 × 10⁶ cells / mL). 4 Cells were seeded in 96-well plates (Corning / Cat: 3610) and incubated at 37°C for 2 hours.

[0228] 1) The IVT RNAs of HER2 CAR-FCE, HER2 CAR-FCE+IFNgR, HER2 CAR-FCE+PI3K, HER2 CAR-FCE+TLR2, HER2 CAR-FCE+TLR4, HER2 CAR-CD40+FCE, HER2 CAR-CD3ζ, HER2 CAR-CD3ζ+IFNgR, HER2 CAR-CD3ζ+PI3K, HER2 CAR-CD3ζ+TLR4, and HER2 CAR-CD40+CD3ζ prepared in Example 1 were transfected into macrophages obtained according to the method in Example 2 (the transfection method was the same as in Example 2). Two days later, the cells were resuspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0229] The negative macrophage group (UTD group) consisted of untransfected RNA macrophages suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until the density reached 1×10⁶ cells / mL. 6 Cells / mL.

[0230] Effector cells (transfected macrophages and negative macrophages) were prepared and added at effector-to-target ratios (cell count ratios) of 5:1, 1:1, 1:5, and 1:10. After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 minutes, and 100 μL of the supernatant was discarded after cell precipitation. 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520). After 5 minutes of reaction, read the values ​​on the microplate reader.

[0231] The results showed that, based on OE19-luciferase target cells, HER2-CAR molecules containing ICD combinations exhibited higher killing activity, with the five ICD combinations of FCE-IFNgR, FCE-PI3K, FCE-TLR2, CD3ζ-IFNgR, and CD3ζ-PI3K showing the best performance. Particularly under conditions of low target-to-cell ratio, the killing activity of HER2-CAR molecules containing ICD combinations was superior to that of CAR molecules containing individual ICDs (see Figure 22).

[0232] 2) The IVT RNAs of PSMA CAR-FCE, PSMA CAR-FCE+IFNgR, PSMA CAR-FCE+PI3K, PSMA CAR-FCE+TLR2, PSMA CAR-FCE+TLR4, PSMA CAR-CD40+FCE, PSMA CAR-CD3ζ, PSMA CAR-CD3ζ+IFNgR, PSMA CAR-CD3ζ+PI3K, PSMA CAR-CD3ζ+TLR4, and PSMA CAR-CD40+CD3ζ prepared in Example 1 were transfected into macrophages obtained according to the method in Example 2 (the transfection method was the same as in Example 2). Two days later, the cells were resuspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶. 6 Cells / mL.

[0233] The negative macrophage group (UTD group) consisted of untransfected RNA macrophages suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until the density reached 1×10⁶ cells / mL. 6 Cells / mL.

[0234] Effector cells (transfected macrophages and negative macrophages) were prepared and added at effector-to-target ratios (cell count ratios) of 5:1, 1:1, 1:5, and 1:10. After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 minutes, and 100 μL of the supernatant was discarded after cell precipitation. 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520). After 5 minutes of reaction, read the values ​​on the microplate reader.

[0235] The results showed that, based on LnCap-luciferase target cells, PSMA-CAR molecules containing ICD combinations exhibited higher killing efficacy than CAR molecules containing a single ICD. Among these, the four ICD combinations of FCE-IFNgR, FCE-PI3K, CD3ζ-IFNgR, and CD3ζ-PI3K showed the best performance. The killing efficacy of PSMA-CAR molecules containing ICD combinations was superior to that of CAR molecules containing a single ICD in each case (see Figure 23).

[0236] 3) The IVT RNAs of GPC3 CAR-FCE, GPC3 CAR-FCE+IFNgR, GPC3 CAR-FCE+PI3K, GPC3 CAR-FCE+TLR2, GPC3 CAR-FCE+TLR4, GPC3 CAR-CD40+FCE, GPC3 CAR-CD3ζ, GPC3 CAR-CD3ζ+IFNgR, GPC3 CAR-CD3ζ+PI3K, GPC3 CAR-CD3ζ+TLR4, and GPC3 CAR-CD40+CD3ζ prepared in Example 1 were transfected into macrophages obtained according to the method in Example 2 (the transfection method was the same as in Example 2). Two days later, the cells were resuspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶. 6 Cells / mL.

[0237] The negative macrophage group (UTD group) consisted of untransfected RNA macrophages suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until the density reached 1×10⁶ cells / mL. 6 Cells / mL.

[0238] Effector cells (transfected macrophages and negative macrophages) were prepared and added at effector-to-target ratios (cell count ratios) of 5:1, 1:1, 1:5, and 1:10. After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 minutes, and 100 μL of the supernatant was discarded after cell precipitation. 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520). After 5 minutes of reaction, read the values ​​on the microplate reader.

[0239] The results showed that, based on HepG2-luciferase target cells, GPC3-CAR molecules containing ICD combinations exhibited higher killing efficacy than CAR molecules containing a single ICD. Among these, the four ICD combinations of FCE-IFNgR, FCE-PI3K, CD3ζ-IFNgR, and CD3ζ-PI3K were the most effective. The killing efficacy of GPC3-CAR molecules containing ICD combinations was superior to that of CAR molecules containing a single ICD in each case (see Figure 24).

[0240] Example 5. Detection of HER2, PSMA, and GPC3 CAR-M molecule expression in adenovirus-transfected monocytes.

[0241] Apheresis blood obtained from a commercial company (with relevant ethical approvals) was centrifuged at 200g for 10 minutes to remove platelets and some red blood cells. The supernatant was discarded, and the cell pellet was mixed with PBS and added to Ficoll (lymphocyte separation medium, purchased from Sigma), then centrifuged at 1200 rpm for 30 minutes. After centrifugation, PBMCs were recovered and washed once with phosphate-buffered saline (PBS) by centrifugation. PBMCs were co-incubated with CD14+ magnetic beads (purchased from Miltenyi) for 15 minutes, and CD14+ cells were separated using a CD14 sorting column (purchased from Miltenyi) to harvest CD14+ monocytes.

[0242] The CAR sequences of FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K were constructed into a plasmid system with an AD5 / F35 vector backbone (purchased from Yunzhou Biotechnology). The plasmid was linearized in vitro using Pac I restriction enzyme, and then 293A cells were infected. Cells were cultured until cytopathic effects (plaques between cells, some cells appearing in chains, etc.) reached 70-80%, at which point the P0 generation virus was harvested. The P0 generation virus was amplified, and when cytopathic effects reached ≥50%, the cells were harvested, lysed, and the virus was collected. After lysis, the virus was purified and dispensed for use.

[0243] 1) Adenoviruses containing different HER2 CAR-ICD molecules (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) were added to monocytes. The negative control group (UTD group) consisted of untransfected monocytes. Cells were harvested on day 2 post-transfection, with at least 1 × 10⁻⁶ cells per group. 6Cells were collected, mixed with PBS, and centrifuged at 400g for 5 minutes. The cell pellet was then mixed with PBS, and HER2 protein (purchased from Acro) (4 μL / test) was added. The cells were incubated at room temperature for 20 minutes, centrifuged at 400g for 5 minutes to remove unbound HER2 protein. After washing, the cells were subjected to flow cytometry analysis of HER2-targeting CAR molecules, and the monocyte viability (monocyte viability was obtained by dividing the number of viable monocytes recovered by the total number of recovered cells) and recovery volume (Figure 28) of each group were analyzed.

[0244] The percentage of CAR molecules expressed on the surface of monocyte membranes was detected by flow cytometry (Figure 31), the MFI of CD80 molecule expression level (Figure 34), and the MFI of CD163 molecule expression level (Figure 37).

[0245] The results showed that, in terms of viability, the various ICD combinations were on par with the negative control group; in terms of recovery, the various ICD combinations were superior to the corresponding FCE or CD3ζ ICD groups alone; at the same time, all ICD combinations had good CAR positive expression levels, which were better than those of ICDs alone; phenotypic information showed that all groups after adenovirus transfection tended towards M1(CD80) + ) polarization, while simultaneously exhibiting M2 (CD163) polarization. + ) resistance.

[0246] 2) Adenoviruses containing different PSMA CAR-ICD molecules (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) were added to monocytes. The negative control group (UTO group) consisted of untransfected monocytes. Cells were harvested on day 2 post-transfection, with at least 1 × 10⁻⁶ cells per group. 6 Cells were collected, mixed with PBS, and centrifuged at 400g for 5 minutes. The cell pellet was then mixed with PBS, and PSMA protein (4 μL / test) was added. The mixture was incubated at room temperature for 20 minutes, centrifuged at 400g for 5 minutes to remove unbound PSMA protein. After washing, the cells were subjected to flow cytometry analysis of PSMA-targeting CAR molecules, and the monocyte viability (Figure 26) and recovery volume (Figure 29) of each group were analyzed.

[0247] The percentage of CAR molecules expressed on the surface of monocyte membranes was detected by flow cytometry (Figure 32), the MFI of CD80 molecule expression level (Figure 35), and the MFI of CD163 molecule expression level (Figure 38).

[0248] The results showed that, in terms of viability, the various ICD combinations were on par with the negative control group; in terms of recovery, the various ICD combinations were superior to the negative control group; at the same time, all ICD combinations had good CAR positive expression levels, which were better than those of ICD alone; phenotypic information showed that all groups after adenovirus transfection tended towards M1(CD80) + ) polarization, while simultaneously exhibiting M2 (CD163) polarization. + ) resistance.

[0249] 3) Adenoviruses containing different GPC3 CAR-ICD molecules (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) were added to monocytes. The negative control group (UTO group) consisted of untransfected monocytes. Cells were harvested on day 2 post-transfection, with at least 1 × 10⁻⁶ cells per group. 6 Cells were collected, mixed with PBS, and centrifuged at 400g for 5 minutes. The cell pellet was then mixed with PBS, and GPC3 protein (4 μL / test) was added. The mixture was incubated at room temperature for 20 minutes, centrifuged at 400g for 5 minutes to remove unbound GPC3 protein. After washing, the cells were subjected to flow cytometry analysis of GPC-targeting CAR molecules, and the monocyte viability (Figure 27) and recovery volume (Figure 30) of each group were analyzed.

[0250] The percentage of CAR molecules expressed on the surface of monocyte membranes was detected by flow cytometry (Figure 33), the MFI of CD80 molecule expression level (Figure 36), and the MFI of CD163 molecule expression level (Figure 39).

[0251] The results showed that, in terms of viability, the various ICD combinations were on par with the negative control group; in terms of recovery, the various ICD combinations were superior to the negative control group; at the same time, all ICD combinations had good CAR positive expression levels, which were better than those of ICD alone; phenotypic information showed that all groups after adenovirus transfection tended towards M1(CD80) + ) polarization, while simultaneously exhibiting M2 (CD163) polarization. + The inhibitory phenotype showed a suppressive effect in various ICDs; in terms of the inhibitory phenotype, all ICDs showed an inhibitory effect in the inhibitory phenotype.

[0252] Example 6. In vitro phagocytosis experiment of HER2, PSMA and GPC3 CAR-M transfected monocytes with adenovirus.

[0253] OE19 cells were resuspended in complete culture medium until a density of 1×10⁻⁶ cells was achieved. 6 Cells / mL.

[0254] 1) Adenoviruses containing different HER2 CAR-ICD molecules (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) were added to monocytes (preparation method as in Example 5). One day after transfecting the monocytes prepared in Example 5 with adenovirus, the cells were suspended in PBS, DiR dye was added (final concentration 5 μM), and incubated at 37°C for 20 minutes. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0255] The negative monocyte group (UTD group) consisted of untransfected adenovirus monocytes, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL.

[0256] Effector cells (transfected monocytes and negative monocytes) and target cells (OE19 cells) were added to culture dishes at a 1:1 ratio (cell number ratio). After co-culturing for 2 hours, the cells were collected, stained with anti-CD11b-FITC (purchased from eBioscience), and analyzed by flow cytometry for DiR-labeled cells in the CD11b+ population. The results are shown in Figure 40: these cells are monocytes that phagocytose OE19 cells, and in terms of phagocytic ability, the combined ICD-CARs are superior to the individual ICD-CARs.

[0257] 2) Adenoviruses containing different PSMA CAR-ICD (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) molecules were added to monocytes (preparation method as in Example 5). One day after transfecting the monocytes prepared in Example 5 with adenovirus, the cells were suspended in PBS, DiR dye was added (final concentration 5 μM), and incubated at 37°C for 20 minutes. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0258] The negative monocyte group (UTD group) consisted of untransfected adenovirus monocytes, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL.

[0259] Effector cells (transfected monocytes and negative monocytes) and target cells (LnCap cells) were added to culture dishes at a 1:1 ratio (cell number ratio). After co-culturing for 2 hours, the cells were collected, stained with anti-CD11b-FITC (purchased from eBioscience), and analyzed by flow cytometry for DiR-labeled cells in the CD11b+ population. The results are shown in Figure 41: These cells are monocytes that phagocytose LnCap cells, and in terms of phagocytic ability, the combined ICD-CARs are superior to the individual ICD-CARs.

[0260] 3) Adenoviruses containing different GPC3 CAR-ICD molecules (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) were added to monocytes (preparation method as in Example 5). One day after transfecting the monocytes prepared in Example 5 with adenovirus, the cells were suspended in PBS, DiR dye was added (final concentration 5 μM), and incubated at 37°C for 20 minutes. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in preheated complete culture medium to a density of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0261] The negative monocyte group (UTD group) consisted of untransfected adenovirus monocytes, suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in pre-warmed complete culture medium until the density was 1×10⁶. 6 Cells / mL.

[0262] Effector cells (transfected monocytes and negative monocytes) and target cells (HepG2 cells) were added to culture dishes at a 1:1 ratio (cell number ratio). After co-culturing for 2 hours, the cells were collected, stained with anti-CD11b-FITC (purchased from eBioscience), and analyzed by flow cytometry for DiR-labeled cells in the CD11b+ population. The results are shown in Figure 42: these cells are monocytes that phagocytose HepG2 cells, and in terms of phagocytic ability, the combined ICD-CARs are superior to the individual ICD-CARs.

[0263] Example 7. In vitro killing experiment of HER2, PSMA and GPC3 CAR-M transfected monocytes by adenovirus.

[0264] OE19-luciferase cells (same as OE19-luciferase in Example 4) were harvested and resuspended in preheated complete culture medium at a cell density of 1×10⁻⁶. 5 Cells / mL. After mixing, dispense 100 μL per well (1 × 10⁻⁶ cells / mL). 4(Each plate was prepared in 96-well plates (Corning / Cat: 3610) and incubated at 37°C for 2 hours.

[0265] 1) Adenoviruses containing different HER2 CAR-ICD molecules (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) were added to monocytes (preparation method as in Example 5). One day after transfecting the monocytes prepared in Example 5 with adenovirus, they were suspended in PBS. The cells were centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium until the density reached 1×10⁻⁶. 6 Cells / mL.

[0266] The negative monocyte group (UTD group) consisted of untransfected adenovirus monocytes suspended in PBS. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium to a density of 1×10⁶ cells / mL. 6 Cells / mL.

[0267] Effector cells (transfected monocytes and negative monocytes) were prepared and added at effector-to-target ratios (cell count ratios) of 5:1, 1:1, 1:5, and 1:10. After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 minutes. 100 μL of the supernatant was discarded after cell precipitation, and 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520), after 5 minutes of reaction, read the value on the microplate reader.

[0268] The results showed that, based on OE19-luciferase target cells, CAR molecules containing ICD combinations exhibited higher killing activity, with the ICD combinations FCE-PI3K and CD3ζ-PI3K2 showing the best killing activity. CAR molecules containing ICD combinations were all superior to CAR molecules containing ICDs alone (see Figure 43).

[0269] 2) Adenoviruses containing different PSMA CAR-ICD (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) molecules were added to monocytes (preparation method as in Example 5). One day after transfecting the monocytes prepared in Example 5 with adenovirus, they were suspended in PBS. The cells were centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium until the density reached 1×10⁻⁶. 6 Cells / mL.

[0270] The negative monocyte group (UTD group) consisted of untransfected adenovirus monocytes suspended in PBS. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium to a density of 1×10⁶ cells / mL. 6 Cells / mL.

[0271] Effector cells (transfected monocytes and negative monocytes) were prepared and added at effector-to-target ratios (cell count ratios) of 5:1, 1:1, 1:5, and 1:10. After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 minutes. 100 μL of the supernatant was discarded after cell precipitation, and 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520), after 5 minutes of reaction, read the value on the microplate reader.

[0272] The results showed that, based on LnCap-luciferase target cells, CAR molecules containing ICD combinations had higher killing power than CAR molecules containing ICDs alone (see Figure 44).

[0273] 3) Adenoviruses containing different GPC3 CAR-ICD molecules (FCE, FCE-IFNgR (IFNR), FCE-PI3K, CD3ζ, CD3ζ-IFNgR, CD3ζ-PI3K) were added to monocytes (preparation method as in Example 5). One day after transfecting the monocytes prepared in Example 5 with adenovirus, they were suspended in PBS. The cells were centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium until the density reached 1×10⁻⁶. 6 Cells / mL.

[0274] The negative monocyte group (UTD group) consisted of untransfected adenovirus monocytes suspended in PBS. The cells were centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium to a density of 1×10⁶ cells / mL. 6 Cells / mL.

[0275] Effector cells (transfected monocytes and negative monocytes) were prepared and added at effector-to-target ratios (cell count ratios) of 5:1, 1:1, 1:5, and 1:10. After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 minutes. 100 μL of the supernatant was discarded after cell precipitation, and 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520), after 5 minutes of reaction, read the value on the microplate reader.

[0276] The results showed that, based on HepG2-luciferase target cells, CAR molecules containing ICD combinations had higher killing power than CAR molecules containing ICDs alone (see Figure 45).

[0277] Example 8. Functional comparison of adenovirus-transfected monocytes and induced macrophages

[0278] 1) After plating CD14+ mononuclear cells obtained according to Example 5, adenoviruses expressing different HER2 CAR-ICD molecules (prepared in the same way as in Example 5) were added, containing different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0279] The negative control group (UTD group) consisted of one group of untransfected adenovirus mononuclear cells.

[0280] Cells were harvested on day 2 (Mono-D state) and day 7 (Macrophage state) after transfection.

[0281] The percentage of CAR molecules expressed on the surface of monocytes and macrophages was detected (method described in Example 5);

[0282] The phagocytic effect on OE19-luciferase target cells was detected (method described in Example 6);

[0283] The killing effect on OE19-luciferase target cells was detected at effector-to-target ratios of 5:1 and 1:1 (see Example 7 for the method).

[0284] The results showed that each ICD combination had good CAR positive expression levels in both monocytes and macrophages (Figure 46). These cells were either monocytes (day 2) or macrophages (day 7) that had phagocytosed OE19. In terms of phagocytic ability, the combined ICD-CAR was superior to the individual ICD-CAR in both monocyte and macrophage cases (Figure 49). In terms of killing ability, in the case of monocytes (day 2), the combined ICD-CAR was superior to the individual ICD-CAR in both high and low target ratios. In the case of macrophages (day 7), the killing level tended to saturate (~100%) under the high target ratio (5:1) and the killing level was also significantly improved (>70%) under the low target ratio (1:1) (Figure 52).

[0285] 2) After plating CD14+ mononuclear cells obtained according to Example 5, adenoviruses expressing different PSMA CAR-ICD molecules (prepared in the same way as in Example 5) were added, containing different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0286] One negative control group (UTD group) consisted of untransfected adenovirus monocytes. Cells were harvested on day 2 and day 7 post-transfection.

[0287] The percentage of CAR molecules expressed on the surface of monocytes and macrophages was detected (method described in Example 5);

[0288] The phagocytic effect on LnCap-luciferase target cells was detected (method described in Example 6);

[0289] The killing effect on LnCap-luciferase target cells was detected at effector-target ratios of 5:1 and 1:1 (see Example 7 for the method).

[0290] The results showed that each ICD combination had good CAR positive expression levels in both monocytes and macrophages (Figure 47); these cells were monocytes or macrophages that phagocytosed LnCap, and in terms of phagocytic ability, the combined ICD-CARs were superior to the individual ICD-CARs (Figure 50); in terms of killing ability, in the cases of monocytes (day 2) or macrophages (day 7), the combined ICD-CARs were superior to the individual ICD-CARs at both high and low efficiency target ratios (Figure 53).

[0291] 3) After plating CD14+ mononuclear cells obtained according to Example 5, adenoviruses expressing different GPC3 CAR-ICD molecules (prepared in the same way as in Example 5) were added, containing different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0292] One negative control group (UTD group) consisted of untransfected adenovirus monocytes. Cells were harvested on day 2 and day 7 post-transfection.

[0293] The percentage of CAR molecules expressed on the surface of monocytes and macrophages was detected (method described in Example 5);

[0294] The phagocytic effect on HepG2-luciferase target cells was detected (method described in Example 6);

[0295] The killing effect on HepG2-luciferase target cells was detected at effector-to-target ratios of 5:1 and 1:1 (see Example 7 for the method).

[0296] The results showed that each ICD combination had good CAR positive expression levels in both monocytes and macrophages (Figure 48); these cells were monocytes or macrophages that phagocytosed HepG2, and in terms of phagocytic ability, the combined ICD-CARs were superior to the individual ICD-CARs (Figure 51); in terms of killing ability, in the cases of monocytes (day 2) or macrophages (day 7), the combined ICD-CARs were superior to the individual ICD-CARs at both high and low efficiency target ratios (Figure 54).

[0297] Example 9. Mononuclear cell exhaustion level after adenovirus transfection

[0298] 1) After the CD14+ mononuclear cells obtained in Example 5 were plated, adenoviruses expressing different HER2 CAR-ICD molecules (prepared in the same way as in Example 5) were added, which contained different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0299] The negative control group (UTD group) consisted of one group of untransfected adenovirus mononuclear cells.

[0300] Cells were harvested on day 2 after transfection (day 2) and co-incubated with OE19-luciferase target cells at E:T = 1:1 for 4 days (day 6). The expression of HER2 CAR, CD11b, CD80 and CD86 molecules was detected by flow cytometry on the harvested cells.

[0301] The results showed that, compared with the corresponding ICD-only group or UTD group, the CAR-M of the ICD combination had a lower depletion capacity under co-incubation conditions with tumor cells (Figure 55); and the CAR-positive expression level of the ICD combination after co-incubation was significantly better than that of the ICD-only group (Figure 58), and it also had a significant advantage in maintaining the M1 macrophage phenotype (CD80 and CD86 expression) compared with the ICD-only group (Figure 61).

[0302] 2) After the CD14+ mononuclear cells obtained in Example 5 were plated, they were divided into 6 experimental groups and adenoviruses expressing different PSMA CAR-ICD molecules (prepared in the same way as in Example 5) were added to each group. These groups contained different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0303] The negative control group (UTD group) consisted of one group of untransfected adenovirus mononuclear cells.

[0304] Cells were harvested on day 2 after transfection and co-incubated with LnCap-luciferase target cells at an E:T ratio of 1:1 for 4 days. The expression of PSMA CAR, CD11b, CD80, and CD86 molecules was detected by flow cytometry in the harvested cells.

[0305] The results showed that, compared with the corresponding single ICD group or UTD group, the CAR-M containing PI3K ICD combination had a lower depletion capacity under co-incubation conditions with tumor cells (Figure 56); and the CAR-positive expression level of the co-incubated ICD combination was significantly better than that of the single ICD group (Figure 59), and it also had a significant advantage in maintaining the M1 macrophage phenotype (CD80 and CD86 expression) compared with the single ICD group (Figure 62).

[0306] 3) After the CD14+ mononuclear cells obtained in Example 5 were plated, adenoviruses expressing different GPC3 CAR-ICD molecules (prepared in the same way as in Example 5) were added, which contained different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0307] The negative control group (UTD group) consisted of one group of untransfected adenovirus mononuclear cells.

[0308] Cells were harvested on day 2 after transfection and co-incubated with HepG2-luciferase target cells at an E:T ratio of 1:1 for 4 days. The expression of PSMA CAR, CD11b, CD80, and CD86 molecules was detected by flow cytometry in the harvested cells.

[0309] The results showed that, compared with the corresponding ICD-only group or UTD group, the CAR-M of the ICD combination had a lower depletion capacity under co-incubation conditions with tumor cells (Figure 57); and the CAR-positive expression level of the ICD combination after co-incubation was significantly better than that of the ICD-only group (Figure 60), and it also had a significant advantage in maintaining the M1 macrophage phenotype (CD80 and CD86 expression) compared with the ICD-only group (Figure 63).

[0310] Example 10. Long-lasting killing effect of adenovirus-transfected monocytes

[0311] 1) After the CD14+ mononuclear cells obtained in Example 5 were plated, adenoviruses expressing different HER2 CAR-ICD molecules (prepared in the same way as in Example 5) were added, which contained different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0312] The negative control group (UTD group) consisted of one group of untransfected adenovirus mononuclear cells.

[0313] Cells were harvested on day 2 post-transfection (day 0) and prepared with OE19-luciferase target cells at effector-to-target ratios of 5:1, 1:1, and 1:5, respectively. Effector cells (transfected monocytes and negative monocytes, 100 μL per well) were added to each well and plated in parallel. After 72 hours, the cells were centrifuged at 500g for 5 min to precipitate the cells. 100 μL of the supernatant was discarded, and complete culture medium containing OE19-luciferase target cells was added to each well (100 μL per well). Luciferase substrate assays were performed on day 2 (48 h), day 3 (72 h before and 72-add after addition), and day 6 (72+72 h).

[0314] Luciferase assay: 96-well white bottom plate was sterilized and centrifuged at 400g for 5 minutes. The substrate reaction solution (Promega / Cat.E250) was prepared, thawed at room temperature, and then 100ml was added. Add 1 vial to Luciferase Assay Buffer Mix Luciferase Assay Substrate (lyophilized) thoroughly; add 50 μL of the prepared solution to each well of the 96-well plate to be tested. After 5 minutes of reaction, the Luciferase Assay reagent was used, and the readings were taken using an ELISA reader.

[0315] The results showed that under the E:T = 5:1 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group exhibited a significant and sustained inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 64). Under the E:T = 1:1 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group also showed a significant and sustained inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 65). Under the E:T = 1:5 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group showed a weaker inhibitory trend in luciferase values, with the ICD-PI3K combination group showing a significant inhibitory effect compared with the corresponding individual ICD group (Figure 66). This indicates that the ICD combination group has the ability to sustainably inhibit the killing of target cells, and under low target-to-cell ratio conditions, its killing and inhibitory effect is significantly better than that of the individual ICD group.

[0316] 2) After the CD14+ mononuclear cells obtained in Example 5 were plated, they were divided into 6 experimental groups and adenoviruses expressing different PSMA CAR-ICD molecules (prepared in the same way as in Example 5) were added to each group. These groups contained different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0317] The negative control group (UTD group) consisted of one group of untransfected adenovirus mononuclear cells.

[0318] Cells were harvested on day 2 post-transfection and prepared with LnCap-luciferase target cells at effector-to-target ratios of 5:1, 1:1, and 1:5, respectively. Effector cells (transfected monocytes and negative monocytes, 100 μL per well) were added to each well and plated in parallel. After 72 hours, the cells were centrifuged at 500g for 5 min to collect the cell pellet. 100 μL of the supernatant was discarded, and 100 μL of complete culture medium containing LnCap-luciferase target cells was added to each well. Luciferase substrate assays were performed on days 2, 3, and 6 post-plate preparation.

[0319] The results showed that under the E:T = 5:1 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group exhibited a significant and sustained inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 67). Under the E:T = 1:1 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group also showed a significant and sustained inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 68). Under the E:T = 1:5 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group showed a weaker inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 69). This indicates that the ICD combination group has the ability to sustainably inhibit the killing of target cells, and its killing inhibition effect is significantly better than that of the individual ICD group.

[0320] 3) After the CD14+ mononuclear cells obtained in Example 5 were plated, they were divided into 6 experimental groups and adenoviruses expressing different GPC3 CAR-ICD molecules (prepared in the same way as in Example 5) were added to each group. These groups contained different ICD combinations FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K.

[0321] The negative control group (UTD group) consisted of one group of untransfected adenovirus mononuclear cells.

[0322] Cells were harvested on day 2 post-transfection and prepared with HepG2-luciferase target cells at effector-to-target ratios of 5:1, 1:1, and 1:5, respectively. Effector cells (transfected monocytes and negative monocytes, 100 μL per well) were added to each well and plated in parallel. After 72 hours, the cells were centrifuged at 500g for 5 min to collect the cell pellet. 100 μL of the supernatant was discarded, and 100 μL of complete culture medium containing HepG2-luciferase target cells was added to each well. Luciferase substrate assays were performed on days 2, 3, and 6 post-plate preparation.

[0323] The results showed that under the E:T = 5:1 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group exhibited a significant and sustained inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 70). Under the E:T = 1:1 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group also showed a significant and sustained inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 71). Under the E:T = 1:5 condition, compared with the UTD group, both the ICD combination group and the corresponding individual ICD group showed a weaker inhibitory trend in luciferase values, with the ICD combination group showing a stronger inhibitory effect than the corresponding individual ICD group (Figure 72). This indicates that the ICD combination group has the ability to sustainably inhibit the killing of target cells, and its killing inhibition effect is significantly better than that of the individual ICD group.

[0324] Example 11. Infiltration and killing of 3D tumor spheres by mononuclear cells after adenovirus transfection

[0325] 1) Using the overexpression modification scheme in Example 4, the OE19 cell line was modified with NLS-mCherry lentivirus (MOI of 10), and the single clones selected by screening in complete medium containing 300 μg / mL of hygromycin (purchased from MCE) (hereinafter referred to as OE19-NLS mCherry) were selected.

[0326] After plating CD14+ monocytes obtained according to Example 5, adenoviruses containing different HER2 CAR-ICD molecules (prepared using the same method as in Example 5) were added. These adenoviruses contained different ICD combinations: FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K. One negative control group (Mock group) consisted of monocytes not transfected with adenovirus.

[0327] Pre-resuscitate OE19-NLS mCherry monoclonal cells until cell confluence reaches >50%, harvest cells, and resuspend them in complete culture medium at a cell density of 5 × 10⁶ cells / mL. 4 / mL. Take a 96-well transparent round-bottom ultra-low adsorption microplate (purchased from Corning / #7007), add 100μL to each well, and incubate in an Incucyte cell culture incubator for 4 days until 3D tumor microspheres are formed.

[0328] Cells were harvested on day 2 post-transfection and added to 96-well U-plates containing OE19-NLS mCherry monoclonal target cells that had formed 3D tumor spheres. Effector cells (transfected monocytes and negative monocytes, 100 μL per well) were added to each well at effector-to-target ratios of 5:1 and 1:1, and the plates were plated in parallel. The plates were then placed in Incucyte for monitoring at 2-hour intervals. After 96 hours, 50 μL of complete culture medium (100 μL per well) was added.

[0329] The results showed that, compared with the Mock group, both the ICD combination group and the ICD group alone had a certain inhibitory effect on OE19-NLS mCHerry (red) tumor spheres. Meanwhile, the ICD combination group showed better infiltration and lysis of the tumor spheres; under E:T = 5:1 or 1:1 conditions, the ICD-PI3K group achieved near-complete eradication (Figure 73).

[0330] 2) Using the overexpression modification scheme in Example 4, the LnCap cell line was modified with NLS-mCherry lentivirus (MOI of 10), and the single clones selected by screening in complete culture medium containing 300 μg / mL of hygromycin (purchased from MCE) (hereinafter collectively referred to as LnCap-NLS mCherry).

[0331] After plating CD14+ monocytes obtained according to Example 5, adenoviruses containing different PSMA CAR-ICD molecules (prepared using the same method as in Example 5) were added, each containing different ICD combinations: FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K. One negative control group (Mock group) consisted of monocytes not transfected with adenovirus.

[0332] LnCap-NLS mCherry monoclonal cells were pre-resuscitated until cell confluence reached >50%, then harvested and resuspended in complete culture medium at a cell density of 5 × 10⁶ cells / mL. 4 / mL. Take a 96-well transparent round-bottom ultra-low adsorption microplate (purchased from Corning / #7007), add 100μL to each well, and incubate in an Incucyte cell culture incubator for 4 days until 3D tumor microspheres are formed.

[0333] Cells were harvested on day 2 post-transfection and added to 96-well U-plates containing LnCap-NLS mCherry monoclonal target cells that had formed 3D tumor spheres. Effector cells (transfected monocytes and negative monocytes, 100 μL per well) were added at effector-to-target ratios of 5:1 and 1:1, and the plates were plated in parallel. The plates were then placed in Incucyte for monitoring at 2-hour intervals. After 96 hours, 50 μL of complete culture medium was added (100 μL per well).

[0334] The results showed that, compared with the Mock group, both the ICD combination group and the ICD group alone had a certain inhibitory effect on LnCap-NLS mCHerry (red) tumor spheres. Meanwhile, the ICD combination group showed better invasive and lytic effects on the tumor spheres (Figure 74).

[0335] 3) Using the overexpression modification scheme in Example 4, the HepG2 cell line was modified with NLS-mCherry lentivirus (MOI of 10), and the single clones selected by screening in complete medium containing 300 μg / mL of hygromycin (purchased from MCE) (hereinafter collectively referred to as HepG2-NLS mCherry) were selected.

[0336] After seeding CD14+ monocytes obtained according to Example 5, adenoviruses containing different GPC3 CAR-ICD molecules (prepared using the same method as in Example 5) were added to the plates. These adenoviruses contained different ICD combinations: FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR, and CD3ζ-PI3K. One negative control group (Mock group) consisted of monocytes not transfected with adenovirus.

[0337] HepG2-NLS mCherry monoclonal cells were pre-resuscitated until cell confluence reached >50%, then harvested and resuspended in complete culture medium at a cell density of 5 × 10⁶ cells / mL. 4 / mL. Take a 96-well transparent round-bottom ultra-low adsorption microplate (purchased from Corning / #7007), add 100μL to each well, and incubate in an Incucyte cell culture incubator for 4 days until 3D tumor microspheres are formed.

[0338] Cells were harvested on day 2 post-transfection and added to 96-well U-plates containing HepG2-NLS mCherry monoclonal target cells that had formed 3D tumor spheres. Effector cells (transfected monocytes and negative monocytes, 100 μL per well) were added to each well at effector-to-target ratios of 5:1 and 1:1, and the plates were plated in parallel. The plates were then placed in Incucyte for monitoring at 2-hour intervals. After 96 hours, 50 μL of complete culture medium was added (100 μL per well).

[0339] The results showed that, compared with the Mock group, both the ICD combination group and the ICD group alone had a certain inhibitory effect on HepG2-NLS mCHerry (red) tumor spheres. Meanwhile, the ICD combination group showed better infiltration and lysis of the tumor spheres (Figure 75).

[0340] Example 12. Comparison of in vitro functions of GPC3 combined with ICD and ICD alone in IVT RNA transfection system.

[0341] The target cells were killed using the HepG2-luci modified cell line from Example 4. The IVT RNAs of GPC3 CAR-FCE, GPC3 CAR-FCE+IFNgR, GPC3 CAR-IFNgR, GPC3 CAR-FCE+PI3K, GPC3 CAR-PI3K, GPC3 CAR-FCE+TLR4, GPC3 CAR-TLR4, GPC3 CAR-CD40+FCE, GPC3 CAR-CD40, GPC3 CAR-CD3ζ, GPC3 CAR-CD3ζ+IFNgR, GPC3 CAR-CD3ζ+PI3K, GPC3 CAR-CD3ζ+TLR4, and GPC3 CAR-CD40+CD3ζ prepared in Example 1 were transfected into macrophages obtained according to the method in Example 2 (the transfection method was the same as in Example 2). Two days later, the cells were resuspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and the cells were resuspended in preheated complete culture medium until the density was 1×10⁻⁶. 6 Cells / mL.

[0342] The negative macrophage group (UTD group) consisted of untransfected RNA macrophages suspended in PBS, centrifuged at 400g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in pre-warmed complete culture medium until the density reached 1×10⁶ cells / mL. 6 Cells / mL.

[0343] Effector cells (transfected macrophages and negative macrophages) were added at effector-target ratios of 5:1 and 1:1. After incubation at 37°C for 48 hours, the cells were centrifuged at 500g for 5 minutes, and 100 μL of the supernatant was discarded after cell precipitation. 50 μL of the prepared solution was added to each well. Luciferase Assay reagent (Promega / Cat: E2520). After 5 minutes of reaction, read the values ​​on the microplate reader.

[0344] The results showed that, based on HepG2-luciferase target cells, under different effector-to-target ratios, GPC3-CAR molecules containing ICD combinations exhibited a significantly enhanced killing ability compared to any single ICD CAR molecule. Among them, the four ICD combinations of FCE-IFNgR, FCE-PI3K, CD3ζ-IFNgR, and CD3ζ-PI3K showed the largest increase in killing ability compared to any single ICD (see Figure 77). This suggests that the CAR structure of combined ICDs has a superior anti-tumor killing effect. Furthermore, the increase in killing ability of the combined ICDs compared to the UTD was greater than the sum of the increases in killing ability of each ICD in the combination compared to the UTD, indicating that the various ICDs in the combined ICDs have a synergistic effect in enhancing killing ability. Other combined ICDs also showed a synergistic effect in enhancing killing ability.

[0345] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of this invention fall within the protection scope of this invention.

Claims

1. An intracellular signal transduction region of a chimeric antigen receptor (CAR) comprising at least two of a1)-a7): a1) FCE or a part thereof; a2) CD3ζ or a part thereof; a3) CD40 or a part thereof; a4) TLR2 or a part thereof; a5) TLR4 or a part thereof; a6) IFNgR or a part thereof; a7) PI3K recruit or a part thereof.

2. The intracellular signal transduction region according to claim 1, characterized in that, The intracellular signal transduction region of the chimeric antigen receptor includes: (i) at least one of a1)-a2); and (ii) at least one of a3)-a7); Preferably, the intracellular signal transduction region of the chimeric antigen receptor comprises: (i) any one of a1)-a2); and (ii) any one of a3)-a7); Preferably, the intracellular signal transduction region of the chimeric antigen receptor comprises: a1) and a3); or a1) and a4); or a1) and a5); or a1) and a6); or a1) and a7); or a2) and a3); or a2) and a5); or a2) and a6); or a2) and a7); Preferably, the intracellular signal transduction region of the chimeric antigen receptor from the N-terminus to the C-terminus includes: a3) and a1); or a1) and a4); or a1) and a5); or a1) and a6); or a1) and a7); or a3) and a2); or a2) and a5); or a2) and a6); or a2) and a7).

3. A chimeric antigen receptor comprising: (a) an extracellular ligand-binding domain having binding affinity for a ligand; (b) a transmembrane domain; and (c) an intracellular signal transduction region as described in any one of claims 1-2.

4. The chimeric antigen receptor according to claim 3, characterized in that, The extracellular ligand-binding domain is selected from the following ligand-binding domains: antibodies, antigen-binding fragments, antibody mimics, and receptors; Preferably, the antibody or the antigen-binding fragment is selected from: single-chain antibodies, monoclonal antibodies, antigen-binding fragments, nanobodies, biantibodies, triantibodies, microantibodies, F(ab')2 fragments, F(ab)v fragments, single-domain antibodies, V... H Structural domain, V L Domain, Fv fragment, VNAR domain and V HH structural domain; Preferably, the ligands are selected from: CD1a, CD1b, CD1c, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, and CD33. , CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD 49f, CD52, CD53, CD54, CD56, CD57, CD61, CD62L, CD63, CD64, CD66b, CD68, CD69, CD70, CD73, CD74, CD79a, CD79b, CD 80. CD83, CD85k, CD86, CD88, CD93, CD94, CD95, CD99, CD103, CD105, CD107a, CD107b, CD114, CD115, CD117, CD122, C D123, CD129, CD133, CD134, CD138, CD141, CD146, CD152, CD158, CD161, CD163, CD183, CD191, CD193, CD194, CD195, CD197, CD203c, CD205, CD207, CD209, CD223, CD235, CD244, CD252, CD267, CD268, CD273, CD276, CD279, CD282, CD284, CD294, CD304, CD305, CD314, CD319, CD326, CD328, CD335, fetal acetylcholine receptor, ADGRE2, alpha-fetoprotein, ALK, BCMA, BDCA3, C3AR, LewisA. Carbonic anhydrase IX, calciretinoin, cancer antigen-125, CCR1, CCR4, CDS, carcinoembryonic antigen, chromogranin, CLEC12A, cytomegalovirus-infected cell antigen, CS-1, CSPG4, cytokeratin, desmin, DLK1, DLL3, EGFRvIII, EGFR and its allotypes, epithelial cell adhesion molecule, epithelial glycoprotein 2, epithelial glycoprotein 40, epithelial membrane protein, ERBB, epithelial tumor antigen, FAP, leaf Acid-binding protein, FcγR1, FcεRIα, FITC, FLT3, FOLR1, FOLR3, prolactin, gangliosides, liquid protein in macrocystic diseases, GD2, GD3, GM2, GM3, glial fibrillary acidic protein, gpA33, glycopeptides, phosphatidylinositol proteoglycan 2, phosphatidylinositol proteoglycan 3, carcinoembryonic antigen, influenza hemagglutinin, human epidermal growth factor receptor 2, HLA-DR, HM1.24, HMB-45 antigen, HPV E6, HPV E7, ICAM-1, IgG, IgD, IgE, IgM, IL-13 receptor α1, integrin, integrin B7, interleukin-13 receptor subunit α-2, κ light chain, kinase insertion domain receptor, λ light chain, LILRB2, Lewis Y, LGR5, Ly49, Ly108, L1 cell adhesion molecule, melanoma-associated antigen, melanoma antigen family A1, protein melanin A, MCSP, c-Met, MICA / B, mesothelin, muscle-specific actin, mesothelin, pyruvate kinase isoenzyme M2 dimer form, mucin 1, mucin 16, myo-D1, Necl-2, neurofilament, NKCSI, NKG2D, neuron-specific enolase, NY-ESO, cancer-testis antigen NY-ESO-1, abnormal p53 protein, PAP, PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, prostaglandins Prostate stem cell antigen, prostate-specific membrane antigen, Ral-B, K-Ras, abnormal ras protein, ROR1, SLAMF7 / CS1, receptor tyrosine protein kinase erb-B2, erb-B3, erb-B4, spermin 17, STEAP1, synaptophysin, tumor-associated glycoprotein 72, TALLA-1, TARP, TEM-8, human telomerase reverse transcriptase, TIM-3, TLR4, TRBC1, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, TYRP1, tyrosinase, vascular endothelial growth factor R2, Vα24, nephroblastoma protein, or any combination thereof; Preferably, the amino acid sequence of the extracellular ligand binding domain is as follows: i1) An amino acid sequence as shown in SEQ ID NO. 2, 3, or 4; or The amino acid sequences shown in i2) and i1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and have the same function.

5. The chimeric antigen receptor according to any one of claims 3-4, characterized in that, The transmembrane domains are selected from the following transmembrane domains: CD4, CD8, CD28, PD-1, OX40, 4-1BB, CTLA-4, CD2, CD3D, CD3E, CD3G, CD3ζ, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150, CD166, CD200R, CD223, CD270, CD272, CD273, CD274, CD278, CD300, CD357, A2aR, ICAM-1, 2B 4. BTLA, DAP10, FcRα, FcRβ, Fyn, GAL9, IL7, IL12, IL15, KIR, KIR2DL4, KIR2DS1, LAG-3, Lck, LAT, LPA5, LRP, NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, SLP-76, SIRPα, pTα, T cell receptor peptide, TIM3, TRIM, ZAP70, or any combination thereof; further comprising a CD8 transmembrane domain; The amino acid sequence of the transmembrane domain is as follows: j1) The amino acid sequence as shown in SEQ ID NO. 6; or The amino acid sequences shown in j2) and j1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and have the same function.

6. The chimeric antigen receptor according to any one of claims 3-5, characterized in that, The chimeric antigen receptor further includes an extracellular hinge domain and / or an extracellular leader domain. Preferably, the extracellular hinge domain is selected from the following hinge domains: CD8, CD28, CD4, IgG, PD-1, CTLA-4, CD2, LFA-1, CD5, CD27, CD70, 4-1BB, OX40, ICOS, IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgE Fc region, IgM Fc region, IgA Fc region, or any combination thereof; further, it is the CD8 extracellular hinge domain; Preferably, the amino acid sequence of the extracellular hinge domain is as follows: k1) The amino acid sequence as shown in SEQ ID NO.5; or The amino acid sequences shown in k2) and k1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and the same function. Preferably, the extracellular leader domain comprises the CD8 extracellular leader domain; Preferably, the amino acid sequence of the extracellular leader domain is as follows: l1) The amino acid sequence as shown in SEQ ID NO.1; or The amino acid sequences shown in l2) and l1) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity and have the same function.

7. The chimeric antigen receptor according to any one of claims 3-6, characterized in that, The chimeric antigen receptor comprises, from the N-terminus to the C-terminus, an extracellular leader domain, an extracellular ligand-binding domain with binding affinity for ligands, an extracellular hinge domain, a transmembrane domain, and an intracellular signal transduction region as described in any one of claims 1-2.

8. A biomaterial relating to the intracellular signal transduction region of the chimeric antigen receptor according to any one of claims 1-2, or the chimeric antigen receptor according to any one of claims 3-7, wherein the biomaterial comprises any one of n1)-n9): n1) The nucleic acid molecule encoding the intracellular signal transduction region of the chimeric antigen receptor as described in any one of claims 1-2, or the chimeric antigen receptor as described in any one of claims 3-7; n2) contains an expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) contains cells containing the carrier described in n4); n9) A cell comprising the intracellular signal transduction region of the chimeric antigen receptor as described in any one of claims 1-2, or the chimeric antigen receptor as described in any one of claims 3-7; Preferably, any one of the vectors in n3)-n4) is a viral vector or a non-viral vector; more preferably, it is a viral vector; even more preferably, it includes an adenovirus vector, an adeno-associated virus vector, or a retrovirus vector. Preferably, any one of the cells (n5)-n9) does not contain reproductive material.

9. A modified immune cell comprising the intracellular signal transduction region of a chimeric antigen receptor as described in any one of claims 1-2, the chimeric antigen receptor as described in any one of claims 3-7, or the nucleic acid molecule, expression cassette, or vector as described in claim 8; Preferably, the immune cells comprise macrophages, monocytes, dendritic cells, T cells, stem cells, regulatory T cells, natural killer cells, γδ T cells, or natural killer T cells; further comprise macrophages, monocytes, dendritic cells, or stem cells; and even further comprise macrophages and / or monocytes. Preferably, compared with unmodified immune cells, the modified immune cells exhibit enhanced effector activity, enhanced tumor killing activity and / or phagocytic activity, enhanced resistance to exhaustion and / or enhanced tumor infiltration capacity. Preferably, the modified immune cells exhibit increased polarization toward the M1 phenotype compared to unmodified immune cells.

10. A pharmaceutical composition comprising the intracellular signal transduction region of a chimeric antigen receptor as described in any one of claims 1-2, the chimeric antigen receptor as described in any one of claims 3-7, the biomaterial as described in claim 8, or the immune cell as described in claim 9; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

11. Use of the intracellular signal transduction region of the chimeric antigen receptor according to any one of claims 1-2, the chimeric antigen receptor according to any one of claims 3-7, the biomaterial according to claim 8, or the immune cell according to claim 9 in the preparation of a medicament for the prevention or treatment of a disease or condition; Preferably, the disease or symptom includes a tumor; Preferably, the tumor comprises at least one of a solid tumor and a hematoma; more preferably, it is a solid tumor. Preferably, the solid tumors include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, adrenocortical carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdomyosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germinal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germinal tumor, gonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, and Merkel cell carcinoma. At least one of the following: cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal and sinus carcinoma, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal carcinoma, retinoblastoma, salivary gland tumor, sarcoma, Cézare syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and single myeloma; further, breast cancer, gastric cancer, ovarian cancer, colon cancer, lung cancer, bladder cancer, prostate cancer, pancreatic cancer, or liver cancer.

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