Islet antigen-specific car-t therapy

Abstract

CAR-CD8 T cells, and methods of making and using the same, are described. The CAR-CD8 T cells are specific for an islet antigen, such as GAD65. IFN-y dominance can be stimulated, and a proinflammatory microenvironment can be created, in a pancreas using the CAR-CD8 T cells.

Description

TITLEIslet Antigen-Specific CAR-T TherapyInventor: Shahnawaz ImamRELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 666.449 filed under 35 U.S.C. § 111(b) on July 1. 2024. the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with no government support. The government has no rights in this invention.SEQUENCE LISTING

[0003] This instant application contains a Sequence Listing submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML document was created on June 24, 2025, is named “420_64590_Sequence_Listing_XML” and the file is 3,342 bytes in size.BACKGROUND

[0004] The incidence of pancreatic cancer continues to rise steadily. Pancreatic cancer remains the third-leading cause of cancer-related deaths and is expected to become the second-leading cause of death by 2030. Despite extensive research, no effective therapy has been identified, and to date, the five-year survival rate in all SEER stages combined is only 13%. Strategies for converting a cold tumor into a hot tumor pave the way to solid tumor immunotherapy.

[0005] Pancreatic ductal adenocarcinoma (PDAC) is an epithelial neoplasm that accounts for more than 90% of all pancreatic malignancies. PDAC is highly resistant to current therapies. Unfortunately, traditional treatments have not been shown to significantly improve survival. Immunotherapy and targeted therapies have not brought the therapeutic revolution reported in other malignancies. Thus, there is a need in the art for new ways to address PDAC.SUMMARY

[0006] Provided is an immunoresponsive cell comprising a chimeric antigen receptor (CAR) that binds to an islet-specific antigen; the CAR comprising (a) an intracellular signaling domain of a CD3^polypeptide, an intracellular signaling domain of a CD28 hinge-transmembrane-intracellular region, and a 4- IBB (CD 137) costimulatory domain, and (b) an extracellular polypeptide comprising a monoclonal antibody (MAb) variable region having an amino acid sequence which binds to the islet-specific antigen; wherein the cell is a CD8+T cell

[0007] In certain embodiments, the islet-specific antigen is GAD65.

[0008] In certain embodiments, the MAb variable region comprises SEQ ID NO: 1 or a sequence having 90% sequence identity to SEQ ID NO: 1.

[0009] In certain embodiments, the MAb variable region comprises SEQ ID NO: 2 or a sequence having 90% sequence identity to SEQ ID NO: 2.

[0010] In certain embodiments, the MAb variable region consists of SEQ ID NO: 1. In certain embodiments, the MAb variable region consists of SEQ ID NO: 2.

[0011] In certain embodiments, the immunoresponsive cell further comprises a spacer between the intracellular signaling domain and the extracellular polypeptide.

[0012] Further provided is a pharmaceutical composition comprising the immunoresponsive cell described herein or a progenitor thereof, and a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0013] Further provided is a method of creating a proinflammatory environment in a pancreas, the method comprising administering to a subject in need thereof an effective amount of a composition comprising the immunoresponsive cell described herein and creating a proinflammatory environment in a pancreas of the subject. In certain embodiments, the subject has a tumor in the pancreas, and the method creates a proinflammatory tumor microenvironment in the subject. In certain embodiments, the MAb variable region comprises SEQ ID NO: 1. In certain embodiments, the MAb variable region comprises SEQ ID NO: 2.

[0014] Further provided is a method of causing IFN-y dominance in a pancreas, the method comprising administering to a subject in need thereof an effective amount of a composition comprising the immunoresponsive cell described herein and causing IFN-y dominance in a pancreas of the subject. In certain embodiments, the MAb variable region comprises SEQ ID NO: 1. In certain embodiments, the MAb variable region comprises SEQ ID NO: 2.

[0015] Further provided is a method of creating a proinflammatory environment in a pancreas of a subject, the method comprising administering to the subject an effective amount of CAR-T cells that bind to an islet-specific antigen, wherein the CAR-T cells are CD8+T cells, and creating a proinflammatory environment in the pancreas of the subject. In certain embodiments, the CAR-T cells include a MAb variable region comprising SEQ ID NO: 1. In certain embodiments, the CAR-T cells include a MAb variable region comprising SEQ ID NO: 2.

[0016] Further provided is a method of creating CAR-N-CD8 T cells that is GAD65-specific, and theadoptive transfer of CAR-N-CD8 T cells is GAD65-specific in GAD65-specific humanized T1D mice, activates them, and converts them into CTLs in GAD65-specific humanized T1D mice. While C57BL / 6J mice express GAD67 in their pancreatic islet, the CAR-N-CD8 T cells failed to activate in C57BL / 6J mice.

[0017] Further provided is a method of creating a Thl response in GAD65-specific humanized T1D mice and in C57BL / 6 mice post-PDAC and post-adaptive transfer of CAR-N-CD8 T cells. The described methods produced significant enrichment in the Thl response in GAD65-specific humanized T1D mice compared to C57BL / 6 mice.

[0018] Further provided is a method of causing IFN-y dominance in the pancreas, the method comprising administering to a subject an effective amount of CAR-T cells that bind to an islet-specific antigen, wherein the CAR-T cells are CD8+ T cells, and causing IFN-y dominance in the pancreas of the subject. In certain embodiments, the islet-specific antigen is GAD65. In certain embodiments, the CAR-T cells include a MAb variable region comprising SEQ ID NO: 1. In certain embodiments, the CAR-T cells include a MAb variable region comprising SEQ ID NO: 2.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] FIG. 1: Illustration of GAD65-CAR-CD8 T cells.

[0021] FIG. 2: Superiority of CAR over naive CD8+. CTLs are significantly higher in CAR-N-CD8 T cells over naive CD8+T cells (n = 3-4). Data are mean ± SEM.

[0022] FIG. 3: GAD65-CAR-N CD8+T cells home to islets, proliferate, and activate CD8+T cells (n = 3). Data are mean ± SEM. FIG. 3 shows generation of human CAR-N-CD8+T cells from peripheral blood of PDAC patients (n = 3). CAR-N-CD8+T cells / naive CD8+T cells expressing green fluorescent protein were co-cultured with cytotoxic CD8+and CD4+T cells in the presence of homologous human pancreatic islets and PDAC nodules, both from the same patients (ex vivo). CAR-N-CD8 T cell / naive CD8 T cells expressing green fluorescent protein were co-cultured with cytotoxic CD8+and CD4+T cells in the presence of homologous human pancreatic islets and PDAC nodules, both from the same patients (ex vivo).

[0023] FIG. 4: Experimental results indicating negligible activation of CAR-N-CD8 T cells in C57BL / 6J mice.

[0024] FIG. 5: Enrichment in the Thl response in GAD65-specific humanized T1D mice compared to C57BL / 6 mice.DETAILED DESCRIPTION

[0025] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains.

[0026] Chimeric antigen receptor (CAR) molecules are described in depth in, for example, U.S. Patent Application Publication No. 2019 / 0031759 Al and U.S. Patent No. 11,766,457, both of which are incorporated herein by reference for all purposes. CARs have been successfully used in the treatment of various leukemias, and in clinical trials for various solid tumors. However, their use in cancer therapy is generally thought to be limited by the number of cancer cells that are not recognized by T cells, mainly due to the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC) expression of cancer cells.

[0027] While there has been a swift increase in cancer treatments that incorporate targeted immunotherapy-based strategies to treat PDAC, the CAR-CD8 T cells approach has thus far not led to significant improvement in overall pancreatic cancer patients’ survival due to 1) lack of targetable antigens, 2) an immunosuppressive tumor microenvironment, and 3) an inability of T cells to efficiently traffic to tumor sites and infiltrate the tumor. In accordance with the present disclosure, islet antigen-specific T cells can be produced on demand against a desired islet autoantigen, thereby creating an islet antigen-specific proinflammatory microenvironment that can shift the immunosuppressive tumor microenvironment into a proinflammatory one.

[0028] One non-limiting example of an islet antigen is glutamic acid decarboxylase (GAD65). Provided herein are GAD65-specific chimeric antigen receptor (CAR) CD8 T cells created by electing immunodominant B-cell paratopes that bind to the surface of the GAD65 antigen either close to the membrane anchoring domain (CAR-N-CD8) or in a hydrophilic region of the monomeric form of the autoantigen (CAR-M-CD8). Islet antigen-specific CAR-N / M-CD8 T cells can recognize the cognate antigen expressed on human pancreatic islets, activate, and become CAR-N / M-cytotoxic CD8 T cells. CAR-N / M-cytotoxic CD8 T cells can create an IFN-y -dominated proinflammatory bias in the pancreatic microenvironment. Combination immunotherapy based on CAR-N / M-CD8 T cells, followed by autologous activated CD4 and CD8 T cell therapy, can invade the tumor-associated barrier and interact with cancer cells.

[0029] The CAR-N / M-CD8 T cells described herein include a chimeric antigen receptor (CAR) having an intracellular signaling domain of a CD3^ polypeptide, 4-1BB (CD137), a co-stimulatory receptor, and an intracellular signaling domain of a CD28 hingc-transmcmbranc-intraccllular region, a spacer, and an extracellular polypeptide having a MAb variable region. 4-1BB (CD137) is a member of the tumor necrosisfactor receptor superfamily and serves as a critical co-stimulatory receptor, primarily expressed on activated CD8+T cells. Following engagement by natural ligand 4-1BBL, 4-1BB transduces the activation signal of NF-kB, which induces preferential expansion and activation of CD8+compared to CD4+T cells.

[0030] In certain non-limiting examples, the MAb variable region is either SEQ ID NO: 1 or SEQ ID NO: 2. “CAR-N” includes the extracellular polypeptide sequence of MDMRVPAQLLGLLLLWLPGAKCDIQLTQSPTFLSASVGDRVTITCRASQGISSYLAWYQQKPGKA PNLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNNYPLTFGGGTKVEIKRPPPP RPPPPRPPPPRQLQLQESGPGLLKPSETLSLTCSVSGGSIGSSSYSWGWIRQPPGKGLEYIGIIYHSGR TYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAMYYCARQVPYQPLLDGGNWFDPWGQGTLVT VSS (SEQ ID NO: 1), and “CAR-M” includes the extracellular polypeptide sequence of MAWTVLLLGLLSHCTGSVTSYVLTHPPSVSVAPGKTGTITCGGSNIGSKSVHWYQQKPGQAPKLVI YYDSDRPSGIPERFSGSTSGNTATLTISSVEAGDEADYYCQVWDSSGDHMVVFFGGTKLTVLPPPP RPPPPRPPPPRQVQLVESGGGVVQPGRSLRLSCAASGLTFSHHGMHWVRQAPGKGLEWVAFISYD ETKKYYVKSVMGRFTIARDNSKNTLYLHLKSLRPDDAAVYYCAKAFSTTIFGVVTYGMDVWGQG TTVIVSS (SEQ ID NO: 2). Optionally, the polypeptide may comprise a sequence of amino acid residues having at least 95% sequence identity with either SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, or a valiant, or a homolog thereof, adapted for treatment. The term “variant” refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 80% sequence identity, more preferably, at least about 90% homology by sequence. The amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the reference amino acid sequence. A “variant” of a protein of interest, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The valiant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software. Optionally, the polypeptide is attached to a detectable or therapeutic moiety.

[0031] The CAR components (expressed by CAR-CD8 cells), as shown in FIG. 1, include intracellular (CD3^, 4-1BB, and CD28), spacer, and extracellular (MAb variable region) components. The expression of the CAR follows the pathway of expression of any cellular protein (DNA->RNA->protein). The different components (polypeptides CD3^, CD8, spacer, and MAb variable region) arc linked together by peptide bonds. Thus, in some embodiments, the CAR-N / M-CD8 T cells are basically made of a CD8+ Tcell (for example, isolated from the peripheral blood of the subject) and a construct (CAR) that recognizes a specific antigen via a MAb variable region.

[0032] The term “spacer” as used herein means a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a “peptide linker” refers to one or more amino acids used to couple two proteins together. Non-limiting examples of suitable spacers include glycine, serine, or threonine. The intracellular signaling domain of the CD28 hinge-transmembrane-intracellular region is linked to the spacer by a peptide bond, and the extracellular polypeptide is linked to the spacer by a peptide bond. The CD3^ polypeptide is also linked to the intracellular signaling domain of CD28 hinge-transmembrane-intracellular region by a peptide bond. Here, third-generation CAR T cells incorporate both 4- IBB and CD28 costimulatory domains to leverage the advantages offered by each domain: the CD28 domain enhances cytotoxicity and proliferation, while the 4-1BB domain enhances memory and persistence.

[0033] The CAR-N / M-CD8 T cells may be administered to a subject as part of a pharmaceutical composition. Pharmaceutical compositions of the present disclosure comprise genetically modified immunoresponsive cells or their progenitors, along with a pharmaceutically acceptable earner. Administration can be autologous or heterologous. For example, immunoresponsive cells, or progenitors, can be obtained from one subject and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells of the present disclosure or their progeny (e.g., in vivo, ex vivo, or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immunoresponsive cells), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0034] The phrases “pharmaceutical” or “pharmacologically acceptable” refer to molecular entities and compositions that produce no adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human. The preparation of a pharmaceutical composition that contains at least one compound or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington’s Pharmaceutical Sciences, 2003, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it is understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0035] A composition disclosed herein may comprise different types of earners depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. Compositions disclosed herein can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosseously, periprosthetically, topically, intramuscularly, subcutaneously, mucosally, intraosseosly, periprosthetically, in utero, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 2003, incorporated herein by reference).

[0036] The actual dosage amount of a composition disclosed herein administered to an animal or human patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient, and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0037] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound (e.g., cell mixture). In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0038] In other non-limiting examples (e.g. weight-based active compound), a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In nonlimiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above.

[0039] In certain embodiments, a composition herein and / or additional agent is formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsules, they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

[0040] In further embodiments, a composition described herein may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example, but not limited to, intravenously, intradermally, intramuscularly, intra-arterially, intrathecally, subcutaneously, or intraperitoneally (U.S. Patents 6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 are each specifically incorporated herein by reference in their entirety).

[0041] Solutions of the compositions disclosed herein (e.g., cellular mixture) may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In most cases, the form must be sterile and fluid to the extent that easy injectability is achieved. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and / or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as, but not limited to, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate or gelatin.

[0042] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent fust rendered isotonic with sufficient saline or glucose for the cell mixture. These particular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed is known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences’’ 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologies standards.

[0043] Sterile injectable solutions are prepared by incorporating the compositions in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized compositions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desir ed ingredient from a previously sterile- filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.

[0044] In other embodiments, the compositions may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and / or via inhalation.

[0045] Pharmaceutical compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream, or powder. Ointments include all oleaginous, adsorption, emulsion, and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones, and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture. Transdermal administration of the compositions may also comprise the use of a “patch.” For example, the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time.

[0046] In certain embodiments, the compositions may be delivered by eye drops, intranasal sprays,inhalation, and / or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described in U.S. Patents 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in their entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Patent 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts and could be employed to deliver the compositions described herein. Likewise, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in U.S. Patent 5,780,045 (specifically incorporated herein by reference in its entirety), and could be employed to deliver the compositions described herein.

[0047] It is further envisioned the compositions disclosed herein may be delivered via an aerosol. The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol for inhalation consists of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. The administration of the aerosol will vary according to subject's age, weight, and the severity and response of the symptoms.

[0048] As demonstrated in the examples herein, the compositions are useful for creating a proinflammatory environment in the pancreas, inducing a proinflammatory tumor microenvironment in the pancreas, and promoting IFN-y dominance in the pancreas.

[0049] The pancreatic tumor microenvironment, which has unique features and differs from those of other cancers, may play a key role in explaining why immunotherapies and targeted therapies have not been as successful against pancreatic cancer as they have been against other cancers. Strategies for converting a cold tumor into a hot tumor pave the way for treating pancreatic cancer. In accordance with the present disclosure, immunotherapy and / or immune cell plasticity can be exploited to create a pro-inflammatory pancreatic microenvironment and reverse the immunosuppressive microenvironment of PD AC.

[0050] Tumor immunity is achieved when a significant percentage of residual CD8+T cells are pushed toward an exhausted / anergic phenotype through immunoediting. Thus, activating CD8 T cells by upregulating IFN-y expression using pancreatic islet-specific CAR-CD8-T cells can break the tolerance.

[0051] Previously, CAR-CD8 T cells were more efficient in controlling cancer than unmodified CD8 T cells. In PDAC, however, CAR-CD8 T cells cannot directly engage tumors with complete inaccessibility of antigen / loss of antigen. The failure to eliminate low antigen-expressing tumors raises questions about the sensitivity of conventional CARs in PDAC. However, islet antigen-specific GAD65-CAR-N / M-CD8 T cells can recognize the cognate antigen expressed on human pancreatic islets, become activated, and become CAR-N / M-cytotoxic CD8 T cells. As shown in the examples herein, CAR-N / M-cytotoxic CD8 Tcells can create IFN-y -dominated proinflammatory bias in the pancreatic microenvironment. Thus, the immunosuppressive tumor microenvironment can be shifted into a proinflammatory one. Proinflammatory cytokines milieu can induce the secretion of proteases to loosen the extracellular matrix and allow tumorspecific CD 8 T cells to invade tumors and interact with pancreatic cancer cells. In other words, the GAD65-CAR-CD8 T cells described herein, which can recognize human immunodominant regions of GAD65, can reset the immunosuppressive pancreatic microenvironment. This is highly advantageous for any PDAC treatment.

[0052] Immune cell plasticity can be exploited to create a pro-inflammatory pancreatic microenvironment and reverse the immunosuppressive microenvironment of PDAC. However, there are barriers to accomplishing this. First, tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) contribute to the formation of an extracellular matrix (ECM). The ECM creates a physical / biochemical barrier for NK and antigen-specific cytotoxic T cells (CTLs). In human ex vivo experimentation, it has been demonstrated that IFN-y dominance can induce plasticity in TAMs, promoting a shift towards the Ml macrophage phenotype and activating NK cells. This IFN-y dominance breaks the ECM barrier, allowing NK and CD8 T cells to interact with cancer cells. Secondly, the collagen barrier needs to be broken: Collagen fibers form tumor cell clusters, preventing CTLs from interacting with cancer cells. CD4 and CD8 T cells are required to create a pro-inflammatory environment and induce plasticity in conventional and unconventional regulatory T cells, thereby shifting them towards T helper-1 (Thl ) differentiation, which disrupts the tumor-associated barrier. Thirdly, increasing the IFN-y expression in the tumor microenvironment (TME) leads to the expression of Programmed death-ligand 1 (PD-1 / PD-L1): PD- L1 expression on cancer cells is mainly regulated by IFN-y dominance in the TME, which induces T cell exhaustion and enables minor immune evasion and thus increases the expression of IFN-y, recruiting more cytotoxic T cells at the tumor site and may lead to the execution of PD-L1 expression. Fourth, infiltrating T cells need to traffic and interact with tumor cells. Breaking the chemokine barriers by IFN-y dominance leads to CXCL9-mediated recruitment of CD8+ T cells to the tumor. IFN-y Fifth, exploiting the plasticity of CD4 T effector cells: In autoimmunity, T cells adopt alternative transcriptional lineages that generate functionally distinct subsets that modulate localized or specific inflammatory sites. Similarly, a proinflammatory milieu mediated by IFN-y induces plasticity in Tregs, shifting them towards a Thl phenotype.

[0053] A combination therapy that increases IFN-y expression in the tumor microenvironment is possible. Systemic immune checkpoint inhibitor antibodies can increase immune cells within tumors that respond to therapy with the secretion of IFN-y and thus increased expression of IFN-y can recruit more T cells at the tumor site.

[0054] CARs have been produced and developed to modify CD8 T cells (CAR-CD8 T) and redirecttheir specificity towards a known autoantigen, specifically GAD65, which is considered a primary islet autoantigen in approximately 95% of Type 1 Diabetes (T1D) patients. Human GAD65-specific CAR-CD8 T cells were produced by selecting immunodominant B-cell paratopes that bind to the surface of the GAD65 antigen either close to the membrane anchoring domain (CAR-N-CD8 T cell) or in a hydrophilic region of the monomeric form of the autoantigen (CAR-M-CD8 T cell). The CAR-CD8 T cells are also able to home to islets, become activated, and proliferate there, consequently activating CD8 T cells in human ex vivo pancreatic islet co-culture experiments, as shown in the examples herein. Impressively, the same GAD65 CAR N / M constructs, when transduced into mice CD8 T cells, can create a proinflammatory pancreatic microenvironment in a humanized transgenic mouse model of T1D, which closely resembles the human pancreas. Specifically, MHC-II molecules in this mouse model are replaced with human DQ8 in all antigen-presenting cells. Additionally, these mice express human GAD65 in pancreatic beta cells and produce GAD65-specific infiltrating immune cells. Therefore, the homing of islet antigen-specific CAR- CD8 T cells in the pancreatic microenvironment creates a proinflammatory bias that activates tumor antigen-specific CD8 T cells and facilitates their invasion of the solid tumor.

[0055] T cells can be endowed with desired antigen-specific recognition using chimeric antigen receptor (CAR) T cells, which exhibit antibody-type specificity. GAD65 is the primary autoantigen in type 1 diabetes (T1D). More than 95% of T1D patients show a B-cell immune response towards the conformational epitope residing on the surface of GAD65. leveraged as a therapeutic advantage.

[0056] Tumor immunity is achieved when a significant percentage of residual CD8+T cells are driven toward an exhausted or anergic phenotype through immunoediting. Thus, activating CD8 T cells by upregulating IFN-y expression using pancreatic islet-specific CAR-CD8-T cells can break tolerance, which is helpful for pancreatic cancer treatments. In previous studies of other CAR-CD8 T cells in pancreatic cancer models, the CAR-CD8 T cells could not directly engage tumors without complete antigen loss, which raises questions about the sensitivity of CARs in PDAC. T-cell receptor-mediated stimulation requires low antigen expression (-200 molecules / target cell), but studies on CAR T cells indicate that the density of antigen must be high on target cells (-2000 molecules / target cell) and low on normal tissues to trigger CAR activation. In accordance with the present disclosure, CAR-T cell therapies can be employed in combination with islet-antigen-specific CAR-CD8 T cells with cytotoxic CD8 and CD4 T cell therapies so as to overcome tumor-associated barriers to immune infiltration.

[0057] GAD65-specific CAR-N / M-CD8 T cells induce plasticity in Tregs, shifting them toward a T effector phenotype, as an immune therapeutic regimen for modulating the immune response in PDAC. Antigen-specific T cells play a key role in breaking peripheral tolerance in cancer. However, patients with PDAC have few circulating antigen-specific CD8 T effector cells. As noted above, there arc several limiting factors for CAR-CD8 T cell efficacy in PDAC, including: (1) a lack of targetable antigens, (2) animmunosuppressive tumor microenvironment, (3) an inability of T cells to efficiently traffic to tumor sites and infiltrate the tumor. Therefore, conventional CAR-CD8 T cells have not led to significant improvement in overall pancreatic cancer patients’ survival. However, the production of antigen-specific T cells against an islet autoantigen allows for the achievement of an islet antigen-specific proinflammatory microenvironment, which shifts the immunosuppressive tumor microenvironment into a proinflammatory one. Proinflammatory cytokines milieu can induce the secretion of proteases to loosen the extracellular matrix and allow CD8 T cells to invade tumors and interact with the pancreatic cancer cells.

[0058] T cells can be endowed with antigen-specific recognition using chimeric antigen T cell receptors (CARs) with antibody-type specificity. In particular, GAD65-CAR-N / M-CD8 T cells have been developed. GAD65 is the primary autoantigen in T1D, with more than 95% of patients showing a B-cell immune response towards the conformational epitope residing on the surface of GAD65. The natural specificity of human monoclonal antibodies towards GAD65-specific epitopes can be turned into a therapeutic advantage. GAD65-specific CAR T cells have been produced by selecting immunodominant B- cell paratopes that bind to the surface of the antigen either close to the membrane anchoring domain (CAR- N-CD8 T cells) or in a hydrophilic region of the monomeric form of the autoantigen (CAR-M-CD8 T cells). Specifically, human CD8 T cells have been transduced with the human GAD65 (hGAD65) specific antibody paratope to create CAR-N / M-CD8 T cells as shown in FIG. 1.|0059| The activation of islet-specific GAD65-CAR-CD8 T cells leads to their conversion into cytotoxic T lymphocytes (CTLs). This can be exploited and is useful in a variety of treatment options for PDAC.

[0060] The activation of CD8+ T cells is an important aspect of the adaptive immune response, enabling the immune system to target and eliminate cells that pose a threat to the body. CD8+ T cells become activated through a series of steps that involve the recognition of specific antigens, interaction with antigen-presenting cells (APCs), and signals from cytokines. A general overview of the process is as follows. First, the activation of CD8 T cells begins with the presentation of antigens. Antigens are protein fragments derived from pathogens (such as viruses or bacteria) or abnormal cells (such as cancer cells). Antigen-presenting cells (APCs), like dendritic cells, play a crucial role in capturing, processing, and presenting these antigens. CD8 T cells have a T-cell receptor (TCR) on their surface, which can specifically recognize and bind to a specific antigen presented on the surface of an APC. The interaction between the TCR and the antigen, along with additional co-stimulatory signals, is necessary for the activation of the CD8 T cell. In addition to TCR binding with the antigen-MHC (major histocompatibility complex) complex on the APC, co-stimulatory signals are important for full activation of the CD8+ T cell. One of the important co-stimulatory molecules is CD28 on the surface of the T cell, which interacts with CD8O / CD86 on the APC. Upon binding of the TCR to the antigen-MHC complex and receiving co-stimulatory signals, intracellular signaling pathways are activated within the CD8 T cell. This includes the activation of kinases and the phosphorylation of various signaling molecules. Activated CD8 T cells undergo clonal expansion, meaning they rapidly divide and proliferate, generating a large population of effector T cells. This expansion ensures a robust immune response against the specific antigen. During clonal expansion, some of the CD8 T cells differentiate into effector cytotoxic T cells, which are equipped to directly kill cells infected with the recognized pathogen or abnormal cells, such as cancer cells. A subset of activated CD8 T cells forms memory T cells. These memory cells persist in the body after the infection is cleared, providing long-term immunity. If the same antigen reappears, memory CD8 T cells can mount a faster and more effective immune response.

[0061] EXAMPLES

[0062] Generation of GAD65-CAR-N / M-CD8 T cells

[0063] GAD65-specific CAR T cells were produced by selecting immunodominant B-cell paratopes that bind to the surface of the antigen either close to the membrane anchoring domain (CAR-N-CD8 T cell) or in a hydrophilic region of the monomeric form of the autoantigen (CAR-M-CD8 T cell). Human CD8 T cells were transduced with the human GAD65 (hGAD65) specific antibody paratope to create CAR-N / M- CD8 T cells as shown in FIG. 1.

[0064] Activation of islet- specific GAD65-CAR-CD8 T cells leads to its conversion into CTLs10065] Human CAR-N-CD8 T cells recognize cognate GAD65 antigen at pancreatic islet 0-cells, activate and express IFN-y in ex vivo co-culture conditions. To assess the functional potency of CAR-N- CD8 T cells, the IFN-y expression of CAR-N-CD8 T cells and unmodified CD8 T cells isolated from the same human patient were compared. This study involved the collection of a small piece of the pancreas (5- 10 cc wedge) from individuals undergoing pancreatectomies. Briefly, islets were isolated using the collagenase method. 10-20 cc of blood was drawn from the same patients a week before the scheduled surgery (FIG. 3, top left). CD8 T cells were isolated and transduced with the GAD65-CAR-N construct using the lentiviral vector system to make CAR-N-CD8 T cells. To test the functional potency of CAR-N- CD8 T cells, the IFN-y expression on CAR-N-CD8 T cells and unmodified CD8 T cells isolated from the same human patient was compared. CAR-N-CD8 T cells / unmodified CD8 T cells were washed with RPMI and cultured with autologous pancreatic islets for 7 days. Flow cytometry demonstrated the superiority of CAR-N-CD8 T cells over unmodified CD 8 T cells to convert into CTLs and showed a significantly higher (**P<0.004) percentage of IFN-y in CAR-N-CD8 T cells vs unmodified CD8 T cells in the pancreatic islet microenvironment (FIG. 2). Thus, CAR-N-CD8 T cells recognize the GAD65 on isolated islets in MHC independent manner, activate themselves, and proliferate, indicating the applicability in creating a proinflammatory microenvironment.

[0066] CAR-N-CD8 T cells recognize endogenous GAD65 in human pancreatic islets and lead to theconversion of unmodified CD8 T cells into CTLs

[0067] The recognition and activation of CAR-CD8 T cells in an ex vivo system comprising homologous pancreatic islets, transduced CAR-N-CD8 T cell, and / or unmodified CD8 T cells and PDAC nodules from the same patient were evaluated as depicted in FIG. 3. Transduced CAR-N-CD8 T cells / unmodified CD8 T cells were again co-transduced with GFP construct for GFP expression. GFP- expressing CAR-N-CD8 T cells and / or unmodified CD8 T cells were washed with RPMI and were cocultured with homologous isolated islets and PDAC nodules for 7 days. Live -cell immunofluorescence microscopy (IncuCyte S3) demonstrated the distinct homing of CAR-N-CD8 T cells to functional islets. The proliferation of CAR-N-CD8 T cells was clearly visible using GFP fluorochrome (yellow) within 48 hrs (FIG. 3). Next, the IFN-y -positive unmodified CD8 T cells (CTLs) were compared by sorting out GFP-CAR-N-CD8 T cells from ex vivo co-culture. Flow cytometry demonstrates that a combination of CAR-N-CD8 T cells and unmodified CD8 T cells significantly converted unmodified CD8 T cells into CTLs in the autologous pancreatic islet and PDAC co-culture microenvironment.

[0068] The GAD65-specific proliferative capacity of CAR-N / M-CD8 T cells was 4-5 times higher than that of GAD65-specific unmodified (normal) CD8 T cells. CAR-N / M-CD8 T cells were functionally stable.

[0069] CAR-N-CD8 T cells recognize endogenous GAD65 in GAD65-specific humanized T1D mice pancreatic islets post-PDAC development, which leads to the conversion of CAR-N-CD8 T cells into CAR- N-CTLs

[0070] It was investigated whether immunotherapy based on CAR-N-CD8 T cells can establish an IFN-y-dominated pancreatic microenvironment in vivo in a GAD65-specific humanized mouse model, breach the tumor barrier, and activate endogenous CD4 and CD8 T cells. It was found that these cells can invade the tumor-associated bander and interact with cancer cells.

[0071] GAD65-specific CAR-N-CD8 T cells were tested in a robust in vivo pancreatic islet GAD65- specific humanized mouse model. A transgenic humanized mouse with a C57BL / 6 background that expresses the human MHC class II gene in antigen-presenting cells and exhibits human [3-cell antigen GAD65 in the pancreatic islets under the control of the rat insulin promoter was developed. This humanized GAD65-specific transgenic mouse model can provide a PDAC model after orthotopically xenografting KPC luciferase cell lines in the pancreas. To achieve this, the KPC luciferase (PDAC) cell line was orthotopically injected into the pancreas of humanized T1D mice / C57BL / 6 mice at 4 weeks of age. Based on power analysis, this experiment was divide 108 mice (48+6 GAD65-specific humanized + 48+6 C57BL / 6J) into six groups, as shown in Table. 1. KPC luciferase cells were harvested from the sub- conflucnt culture. The cells were washed once in a scrum-free medium and suspended in HBSS. Only suspensions of single cells with >90% viability were used for injection. The pancreas of anesthetizedmice was exposed through a midline laparotomy incision and by retraction of the spleen. KPC-luciferase cells (1.0 x 106) in 50 pl of HBSS containing 1% (v / v) Matrigel were injected into the parenchyma of the pancreas with 27-gauge hypodermic needle. The abdominal wound was closed by suture, followed by sterile clips. These mice developed pancreatic ductal adenocarcinoma (PDAC) after 4 to 5 weeks. The tumor growth was monitored weekly with IVIS spectrum (Caliper Life Sciences), aiding in noninvasive longitudinal imaging of fluorescent cells and tissues in small animals. The uniqueness of this model is that PDAC in humans always precedes brittle diabetes (fluemating blood glucose levels as in Type 1 diabetes). Progression of PDAC in the developing diabetes condition was observed in the mouse model. Prior published work has not provided a PDAC model that simulates both the diabetic phenotype and PDAC simultaneously, as observed in humans.

[0072] Since mice (transgenic for human GAD65) express GAD65 at islet [3-cells, a study was performed to generate CAR-N-CD8 T cells in GAD65-specific humanized T1D mice and C57BL / 6 mice. The lentiviral GAD65-CAR-N construct was transduced in mice CD8 T cells, and CAR-N-CD8 T cells were generated. These CAR-N-CD8 T cells were further transduced with GPF lentiviral constructs for segregating CAR-CD8 T cells / CAR-CTLs from other cells. 5 million CAR-N-CD8 T cells were injected in the tail vein of GAD65-specific humanized T1D mice / C57BL / 6 mice that developed pancreatic ductal adenocarcinoma (PDAC) after KPC luciferase. The percentage of CAR-N-CTLs conversion was analyzed. The mice were sacrificed after four days, and PBMCs were isolated from blood. Flow cytometry analysis revealed that the CAR-N-CTLs were 4-5 times more abundant in GAD65-specific humanized T1D mice than in C57BL / 6 mice. Most excitingly, CAR-N-CD8 T cells demonstrated functional stability.

[0073] Table 1:

[0074] Experiments were performed to verify if islet antigen GAD65-specific CAR-N-CD8 T cells can create an IFN-y- dominated pancreatic microenvironment in GAD65-specific humanized T1D mice / C57BL / 6J mice with established PDAC as described above. After confirmation of the PDAC in C57BL / 6 and GAD65-specific humanized T1D mice, 5 million CAR-N-CD8 T-cells / naive CD8 T- cells / saline were adoptively transferred through the tail vein.10075] FIG. 4 shows experimental results indicating negligible activation of CAR-N-CD8 T cells inC57BL / 6J mice. While C57BL / 6J mice express GAD67 in their pancreatic islet, the CAR-N-CD8 T cells failed to activate in C57BL / 6J mice. Experimental results indicated that CAR-N-CD8 T cells are GAD65- specific, become activated, and are converted into CTLs in GAD65-specific humanized T1D mice. While C57BL / 6J mice express GAD67 in their pancreatic islet, the CAR-N-CD8 T cells failed to activate in C57BL / 6J mice.

[0076] FIG. 5 shows enrichment in the Thl response in GAD65-specific humanized T1D mice compared to C57BL / 6 mice. The Thl response was tested in GAD65-specific humanized T1D mice and in C57BL / 6 mice post-PDAC and post-adaptive transfer of CAR-N-CD8 T cells. A significant enrichment in the Thl response in GAD65-specific humanized T1D mice was observed compared to C57BL / 6 mice.

[0077] Certain embodiments of the compositions and methods disclosed herein are defined in the above examples. It should be understood that these examples, while indicating particular embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the compositions and methods described herein to various usages and conditions. Various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the disclosure. Additionally, various modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof.

Claims

CLAIMSWhat is claimed is:

1. An immunoresponsive cell comprising: a chimeric antigen receptor (CAR) that binds to an islet-specific antigen; the CAR comprising(a) an intracellular signaling domain of a CD3i( polypeptide, an intracellular signaling domain of a CD28 hinge-transmembrane-intracellular region, and a 4- IBB (CD 137) costimulatory domain, and(b) an extracellular polypeptide comprising a monoclonal antibody (MAb) variable region having an amino acid sequence which binds to the islet-specific antigen; wherein the cell is a CD8 T cell.

2. The immunoresponsive cell of claim 1, wherein the islet-specific antigen is GAD65.

3. The immunoresponsive cell of claim 1, wherein the MAb variable region comprises SEQ ID NO: 1 or a sequence having 90% sequence identity to SEQ ID NO: 1.

4. The immunoresponsive cell of claim 1 , wherein the MAb variable region comprises SEQ ID NO: 2 or a sequence having 90% sequence identity to SEQ ID NO: 2.

5. The immunoresponsive cell of claim 1, wherein the MAb variable region consists of SEQ ID NO: 1.

6. The immunoresponsive cell of claim 1, wherein the MAb variable region consists of SEQ ID NO: 2.

7. The immunoresponsive cell of claim 1, further comprising a spacer between the intracellular signaling domain and the 4- IBB (CD 137) costimulatory domain, and the extracellular polypeptide.

8. A pharmaceutical composition comprising: the immunoresponsive cell of claim 1 or a progenitor thereof; and a pharmaceutically acceptable carrier, diluent, or adjuvant.

9. A method of creating a proinflammatory environment in a pancreas, the method comprising administering to a subject in need thereof an effective amount of the composition of claim 1 and creating a proinflammatory environment in a pancreas of the subject.

10. The method of claim 9, wherein the subject has a tumor in the pancreas, and the method creates a proinflammatory tumor microenvironment in the subject.

11. The method of claim 9, wherein the MAb variable region comprises SEQ ID NO: 1.

12. The method of claim 9, wherein the MAb variable region comprises SEQ ID NO: 2.

13. A method of causing IFN-y dominance in a pancreas, the method comprising administering to a subject in need thereof an effective amount of the composition of claim 1 and causing IFN-y dominance in a pancreas of the subject.

14. A method of creating a proinflammatory environment in a pancreas of a subject, the method comprising administering to the subject an effective amount of CAR-T cells that bind to an isletspecific antigen, wherein the CAR-T cells are CD8 T cells, and creating a proinflammatory environment in the pancreas of the subject.

15. The method of claim 14, wherein the CAR-T cells include a MAb variable region comprising SEQ ID NO: 1.

16. The method of claim 14, wherein the CAR-T cells include a MAb variable region comprising SEQ ID NO: 2.

17. A method of creating CAR-N / M-CD8 T cells, wherein the CAR-N / M-CD8 T cells are GAD65-specific, and adoptive transfer of CAR-N / M-CD8 T cells is GAD65-specific in GAD65-specific humanized T1D mice, the method comprising: activating the CAR-N / M-CD8 T cells; and converting the CAR-N / M-CD8 T cells into CTLs in GAD65-specific humanized T1D mice.

18. A method of creating Thl response in GAD65-specific humanized T1D mice and in C57BL / 6 mice post-PDAC and post-adaptive transfer of CAR-N / M-CD8 T cells.

19. A method of causing IFN-y dominance in a pancreas, the method comprising administering to a subject an effective amount of CAR-T cells that bind to an islet-specific antigen,wherein the CAR-T cells are CD8+ T cells, and causing IFN-y dominance in the pancreas of the subject.

20. The method of claim 19, wherein the islet-specific antigen is GAD65.

21. The method of claim 19, wherein the CAR-T cells include a MAb variable region comprising SEQ ID NO: 1.

22. The method of claim 19, wherein the CAR-T cells include a MAb variable region comprising SEQ ID NO: 2.

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