Prophylactic and therapeutic methods for managing diarrhea associated with cell therapy

Abstract

Methods and compositions relate to managing chimeric antigen receptor T (CAR T) cell therapy-associated diarrhea in human subjects undergoing such therapy for colorectal cancer (CRC) are disclosed. The CAR T cells target a CRC-associated antigen, such as Guanylate Cyclase C (GCC) or Carcinoembryonic Antigen (CEA). The methods comprise administering a prophylactic regimen to the subject post-infusion of CAR T cells. This regimen includes agents such as vedolizumab, infliximab, or prophylactic budesonide. Subjects are monitored for diarrhea development and severity based on predefined clinical criteria, including stool frequency or stool volume. The methods may further involve a tiered therapeutic algorithm for treating occurring diarrhea, potentially utilizing corticosteroids or antithymocyte globulin (ATG). These approaches aim to reduce the incidence, duration, and severity of CAR T induced diarrhea, thereby improving patient safety and treatment tolerability.

Description

Attorney Docket: I071-0123PCT / SDS1.0133PCT   PROPHYLACTIC AND THERAPEUTIC METHODS FOR MANAGING DIARRHEA ASSOCIATED WITH CELL THERAPY CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 665,957, filed June 28, 2024; U.S. Provisional Application No.63 / 778,022, filed March 26, 2025; and U.S. Provisional Application No.63 / 783,812, filed April 4, 2025, all of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] The present disclosure relates to compositions and methods for managing gastrointestinal toxicities, such as diarrhea, associated with chimeric antigen receptor T (CAR T) cell therapy, particularly for cancer treatment. BACKGROUND

[0003] CAR T cell therapies represent a promising advancement in oncology. For example, CAR T cell therapies targeting Guanylate Cyclase C (GCC) for treating relapsed or refractory metastatic colorectal cancer (mCRC) have been developed. While offering therapeutic potential, such therapies can present a significant challenge related to their target antigen. In the case of GCC-targeted therapies, the antigen is not only expressed on colorectal cancer cells but also on the luminal aspect of normal enterocytes in the intestinal tract. This "on-target, off-tumor" engagement frequently leads to gastrointestinal toxicity, predominantly diarrhea.

[0004] Clinical trial experience with such therapies has underscored the prevalence and severity of this adverse event, with a high percentage of treated subjects experiencing diarrhea, a notable portion of which were severe (e.g., Grade 3). This diarrhea can significantly impact patient well-being, potentially compromising fluid and metabolic balance and often necessitating hospitalization.

[0005] Furthermore, standard criteria for grading diarrhea, such as the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI CTCAE), which primarily assess stool frequency relative to baseline, have proven inadequate in this context. These criteria can underestimate the severity of high-volume diarrhea, potentially leading to delayed or insufficient medical intervention. Conventional management strategies for immunotherapy-related diarrhea are often reactive. The predictable and potentially severe nature of diarrhea induced by certain CAR T therapies highlight a need for proactive and specifically tailored management approaches to mitigate its impact and improve the safety and tolerability of the therapy. SUMMARY

[0006] The present disclosure relates to methods for managing CAR T cell therapy-associated diarrhea. In aspects, the methods are for a human subject undergoing CAR T cell therapy for colorectal cancer (CRC), wherein the CAR T cells target a CRC-associated antigen. Aspects of the disclosure involve administering a prophylactic regimen to the subject after infusion of the CAR T cells. The prophylactic regimen may comprise agents such as vedolizumab, infliximab, or prophylactic budesonide. The methods can further 1  Attorney Docket: I071-0123PCT / SDS1.0133PCT   include monitoring the subject for the development and severity of diarrhea based on predefined clinical criteria, which may include stool frequency or stool volume, after the CAR T cell therapy.

[0007] Further aspects of the disclosure relate to methods designed to reduce the incidence, duration, and severity of CAR T cell therapy-induced diarrhea. Such methods can improve patient safety and treatment tolerability. In some approaches, if diarrhea occurs and meets certain predefined clinical criteria based on severity, the method may further comprise administering at least one therapeutic agent to the subject, potentially according to a tiered therapeutic algorithm. The CRC-associated antigen targeted by the CAR T cells may include, for example, GCC or Carcinoembryonic Antigen (CEA).

[0008] This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The Detailed Description is described with reference to the accompanying figures. The use of the same reference numbers in different figures indicates similar or identical items.

[0010] FIG. 1A illustrates the actual clinical course for Patient 104-1031, depicting the diarrhea grade on a scale of 0 to 5 over days -3 to 30 post-infusion of CAR T cells. The figure also indicates the timing of administration of various therapeutic agents, including DEX (Dexamethasone), MP (Methylprednisolone), LPM (Loperamide), BUD (Budesonide), IFX (Infliximab), and DAS (Dasatinib).

[0011] FIG.1B illustrates a contemplated revised management timeline for Patient 104-1031, depicting the potential diarrhea grade response over days -3 to 30 post-infusion of CAR T cells under a modified treatment plan. The timing of administration of therapeutic agents in this hypothetical scenario includes MP, LPM, BUD, VDZ (Vedolizumab), IFX, ATG (Antithymocyte Globulin), and AB (Antibiotics). A dashed vertical line around day 5 may indicate a point of intervention or assessment in the revised plan.

[0012] FIG. 2A illustrates the actual clinical course for Patient 101-1052, showing the diarrhea grade over days 0 to 30 post-infusion of CAR T cells. The figure also details the timing of administration of numerous therapeutic and supportive agents, including DEX, MP, PRED (Prednisone), DAS, APAP (Acetaminophen / Paracetamol), SXT (Sulfamethoxazole / Trimethoprim), LVFX (Levofloxacin), CAZ (Ceftazidime), ACV (Acyclovir), LPM, OCT (Octreotide), and IFX.

[0013] FIG. 2B illustrates a contemplated revised management timeline for Patient 101-1052, showing the potential diarrhea grade response over days 0 to 30 post-infusion of CAR T cells under a modified treatment plan. Indicated therapeutic agents in this hypothetical scenario include DEX, MP, PRED, DAS, VDZ, BUD, BUD RF (Budesonide Rectal Foam), IFX, ATG, NPO (Nothing by Mouth), as well as supportive medications such as APAP, SXT, LVFX, CAZ, ACV, LPM, and OCT.

[0014] FIG.2C presents the body temperature profile for Patient 101-1052, recorded in degrees Celsius, over days 0 to 30 post-CAR T cells infusion.

[0015] FIG. 3A illustrates the actual clinical course for Patient 102-1005, depicting the diarrhea grade over days -3 to 30 post-infusion of CAR T cells. The figure indicates the timing of administration of therapeutic and supportive agents, including MP, DAS, APAP, LVFX, CIP (Ciprofloxacin), SXT, FLCZ 2  Attorney Docket: I071-0123PCT / SDS1.0133PCT   (Fluconazole), ACV, IFX, LPM, DPH-ATR (Diphenoxylate-Atropine), OCT, CHOL (Cholestyramine), and TPN (Total Parenteral Nutrition).

[0016] FIG.3B illustrates a contemplated revised management timeline for Patient 102-1005, depicting the potential diarrhea grade response over days -3 to 30 post-infusion of CAR T cells under a modified treatment plan. Indicated therapeutic agents in this hypothetical scenario include MP, DAS, VDZ, BUD, BUD RF, IFX, ATG, NPO, TPN, as well as supportive medications such as APAP, LVFX, CIP, SXT, FLCZ, ACV, LPM, DPH-ATR, OCT, and CHOL.

[0017] FIG.3C presents the body temperature profile for Patient 102-1005, recorded in degrees Celsius, over days -3 to 30 post-infusion of CAR T cells.

[0018] FIG. 4A illustrates the actual clinical course for Patient 104-1013, depicting the diarrhea grade over days -3 to 30 post-infusion of CAR T cells. The figure indicates the timing of administration of various agents, including TCZ (Tocilizumab), ALP (Alprazolam), APAP, MEM (Memantine), VAN (Vancomycin), ACV, LPM, DPH-ATR, and IFX.

[0019] FIG.4B illustrates a contemplated revised management timeline for Patient 104-1013, depicting the potential diarrhea grade response over days -3 to 30 post-infusion of CAR T cells under a modified treatment plan. Indicated therapeutic agents in this hypothetical scenario include MP, TCZ, VDZ, BUD, BUD RF, IFX, ATG, DAS, as well as other supportive medications including ALP, APAP, MEM, VAN, ACV, LPM, and DPH-ATR.

[0020] FIG.4C presents the body temperature profile for Patient 104-1013, recorded in degrees Celsius, over days -3 to 28 post-infusion of CAR T cells.

[0021] FIG. 5A illustrates the actual clinical course for Patient 104-1003, depicting the diarrhea grade over days -3 to 30 post-infusion of CAR T cells. The figure indicates the timing of administration of various agents, including DAS, TCZ, DPH (Diphenhydramine), APAP, LVFX, SXT, FLCZ, ACV, LPM, OND (Ondansetron), LIDO (Lidocaine), LIDO Patch, TRAM (Tramadol), and SUM (Sumatriptan).

[0022] FIG.5B illustrates a contemplated revised management timeline for Patient 104-1003, depicting the potential diarrhea grade response over days -3 to 30 post-infusion of CAR T cells under a modified treatment plan. Indicated therapeutic agents in this hypothetical scenario include DEX, TCZ, VDZ, BUD, BUD RF, IFX, ATG, DPH, APAP, LVFX, SXT, FLCZ, ACV, LPM, OND, LIDO, LIDO Patch, TRAM, and SUM.

[0023] FIG.5C presents the body temperature profile for Patient 104-1003, recorded in degrees Celsius, over days -3 to 29 post-infusion of CAR T cells.

[0024] FIG.6 illustrates the timeline of diarrhea frequency ("times") for Patient 101-1110 over days 0 to 39 post-infusion of CAR T cells, and compares the actual administration of ATG (0.75 mg / kg) under a previous protocol with the expected (earlier) administration of ATG (0.75 mg / kg) under an improved protocol. An annotation indicates that under the improved protocol, "ATG usage will be moved up by 9 days".

[0025] FIG. 7A illustrates the actual clinical course for Patient 101-1114, depicting the diarrhea grade over days -3 to 30 CAR T post-infusion of CAR T cells. The figure indicates the timing of administration 3  Attorney Docket: I071-0123PCT / SDS1.0133PCT   of MP (with a taper period noted), DAS (administered QD (administered once a day) and BID (administered two times daily), IFX, BUD, and VDZ.

[0026] FIG.7B illustrates a contemplated revised management timeline for Patient 101-1114, depicting the potential diarrhea grade response over days -3 to 30 post-infusion of CAR T cells. The indicated therapeutic agents and their timing appear identical to those in FIG.7A, representing the course potentially under a refined or optimized protocol implementation.

[0027] FIG. 8A illustrates the actual clinical course for Patient 104-1039, depicting the diarrhea grade over days -3 to 30 post-infusion of CAR T cells. The figure indicates the timing of administration of VDZ and BUD.

[0028] FIG.8B illustrates a contemplated revised management timeline for Patient 104-1039, depicting the potential diarrhea grade response over days -3 to 30 post-infusion of CAR T cells under a modified treatment plan. Indicated therapeutic agents in this hypothetical scenario include IFX, MP, BUD, and VDZ.

[0029] In these figures, Corticosteroids listed include DEX, which is Dexamethasone; MP, which is Methylprednisolone; and PRED, which is Prednisone. Antibacterial Agents include SXT, identified as Sulfamethoxazole-trimethoprim / Cotrimoxazole; LVFX, which is Levofloxacin; CIP, standing for Ciprofloxacin; CAZ, which is Ceftazidime; MEM, identified as Meropenem; and VAN, which is Vancomycin. Antifungi Agents include FLCZ, which is Fluconazole. Antivirus Agents include ACV, which is Acyclovir. Antidiarrheal agents listed are LPM, identified as Loperamide, and OCT, which is Octreotide. Antipyretics include APAP, which is Acetaminophen. Antiemetics include OND, which is Ondansetron. Other codes and full drug names include ALP for Alprazolam; IFX for Infliximab; TCZ for Tocilizumab; ATG for Anti-thymocyte globulin; VDZ for Vedolizumab; another entry for CAZ as Ceftazidime (also listed under Antibacterial Agents); DPH-ATR for Diphenoxylate-atropine; BUD for Budesonide; BUD RF for Budesonide Rectal Foam; DAS for Dasatinib; AB for Antibody such as CD38, CD52, and / or CD25; CHOL for Cholestyramine; DPH for Diphenhydramine; LIDO for Lidocaine; LIDO Patch for Lidocaine 5% patch; TRAM for Tramadol; and SUM for Sumatriptan. DETAILED DESCRIPTION

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any method and material similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

[0031] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0032] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. 4  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0033] The term “activation,” as used herein, refers to the state of a cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0034] The term “antibody” is used in the broadest sense and refers to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. The antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, and Fv, Fab, Fab’ and F(ab’)2and fragments, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0035] The term “antibody fragments” refers to a portion of a full length antibody, for example, the antigen binding or variable region of the antibody. Other examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

[0036] The term “Fv” refers to the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanates six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv including only three complementarity determining regions (CDRs) specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site (the dimer).

[0037] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two major antibody light chain isotypes.

[0038] The term “synthetic antibody” refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term also includes an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and the expression of the DNA molecule to obtain the antibody, or to obtain an amino acid encoding the antibody. The synthetic DNA is obtained using technology that is available and well known in the art.

[0039] The term “antigen” refers to a molecule that provokes an immune response, which may involve either antibody production, or the activation of specific immunologically-competent cells, or both. Antigens include any macromolecule, including all proteins or peptides, or molecules derived from recombinant or 5  Attorney Docket: I071-0123PCT / SDS1.0133PCT   genomic DNA. For example, DNA including a nucleotide sequence or a partial nucleotide sequence encoding a protein or peptide that elicits an immune response, and therefore, encodes an “antigen” as the term is used herein. An antigen need not be encoded solely by a full-length nucleotide sequence of a gene. An antigen can be generated, synthesized, or derived from a biological sample including a tissue sample, a tumor sample, a cell, or a biological fluid.

[0040] The term “anti-tumor effect” as used herein, refers to a biological effect associated with a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, decrease in tumor cell proliferation, decrease in tumor cell survival, an increase in life expectancy of a subject having tumor cells, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies in the prevention of the occurrence of tumor in the first place.

[0041] The term “autoantigen” or “self-antigen” refers to an antigen mistakenly recognized by the immune system as being foreign. Auto-antigens include cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0042] The term “autologous” is used to describe a material derived from a subject which is subsequently re-introduced into the same subject.

[0043] The term “allogeneic” is used to describe a graft derived from a different subject of the same species. As an example, a donor subject may be related or unrelated to the recipient subject, but the donor subject has immune system markers which are similar to the recipient subject.

[0044] The term “xenogeneic” is used to describe a graft derived from a subject of a different species. As an example, the donor subject is from a different species than a recipient subject and the donor subject and the recipient subject can be genetically and immunologically incompatible.

[0045] The term “cancer” is used to refer to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

[0046] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may include non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may include solid tumors. Types of cancers to be treated with the CARs of the disclosure include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies, e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0047] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic 6  Attorney Docket: I071-0123PCT / SDS1.0133PCT   (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0048] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0049] A solid tumor antigen is an antigen expressed on a solid tumor. In embodiments, solid tumor antigens are also expressed at low levels on healthy tissue. Examples of solid tumor antigens and their related disease tumors are provided in Table 1. Table 1 Solid Tumor antigen Disease Tumor PRLR Breast Cancer7  Attorney Docket: I071-0123PCT / SDS1.0133PCT   SIGLEC15 Urothelial Cancer SLC6A3 Renal Cancer KISS1R R l C, , mprise,” “includes” and “including” will be understood to imply the inclusion of a stated step or element (ingredient or component) or group of steps or elements (ingredients or components) but not the exclusion of any other step or element or group of steps or elements.

[0051] The phrase “consisting of” is meant to include, and is limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements or steps are required or mandatory and that no other elements may be present.

[0052] The phrase “consisting essentially of” is meant to include any element listed after the phrase and can include other elements or steps that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements or steps. Thus, the phrase “consisting essentially of” indicates that the listed elements or steps are required or mandatory, but that other elements or steps are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements or steps. In embodiments, those elements or steps that do not affect an embodiment are those elements or 8  Attorney Docket: I071-0123PCT / SDS1.0133PCT   steps that do not alter the embodiment’s ability in a statistically significant manner to perform a function in vitro or in vivo, such as killing cancer cells in vitro or in vivo.

[0053] The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules or there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0054] The term “corresponds to” or “corresponding to” refers to (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.

[0055] The term “co-stimulatory ligand” refers to a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including at least one of proliferation, activation, differentiation, and other cellular responses. A co-stimulatory ligand can include B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, a ligand for CD7, an agonist or antibody that binds the Toll ligand receptor and a ligand that specifically binds B7-H3. A co-stimulatory ligand also includes, inter alia, an agonist or an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83.

[0056] The term “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as proliferation. Co-stimulatory molecules include an MHC class I molecule, BTLA, and a Toll-like receptor.

[0057] The term “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0058] The terms “co-stimulatory signaling region”, “co-stimulatory domain”, and “co-stimulation domain” are used interchangeably to refer to one or more additional stimulatory domain in addition to a stimulatory or signaling domain such as CD3 zeta. The terms “stimulatory” or “signaling” domain (or region) are also used interchangeably, when referring, for example, to CD3 zeta, the primary signaling 9  Attorney Docket: I071-0123PCT / SDS1.0133PCT   domain. In embodiments, the co-stimulatory signaling domain and the stimulatory signaling domain can be on the same molecule or different molecules in the same cell.

[0059] The terms “disease” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out), and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians. The term “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate. In contrast, a “disorder” in a subject is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0060] The term “effective” refers to adequate to accomplish a desired, expected, or intended result. For example, an “effective amount” in the context of treatment may be an amount of a compound sufficient to produce a therapeutic or prophylactic benefit.

[0061] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence (except that a “T” is replaced by a “U”) and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0062] The term “exogenous” refers to a molecule that does not naturally occur in a wild-type cell or organism but is typically introduced into the cell by molecular biological techniques. Examples of exogenous polynucleotides include vectors, plasmids, and / or man-made nucleic acid constructs encoding the desired protein. With regard to polynucleotides and proteins, the term “endogenous” or “native” refers to naturally-occurring polynucleotide or amino acid sequences that may be found in a given wild-type cell or organism. Also, a particular polynucleotide sequence that is isolated from a first organism and transferred to a second organism by molecular biological techniques is typically considered an “exogenous” polynucleotide or amino acid sequence with respect to the second organism. In specific embodiments, polynucleotide sequences can be “introduced” by molecular biological techniques into a microorganism that already contains such a polynucleotide sequence, for instance, to create one or more additional copies of an otherwise naturally-occurring polynucleotide sequence, and thereby facilitate overexpression of the encoded polypeptide.

[0063] The term “expression” refers to the transcription and / or translation of a particular nucleotide sequence driven by its promoter. The term “overexpression” refers to the production of a gene product in 10  Attorney Docket: I071-0123PCT / SDS1.0133PCT   transgenic organisms or cells that exceeds levels of production in normal or non-transformed organisms or cells.

[0064] The term “expression vector” refers to a vector including a recombinant polynucleotide including expression control (regulatory) sequences operably linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses (AAV)) that incorporate the recombinant polynucleotide.

[0065] The term “homologous” refers to sequence similarity or sequence identity between two polypeptides or between two polynucleotides when a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. A comparison is made when two sequences are aligned to give maximum homology.

[0066] The term “immunoglobulin” or “Ig,” refers to a class of proteins, which function as antibodies. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to the allergen.

[0067] The term “isolated” refers to a material that is substantially or essentially free from components that normally accompany it in its native state. The material can be a cell or a macromolecule such as a protein or nucleic acid. For example, an “isolated polynucleotide,” as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and / or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.

[0068] The term “substantially purified” refers to a material that is substantially free from components that normally associated with it in its native state. For example, a substantially purified cell refers to a cell 11  Attorney Docket: I071-0123PCT / SDS1.0133PCT   that has been separated from other cell types with which it is normally associated in its naturally occurring or native state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to a cell that has been separated from the cells with which they are naturally associated in their natural state. In embodiments, the cells are cultured in vitro. In embodiments, the cells are not cultured in vitro.

[0069] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0070] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0071] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. Moreover, the use of lentiviruses enables integration of the genetic information into the host chromosome resulting in stably transduced genetic information. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0072] The term “modulating,” refers to mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0073] Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0074] The term “under transcriptional control” refers to a promoter being operably linked to and in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0075] The term “overexpressed” tumor antigen or “overexpression” of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area such as a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art. 12  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0076] The term “parenteral administration” of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intrasternal injection, or infusion techniques.

[0077] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, such as a mammal, for example, a human or any living organism amenable to the methods described herein. In embodiments, the patient, subject, or individual is a human or mammal. In embodiments, the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, and animals such as dogs, cats, mice, rats, and transgenic species thereof.

[0078] A subject in need of treatment or in need thereof includes a subject having a disease, condition, or disorder that needs to be treated. A subject in need thereof also includes a subject that needs treatment for prevention of a disease, condition, or disorder. Accordingly, the subject can also be in need of prevention of a disease condition or disorder. In embodiments, the disease is cancer.

[0079] The term “polynucleotide” or “nucleic acid” refers to mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes all forms of nucleic acids including single and double stranded forms of nucleic acids.

[0080] The terms “polynucleotide variant” and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions, and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide or has increased activity in relation to the reference polynucleotide (i.e., optimized). Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages in between, e.g., 90%, 95%, or 98%) sequence identity with a reference polynucleotide sequence described herein. The terms “polynucleotide variant” and “variant” also include naturally- occurring allelic variants and orthologs.

[0081] The terms “polypeptide,” “polypeptide fragment,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. In embodiments, polypeptides may include enzymatic polypeptides, or “enzymes,” which typically catalyze (i.e., increase the rate of) various chemical reactions. 13  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0082] The term “polypeptide variant” refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In embodiments, the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or replaced with different amino acid residues.

[0083] The term “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. The term “expression control (regulatory) sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0084] "NFAT promoter" refers to one or more NFAT binding sites or motifs linked to a minimal promoter of any gene expressed by T cells. In embodiments, the minimal promoter of a gene expressed by T cells is a minimal human IL-12 promoter. NFAT (nuclear factor of activated T cells) are transcription factors. Examples of NFAT transcription factors include NFAT1, NFAT2, NFAT3, NFAT4, and NFAT5. These transcription factors bind NFAT binding sites or motifs in the NFAT promoter. The NFAT promoter (or a functional portion or functional variant thereof) can comprise any number of binding motifs, e.g., at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs. In embodiments, the NFAT promoter comprises six NFAT binding motifs. In an especially preferred embodiment, the NFAT promoter nucleotide sequence comprises or consists of a functional portion or functional variant thereof.

[0085] The NFAT promoter (or a functional portion or functional variant thereof) is operatively associated with the nucleotide sequence encoding IL-12 (or a functional portion or functional variant thereof). "Operatively associated with" means that the nucleotide sequence encoding IL-12 (or a functional portion or functional variant thereof) is transcribed into IL-12 mRNA when the NFAT protein binds to the NFAT promoter sequence (or a functional portion or functional variant thereof). Without being bound to a particular theory, it is believed that NFAT is regulated by a calcium signaling pathway. In particular, it is believed that TCR stimulation (by, e.g., an antigen) and / or stimulation of the calcium signaling pathway of the cell (by, e.g., PMA / Ionomycin) increases intracellular calcium concentration and activates calcium channels. It is believed that the NFAT protein is then dephosporylated by calmodulin and translocated to the nucleus where it binds the NFAT promoter sequence (or a functional portion or functional variant thereof) and activates downstream gene expression. By providing an NFAT promoter (or a functional portion or functional variant thereof) that is operatively associated with the nucleotide sequence encoding IL-12 (or a functional portion or functional variant thereof), the nucleic acids described herein 14  Attorney Docket: I071-0123PCT / SDS1.0133PCT   advantageously make it possible to express IL-12 (or a functional portion or functional variant thereof) only when the host cell including the nucleic acid is stimulated by, e.g., PMA / Ionomycin and / or an antigen. More information can be found at US Patent No: 8,556,882, which is incorporated by the reference.

[0086] The term "corticosteroid" refers to a class of steroid hormones that are produced in the adrenal cortex of vertebrates, as well as the synthetic analogues of these hormones. Examples include, but are not limited3 to, budesonide and methylprednisolone. The term "diarrhea" refers to a condition characterized by frequent loose or watery stools. Severity can be defined by criteria such as stool frequency or stool volume. The term "infliximab" refers to a chimeric monoclonal antibody biologic drug that works against tumor necrosis factor alpha (TNF-α) and is used to treat autoimmune diseases and inflammatory conditions. The term "methylprednisolone" refers to a synthetic glucocorticoid or corticosteroid drug. The term "monitoring" refers to the process of observing, checking, or keeping a continuous record of something, such as a subject's clinical status or response to therapy. "On or about" when referring to a day post-infusion (e.g., "on or about Day 3") means the specified day, or one day before or after the specified day. The term "post-infusion" refers to the time period after the administration of CAR T cells to a subject. The term "prophylactic budesonide" refers to the administration of budesonide for the purpose of preventing the occurrence of diarrhea. The term "prophylactic regimen" refers to a course of medical treatment administered to prevent the occurrence of a disease or condition. The term "stool frequency" refers to the number of bowel movements, specifically loose or watery stools, a subject has within a defined period, typically 24 hours. The term "stool volume" refers to the total amount of loose or watery stool produced by a subject within a defined period, typically 24 hours, often measured in milliliters (mL). The term "therapeutic budesonide" refers to the administration of budesonide for the purpose of treating existing diarrhea. This can include different forms or dosages than prophylactic budesonide. The term "vedolizumab" refers to a humanized monoclonal antibody that binds to α4β7 integrin and is used in the treatment of inflammatory bowel diseases. As demonstrated by the clinical experiences detailed herein, the prophylactic regimen may be tailored to the patient, and in various embodiments, may comprise the administration of vedolizumab, infliximab, or budesonide, either individually or in various combinations.

[0087] The term “predefined clinical criteria,” as used herein, refers to a set of specific standards or conditions established before or during a monitoring period to assess a particular outcome, such as patient eligibility for therapy, risk status, or the development and severity of a toxicity like diarrhea. These criteria can include parameters such as stool frequency, stool volume, and laboratory or biomarker values. For example, in various embodiments described herein, the predefined clinical criteria for grading the severity of diarrhea are based on stool frequency and / or stool volume and may be defined as follows: mild diarrhea may be defined as 2 to 3 loose or watery stools per 24 hours or less than 600 mL of stool volume per 24 hours; moderate diarrhea may be defined as 4 to 6 loose or watery stools per 24 hours or a stool volume of 600 mL to 1200 mL per 24 hours; and severe diarrhea may be defined as 7 or more loose or watery stools per 24 hours, or a stool volume greater than 1200 mL per 24 hours, or any milder diarrhea that is not controlled by other therapeutic agents. In some contexts, the criteria may also define life-threatening 15  Attorney Docket: I071-0123PCT / SDS1.0133PCT   gastrointestinal events, which can include signs of perforation, ischemia, necrosis, bleeding, toxic megacolon, or hemodynamic collapse.

[0088] The term “predefined clinical criteria” as used herein also encompasses other objective standards detailed in the specification, such as laboratory value thresholds for patient eligibility (e.g., C-reactive protein (CRP) ≤21 mg / L, serum ferritin not exceeding two times the upper limit of normal, and Absolute Neutrophil Count (ANC) ≥1,000 / µL) and biomarker thresholds used for risk stratification or to guide intervention (e.g., Interleukin-6 (IL-6) >50 pg / mL or Interferon-gamma (IFN-γ) >500 pg / mL). Furthermore, the term can encompass criteria used for ongoing patient monitoring, which may include regular assessment of a complete blood count with differential (CBC w / diff), investigations to rule out an infectious cause for any observed symptoms, and the close tracking of fluid intake and output volumes.

[0089] While certain embodiments describe specific day-based schedules for prophylactic interventions, which represent an exemplary and reproducible protocol, it is understood that the optimal timing may vary between patients. Therefore, in other embodiments, the administration of therapeutic agents is tied to clinical or biomarker events, such as the onset of fever or the crossing of a predefined cytokine threshold, as described herein. This allows for a more personalized approach that accounts for individual patient responses.

[0090] The term "bind," "binds," or "interacts with" refers to a molecule recognizing and adhering to a second molecule in a sample or organism but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. The term “specifically binds,” as used herein with respect to an antibody, refers to an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as being specific. In another example, an antibody that specifically binds an antigen may also bind different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as being specific. In some instances, the terms “specific binding” or “specifically binding,” can be used to describe the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds a specific protein structure rather than to any protein. If an antibody is specific for epitope “A,” the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0091] A “binding protein” is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA- binding protein) and / or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding, and protein-binding activity. 16  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0092] A “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

[0093] Zinc finger binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein. Further, a Zinc finger binding domain may be fused a DNA-cleavage domain to form a Zinc finger nuclease (ZFN) targeting a specific desired DNA sequence. For example, a pair of ZFNs (e.g., a ZFN-left arm and a ZFN-right arm) may be engineered to target and cause modifications of specific desired DNA sequences (e.g., TRAC genes).

[0094] “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.

[0095] A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist. For example, the sequence 5′ GAATTC 3′ is a target site for the Eco RI restriction endonuclease.

[0096] A “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be the same chemical type of molecule or can be different chemical types of molecules. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP DNA-binding domain and one or more activation domains) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra). Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex- forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.

[0097] Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation can also be involved in the expression of the protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.

[0098] “Modulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a ZFP as described herein. Thus, gene inactivation may be partial or complete.

[0099] A “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non- coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. 17  Attorney Docket: I071-0123PCT / SDS1.0133PCT   Binding can be for the purposes of targeted DNA cleavage and / or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.

[0100] By “statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of statistical significance include the p-value, which is the frequency or probability with which the observed event would occur if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less. A “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” or a physiologically significant amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.61.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) an amount or level described herein.

[0101] The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β, and / or reorganization of cytoskeletal structures. CD3 zeta is not the only suitable primary signaling domain for a CAR construct with respect to the primary response. For example, back in 1993, both CD3 zeta and FcRy were shown as functional primary signaling domains of CAR molecules. Eshhar et al., "Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T cell receptors" PNAS, 1993 Jan 15;90(2):720-4, showed that two CAR constructs in which an scFv was fused to "either the FcR gamma chain or the CD3 complex chain" triggered T cell activation and target cell. Notably, as demonstrated in Eshhar et al., CAR constructs containing only the primary signaling domain CD3 zeta or FcR gamma are functional without the co-presence of co-stimulatory domains. Additional non-CD3 zeta based CAR constructs have been developed over the years. For example, Wang et al. (,"A Chimeric Antigen Receptor (CAR) Based Upon a Killer Immunoglobulin-Like Receptor (KIR) Triggers Robust Cytotoxic Activity in Solid Tumors" Molecular Therapy, vol.22, no. Suppl.1, May 2014, page S57) tested a CAR molecule in which an scFv was fused to "the transmembrane and cytoplasmic domain of' a killer immunoglobulin-like receptor (KIR). Wang et al. reported that, "a KIR-based CAR targeting mesothelin (SS 1-KIR) triggers antigen-specific cytotoxic activity and cytokine production that is comparable to CD3~-based CARs." A second publication from the same group, Wang et al. ("Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors" Cancer Immunol Res.2015 Jul;3(7):815-26) showed that a CAR molecule in which "a single-chain variable 18  Attorney Docket: I071-0123PCT / SDS1.0133PCT   fragment for antigen recognition was fused to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR)" functioned both in vitro and in vivo "when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor."

[0102] The term “stimulatory molecule” refers to a molecule on a T cell that specifically binds a cognate stimulatory ligand present on an antigen presenting cell. For example, a functional signaling domain derived from a stimulatory molecule is the zeta chain associated with the T cell receptor complex. The stimulatory molecule includes a domain responsible for signal transduction.

[0103] The term “stimulatory ligand” refers to a ligand that when present on an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the like.) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a cell, for example a T cell, thereby mediating a primary response by the T cell, including activation, initiation of an immune response, proliferation, and similar processes. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0104] The term “therapeutic” refers to a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state or alleviating the symptoms of a disease state.

[0105] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or another clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0106] The term “treat a disease” refers to the reduction of the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0107] The term “transfected” or “transformed” or “transduced” refers to a process by which an exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0108] The term “vector” refers to a polynucleotide that comprises an isolated nucleic acid that can be used to deliver the isolated nucleic acid to the interior of a cell. The cell can be an in vitro cell or an in vivo cell in a subject. Numerous vectors are known in the art including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term also includes non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and others. For example, lentiviruses are complex retroviruses, which, in 19  Attorney Docket: I071-0123PCT / SDS1.0133PCT   addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Lentiviral vectors are well known in the art. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2, and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu, and nef are deleted making the vector biologically safe.

[0109] In embodiments, a polynucleotide encoding the antigen binding molecule and / or therapeutic agent(s) can be used to implement techniques described herein. The method or use includes: providing a viral particle (e.g., AAV, lentivirus or their variants) comprising a vector genome, the vector genome comprising the polynucleotide, wherein the polynucleotide is operably linked to an expression control element conferring transcription of the polynucleotide; and administering an amount of the viral particle to the subject such that the polynucleotide is expressed in the subject. In embodiments, the AAV preparation may include AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids. More information of the administration and preparation of the viral particle may be found at the US Patent NO: 9840719 and Milani et al., Sci. Transl. Med. 11, eaav7325 (2019) 22 May 2019, which are incorporated herein by reference. In embodiments, the polynucleotide may integrate into the genome of the modified cell and the progeny of the modified cell will also express the polynucleotide, resulting in a stably transfected modified cell. In embodiments, the modified cell expresses the polynucleotide encoding the CAR but the polynucleotide does not integrate into the genome of the modified cell such that the modified cell expresses the transiently transfected polynucleotide for a finite period of time (e.g., several days), after which the polynucleotide is lost through cell division or other factors. For example, the polynucleotide is present in the modified cell in a recombinant DNA construct, in an mRNA, or in a viral vector, and / or the polynucleotide is an mRNA, which is not integrated into the genome of the modified cell. In embodiments, the vector is a lentivirus. In embodiments, the lentivirus can be packaged with a particle (e.g., a Nano particle) such as to be released for a predetermined time and directly infused to the subject such that the lentivirus can be transferred to T cells of the subject.

[0110] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0111] The T cell response in a subject refers to cell-mediated immunity associated with a helper, killer, regulatory, and other types of T cells. For example, T cell response may include activities such as assistance to other white blood cells in immunologic processes and identifying and destroying virus-infected cells and tumor cells. T cell response in the subject may be measured via various indicators such as the number of 20  Attorney Docket: I071-0123PCT / SDS1.0133PCT   virus-infected cells and / or tumor cells that T cells kill, an amount of cytokines that T cells release, for example, in co-culturing with virus-infected cells and / or tumor cells, a level of proliferation of T cells in the subject, a phenotype change of T cells (e.g., changes to memory T cells), and the longevity or lifespan of T cells in the subject.

[0112] In embodiments, in vitro killing assay may be performed by measuring the killing efficacy of CAR T cells by co-culturing CAR T cells with antigen-positive cells. CAR T cells may be considered to have killing effect on the corresponding antigen-positive cells by showing a decrease in the number of corresponding antigen-positive cells co-cultured with CAR T cells and an increase in the release of cytokines such as IFN-γ, TNF-α, and the like, as compared to control cells that do not express the corresponding antigen. Further, in vivo antitumor activity of the CAR T cells may be tested. For example, xenograft models can be established using the antigens described herein in immunodeficient mice. Heterotransplantation of human cancer cells or tumor biopsies into immunodeficient rodents (xenograft models) has, for the past two decades, constituted the major preclinical screen for the development of novel cancer therapeutics (Song et al., Cancer Res. PMC 2014 Aug 21, and Morton et al., Nature Protocols, 2, - 247 - 250 (2007)). To evaluate the anti-tumor activity of CAR T cells in vivo, immunodeficient mice bearing tumor xenografts were evaluated for CAR T cell anti-tumor activity, for example, a decrease in mouse tumors and / or mouse blood cytokines, such as IFN-γ, TNF-α, and the like.

[0113] The term “chimeric antigen receptor” or alternatively a “CAR” refers to a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain (e.g., cytoplasmic domain) including an intracellular signaling domain. In embodiments, the domains in the CAR polypeptide are on the same polypeptide chain, for example, comprising a chimeric fusion protein. In embodiments, the domains of the CAR polypeptide are not on the same molecule, for example, not contiguous with each other, or are on different polypeptide chains.

[0114] In embodiments, the intracellular signaling domain may include a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule as described herein. In embodiments, the intracellular signaling domain includes a functional signaling domain derived from a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In embodiments, the intracellular signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule. The co-stimulatory signaling region refers to a portion of the CAR including the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules can include cell surface molecules for inducing an efficient response from the lymphocytes (in response to an antigen).

[0115] Between the extracellular domain and the transmembrane domain of the CAR, there can be incorporated a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the cytoplasmic domain in the polypeptide chain. A spacer domain may include up to 300 amino acids, 10 to 100 amino acids, or 25 to 50 amino acids.

[0116] The extracellular domain of a CAR may include an antigen binding domain (e.g., a scFv, a single domain antibody, or TCR, such as a TCR alpha binding domain or a TCR beta binding domain), that targets 21  Attorney Docket: I071-0123PCT / SDS1.0133PCT   a specific tumor marker (e.g., a tumor antigen). Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T cell mediated immune responses. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, CEA, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. For example, when the antigen that the CAR binds is CD19, the CAR thereof is referred to as CD19 CAR. 19CAR, CD19CAR, CD19 CAR, or CD19-CAR), which is a CAR molecule that includes an antigen binding domain that binds CD19.

[0117] In embodiments, the extracellular ligand-binding domain comprises a scFv comprising the light chain variable (VL) region and the heavy chain variable (VH) region of a target antigen-specific monoclonal antibody joined by a flexible linker. Single chain variable region fragments are made by linking light and / or heavy chain variable regions by using a short linking peptide (Bird et al., Science 242:423-426, 1988). An example of a linking peptide is the GS linker having the amino acid sequence (GGGGS)3(SEQ ID: 2), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). In general, linkers can be short, flexible polypeptides comprising about 20 or fewer amino acid residues. Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports. The single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect, or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resultant scFv can be isolated using standard protein purification techniques known in the art.

[0118] In embodiments, the tumor antigen includes HER2, CD19, CD20, CD22, Kappa or light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3 / 4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor α 2, IL-11 receptor α, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-α, CD44v7 / 8, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8, or viral-associated antigens expressed by a tumor. In embodiments, the binding element of the CAR includes any antigen binding moiety that when bound to its cognate antigen, affects a tumor cell such that the tumor cell fails to grow, decrease in size, or dies.

[0119] The CAR can be a bispecific CAR. For example, the two antigen binding domains are on the same CAR (a bispecific CAR or tandem CAR (tanCAR)), on different CAR molecules, or on a CAR and T cell receptor (TCR). A single CAR can include two different antigen binding domains, or the two different 22  Attorney Docket: I071-0123PCT / SDS1.0133PCT   antigen binding domains are each on a separate CAR. The CAR can have more than two antigen binding domains, for example, a multispecific CAR. The antigen binding domains of the multispecific CAR can be on the same CAR or on separate CAR, such as one antigen binding domain on each CAR.

[0120] In embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises an intracellular domain of a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof.

[0121] In embodiments, the intracellular domain comprises a CD3 zeta signaling domain. Embodiments relate to a vector comprising the isolated nucleic acid sequence described herein. Embodiments relate to an isolated cell comprising the isolated nucleic acid sequence described herein.

[0122] The cells, including CAR cells and modified cells, described herein can be derived from a stem cell. The stem cells may be adult stem cells, embryonic stem cells, or non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. The cells can also be a dendritic cell, a NK-cell, a B-cell, or a T cell selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, and helper T lymphocytes. In embodiments, the cells can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. Prior to expansion and genetic modification of the cells described herein, a source of cells may be obtained from a subject through a variety of non-limiting methods. T cells may be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In embodiments, any number of T cell lines available and known to those skilled in the art, can be used. In embodiments, the cells may be derived from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In embodiments, the cells are part of a mixed population of cells which present different phenotypic characteristics.

[0123] A population of cells refers to a group of two or more cells. The cells of the population could be the same, such that the population is a homogenous population of cells. The cells of the population could be different, such that the population is a mixed population or a heterogeneous population of cells. For example, a mixed population of cells could include modified cells comprising a first CAR and cells comprising a second CAR, wherein the first CAR and the second CAR bind different antigens.

[0124] The term “stem cell” refers to any type of cell which has the capacity for self-renewal and the ability to differentiate into other kind(s) of cell. For example, a stem cell gives rise either to two daughter stem cells (as occurs in vitro with embryonic stem cells in culture) or to one stem cell and a cell that undergoes differentiation (as occurs e.g., in hematopoietic stem cells, which give rise to blood cells). Different categories of stem cells may be distinguished on the basis of their origin and / or on the extent of their capacity for differentiation into other types of cells. Stem cells can include embryonic stem (ES) cells (i.e., pluripotent stem cells), somatic stem cells, induced pluripotent stem cells, and any other types of stem cells. 23  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0125] Pluripotent embryonic stem cells can be found in the inner cell mass of a blastocyst and have high innate capacity for differentiation. For example, pluripotent embryonic stem cells have the potential to form any type of cell in the body. When grown in vitro for long periods of time, ES cells maintain pluripotency, and progeny cells retain the potential for multilineage differentiation.

[0126] Somatic stem cells can include fetal stem cells (from the fetus) and adult stem cells (found in various tissues, such as bone marrow). These cells have been regarded as having a capacity for differentiation lower than that of the pluripotent ES cells – with the capacity of fetal stem cells being greater than that of adult stem cells; they apparently differentiate into only a limited number of different types of cells and have been described as multipotent. “Tissue-specific” stem cells normally give rise to only one type of cell. For example, embryonic stem cells can differentiate into blood stem cells (e.g., Hematopoietic stem cells (HSCs)), which can further differentiate into various blood cells (e.g., red blood cells, platelets, white blood cells, etc.).

[0127] Induced pluripotent stem cells (iPS cells or iPSCs) can include a type of pluripotent stem cell artificially derived from a non-pluripotent cell (e.g., an adult somatic cell) by inducing expression of specific genes. Induced pluripotent stem cells are similar to naturally occurring pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability. Induced pluripotent cells can be isolated from adult stomach, liver, skin, and blood cells.

[0128] In embodiments, the CAR cells, the modified cell, or the cell is a T cell, a NK cell, a macrophage, or a dendritic cell. For example, the CAR cells, the modified cell, or the cell is a T cell.

[0129] T cells, or T lymphocytes, are a type of white blood cell of the immune system. There are various types of T cells including T helper (TH) cells, cytotoxic T (TC) cells (T killer cells, killer T cells), natural killer T (NKT) cells, memory T (Tm) cells, regulatory T (Treg) cells, and gamma delta T (γδ T) cells.

[0130] T helper (TH) cells assist other lymphocytes, for example, activating cytotoxic T cells and macrophages and maturation of B cells into plasma cells and memory B cells. These T helper cells express CD4 glycoprotein on their surface and are also known as CD4+ T cells. Once activated, these T cells divide rapidly and secrete cytokines.

[0131] Cytotoxic T (TC) cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. They express CD8 protein on their surface. Cytotoxic T cell release cytokines.

[0132] Natural Killer T (NKT) cells are different from natural killer cells. NKT cells recognize glycolipid antigens presented by CD1d. Once activated, NKT cells produce cytokine and release cell killing molecules.

[0133] Memory T (Tm) cells are long-lived and can expand to large number of effector T cells upon re- exposure to their cognate antigen. Tm cells provide the immune system with memory against previously encountered pathogens. There are various subtypes of Tm cells including central memory T (TCM) cells, effector memory T (TEM) cells, tissue resident memory T (TRM) cells, and virtual memory T cells. Tm cells are either CD4+ or CD8+ and usually CD45RO. 24  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0134] Regulatory T (Treg) cells shut down T cell mediated immunity at the end of an immune reaction and suppress autoreactive T cells that escaped the process of negative selection in the thymus. Subsets of Treg cells include thymic Treg and peripherally derived Treg. Both subsets of Treg require the expression of the transcription factor FOXP3.

[0135] Gamma delta T (γδ T) cells are a subset of T cells that possess a γδ T cell receptor (TCR) on the cell surface, as most T cells express the αβ TCR chains. γδ T cells are less common in human and mice and are mainly found in the gut mucosa, skin, lung, and uterus. They are involved in the initiation and propagation of immune responses.

[0136] In embodiments, the antigen binding molecule is a T Cell Receptor (TCR). In embodiments, the TCR is modified TCR. In embodiments, the TCR is derived from spontaneously occurring tumor-specific T cells in patients. In embodiments, the TCR binds a tumor antigen. In embodiments, the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1. In embodiments, the TCR comprises TCRγ and TCRδ chains or TCRα and TCRβ chains.

[0137] In embodiments, a T cell clone that expresses a TCR with high affinity for the target antigen may be isolated. In embodiments, tumor-infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) may be cultured in the presence of antigen-presenting cells (APCs) pulsed with a peptide representing an epitope known to elicit a dominant T cell response when presented in the context of a defined HLA allele. High-affinity clones may be then selected on the basis of MHC–peptide tetramer staining and / or the ability to recognize and lyse target cells pulsed with low titrated concentrations of cognate peptide antigen. After the clone has been selected, the TCRα and TCRβ chains or TCRγ and TCRδ chains are identified and isolated by molecular cloning. For example, for TCRα and TCRβ chains, the TCRα and TCRβ gene sequences are then used to generate an expression construct that ideally promotes stable, high-level expression of both TCR chains in human T cells. The transduction vehicle (e.g., a gammaretrovirus or lentivirus) may be then generated and tested for functionality (antigen specificity and functional avidity) and used to produce a clinical lot of the vector. An aliquot of the final product is then used to transduce the target T cell population (generally purified from patient PBMCs), which is expanded before infusion into the subject.

[0138] In embodiments, the APCs include dendritic cells, macrophages, Langerhans cells and B cells, or T cells.

[0139] In embodiments, the binding element of the CAR may include any antigen binding moiety that when bound to its cognate antigen, affects a tumor cell for example, it kills the tumor cell, inhibits the growth of the tumor cell, or promotes death of the tumor cell.

[0140] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically, rather than cloned. 25  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0141] The embodiments of the present disclosure further relate to vectors in which a nucleic acid described herein is inserted. Vectors can be derived from retroviruses such as the lentiviruses that are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0142] Viruses can be used to deliver nucleic acids into a cell in vitro and in vivo (in a subject). Examples of viruses useful for delivery of nucleic acids into cells include retrovirus, adenovirus, herpes simplex virus, vaccinia virus, and adeno-associated virus.

[0143] There also exist non-viral methods for delivering nucleic acids into a cell, for example, electroporation, gene gun, sonoporation, magnetofection, and the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles.

[0144] The expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to one or more promoters and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration into eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0145] Additional information related to expression of synthetic nucleic acids encoding CARs and gene transfer into mammalian cells is provided in U.S. Pat. No. US8,906,682, incorporated by reference in its entirety.

[0146] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0147] When “an immunologically effective amount”, “an anti-tumor effective amount”, “a tumor- inhibiting effective amount”, “therapeutic amount”, or “effective amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104to 109cells / kg body weight, preferably 105to106cells / kg body weight, including all integer values within those ranges. T cell compositions can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly. In embodiments, activated T cells are administered to a subject and then subsequently blood is redrawn (or have apheresis performed). T cells are collected, expanded, and reinfused into the subject. 26  Attorney Docket: I071-0123PCT / SDS1.0133PCT   This process can be carried out multiple times every few weeks. In embodiments, T cells can be activated from blood draws of from 10 cc to 400 cc. In embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multiple blood draw / multiple reinfusion protocols, certain populations of T cells can be selected.

[0148] The administration of the pharmaceutical compositions described herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The pharmaceutical compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (i. v.), or intraperitoneally. In embodiments, the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In embodiments, the T cell compositions of the present disclosure are administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection. In embodiments of the present disclosure, cells activated and expanded using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present disclosure may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993; Isoniemi (supra)). In embodiments, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In embodiments, the cell compositions of the present disclosure are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan®. For example, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present disclosure. In embodiments, expanded cells are administered before or following surgery.

[0149] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices by a physician depending on various factors. 27  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0150] In embodiments, the population of cells described herein is used in autologous CAR T cell therapy. In embodiments, the CAR T cell therapy is allogenic CAR T cell therapy, TCR T cell therapy, and NK cell therapy.

[0151] Embodiments relate to an in vitro method for preparing modified cells. The method may include obtaining a sample of cells from the subject. For example, the sample may include T cells or T cell progenitors. The method may further include transfecting the cells with a DNA encoding at least a CAR, culturing the population of CAR cells ex vivo in a medium that selectively enhances proliferation of CAR- expressing T cells.

[0152] In embodiments, the sample is a cryopreserved sample. In embodiments, the sample of cells is from umbilical cord blood or a peripheral blood sample from the subject. In embodiments, the sample of cells is obtained by apheresis or venipuncture. In embodiments, the sample of cells is a subpopulation of T cells.

[0153] As used herein, the term “gene fusion” refers to the fusion of at least a portion of a gene to at least a portion of an additional gene. The gene fusion need not include entire genes or exons of genes. In some instances, gene fusion is associated with alternations in cancer. A gene fusion product refers to a chimeric genomic DNA, a chimeric messenger RNA, a truncated protein or a chimeric protein resulting from a gene fusion. The gene fusion product may be detected by various methods described in U.S. Patent 9,938,582, which is incorporated as a reference herein. A “gene fusion antigen” refers to a truncated protein or a chimeric protein that results from a gene fusion. In embodiments, an epitope of a gene fusion antigen may include a part of the gene fusion antigen or an immunogenic part of another antigen caused by the gene fusion. In embodiments, the gene fusion antigen interacts with, or is part of, cell membranes.

[0154] In embodiments, detection of mRNA and protein expression levels of a target molecules (e.g., CARs and cytokines) in cells, such as human cells, may be performed using experimental methods such as qPCR and FACS. Further, target molecules specifically expressed in the corresponding tumor cells with very low expression or undetectable expression in normal tissue cells may be identified.

[0155] In embodiments, In Vitro Killer Assay as well as killing experiment of CAR T Cells Co-Cultured with Antigen-Positive Cells can be performed. CAR T cells can exhibit a killing effect on the corresponding antigen-positive cells, a decrease in the number of corresponding antigen-positive cells co-cultured with CAR T cells, and an increase in the release of IFN-γ, TNF-α, etc. as compared to control cells that did not express the corresponding antigen.

[0156] In embodiments, In Vivo Killer Assay can be performed. For example, mice may be transplanted with corresponding antigen tumor cells, and tumorigenic, transfusion of CAR T cells, and a decrease in mouse tumors and mouse blood IFN-γ, TNF-α, and other signals can be detected.

[0157] Embodiments relate to a method of eliciting and / or enhancing T cell response in a subject having a solid tumor or treating a solid tumor in the subject, the method comprising administering an effective amount of T cells comprising the CAR described herein. In embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises an intracellular domain of a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, 28  Attorney Docket: I071-0123PCT / SDS1.0133PCT   lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof. In embodiments, the intracellular domain comprises a CD3 zeta signaling domain.

[0158] Embodiments relate to a vector comprising the isolated nucleic acid described herein. Embodiments relate to an isolated cell comprising the isolated nucleic acid sequence described herein. Embodiments relate to a composition comprising a population of T cells comprising the CAR described herein. Embodiments relate to a CAR encoded by the isolated nucleic acid sequence described herein. Embodiments relate to a method of eliciting and / or enhancing T cell response in a subject or treating a tumor of the subject, the method comprising: administering an effective amount of T cell comprising the CAR described herein.

[0159] In embodiments, the CAR molecules described herein comprise one or more complementarity- determining regions (CDRs) for binding an antigen of interest. CDRs are part of the variable domains in immunoglobulins and T cell receptors for binding a specific antigen. There are three CDRs for each variable domain. Since there is a variable heavy domain and a variable light domain, there are six CDRs for binding an antigen. Further since an antibody has two heavy chains and two light chains, an antibody can have twelve CDRs altogether for binding antigens.

[0160] In embodiments, the modified cells described herein includes a CAR molecule comprising at least two different antigen binding domains. The CAR molecule can be a bispecific CAR molecule. For example, the two antigen binding domains can be on the same CAR molecule, on different CAR molecules, or on a CAR molecule and T cell receptor (TCR). A single CAR can include at least two different antigen binding domains, or the two different antigen binding domains are each on a separate CAR molecule. The at least two different antigen binding domains can be on the same CAR molecule or different CAR molecules, but in the same modified cell. Moreover, the at least two different antigen binding domains can be on a CAR molecule and a T cell receptor in the same modified cell. In embodiments, the bispecific CAR molecule can include a binding domain binding an antigen of WBC (e.g., CD19) and a binding domain binding a solid tumor antigen. In embodiments, the bispecific CAR molecule may include two binding domains binding two different solid tumor antigens.

[0161] In embodiments, the at least two different antigen binding domains are on different CAR molecules which are expressed by different modified cells. Further, the one or more different antigen binding domains are on a CAR molecule and a T cell receptor, which are expressed by different modified cells.

[0162] The embodiments of the present disclosure further relate to vectors in which a DNA encoding a desired molecule of the present disclosure can be inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non- proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0163] Pharmaceutical compositions of the present disclosure can be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will 29  Attorney Docket: I071-0123PCT / SDS1.0133PCT   be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials. As an example, pharmaceutical compositions disclosed herein include nucleic acids encoding CAR or vectors described herein and a pharmaceutically acceptable carrier.

[0164] The term "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. Federal or a state government or the EMA (European Medicines Agency) or listed in the U.S. Pharmacopeia (United States Pharmacopeia- 33 / National Formulary-28 Reissue, published by the United States Pharmacopeial Convention, Inc., Rockville Md., publication date: April 2010) or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0165] The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origins, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. For the use of (further) excipients and their use see also "Handbook of Pharmaceutical Excipients", fifth edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0166] The administration of the pharmaceutical compositions described herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i. v.) injection, or intraperitoneally. In embodiments, the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present disclosure are preferably administered by i.v. injection. The compositions of T cells can be injected directly into a tumor, lymph node, or site of infection. In embodiments of the present disclosure, cells activated and expanded using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously, or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir, and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In embodiments, the T cells of the present disclosure can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either 30  Attorney Docket: I071-0123PCT / SDS1.0133PCT   the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor-induced signaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993; Isoniemi (supra)). In embodiments, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously, or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In embodiments, the cell compositions of the present disclosure are administered following B-cell ablative therapy, such as agents that react with CD20, e.g., Rituxan. For example, subjects can undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present disclosure. In other embodiments, expanded cells are administered before or following surgery.

[0167] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices by a physician, depending on various factors.

[0168] When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor- inhibiting effective amount,” “therapeutic amount,” or “effective amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, the extent of infection or metastasis, and condition of the patient (subject). It can be stated that a pharmaceutical composition comprising the T cells described herein can be administered at a dosage of 104to 109cells / kg body weight, preferably 105to106cells / kg body weight, including all integer values within those ranges. T cell compositions can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly. In embodiments, it can be desired to administer activated T cells to a subject and then subsequently redraw the blood (or have apheresis performed), collect the activated and expanded T cells, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In embodiments, T cells can be activated from blood draws of from 10 cc to 400 cc. In embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Not to be bound by theory, certain populations of T cells can be selected using this multiple blood draw / multiple reinfusion protocols.

[0169] Embodiments of the present disclosure relate to methods for managing inflammatory diarrhea associated with immune therapy (e.g, CAR T therapy). The methods are based on clinical insights into the mechanisms of immune therapy associated diarrhea, including the recognition of distinct inflammatory 31  Attorney Docket: I071-0123PCT / SDS1.0133PCT   pathways and the utility of stool volume-based severity grading. The methods provide for prophylactic and therapeutic interventions to reduce the incidence and severity of this toxicity.

[0170] Immunotherapy is a type of medical treatment that utilizes a person's own immune system to fight diseases, such as cancer, by stimulating or altering its function to better find and attack targeted cells. This field includes several major approaches. Immune checkpoint inhibitors, for example, are drugs that block proteins, such as PD-1, which cancer cells can use to hide from the immune system. Another major type is antibody therapy using monoclonal antibodies, which are lab-made proteins designed to attach to specific targets on cancer cells, marking them for destruction. Adoptive cell therapies, such as CAR T cell therapy, represent another key strategy where a patient's own immune cells (T-cells) are collected, genetically modified to better recognize cancer, and then reinfused to fight the disease. Because the cells are genetically altered, CAR T therapy is also considered a form of cell-based gene therapy. Other forms of immunotherapy further expand the ways the immune system can be harnessed. Cancer treatment vaccines, for instance, are designed to boost the immune system's response to cancer cells already present in the body. Immune system modulators, such as cytokines, work more broadly to enhance the body's overall immune response against cancer. Additionally, oncolytic virus therapy uses viruses that are genetically modified to specifically infect and destroy cancer cells while sparing healthy ones.

[0171] In one aspect, the disclosure provides a method for reducing the incidence or severity of inflammatory diarrhea in a human subject receiving a CAR T therapy. The method may comprise administering to the subject a prophylactically effective amount of at least one agent selected from a gut- selective integrin inhibitor and / or a tumor necrosis factor-alpha (TNF-α) inhibitor. This prophylactic administration is performed after the subject has received an infusion of CAR T cells but before the subject develops significant inflammatory diarrhea, for example, prior to the onset of grade 2 or higher diarrhea as graded by standard criteria such as the CTCAE and / or volume-based criteria like MAGIC. This preemptive approach is designed to interrupt the gut-specific inflammatory cascade before it leads to severe mucosal injury.

[0172] In some embodiments, the at least one agent administered prophylactically is a gut-selective integrin inhibitor. The gut-selective integrin inhibitor may function by blocking the trafficking of inflammatory lymphocytes to the gut mucosa. In a particular embodiment, the gut-selective integrin inhibitor is an inhibitor of α4β7 integrin. A specific, non-limiting example of an α4β7 integrin inhibitor suitable for use in the methods described herein is vedolizumab. The administration of an agent like vedolizumab is intended to specifically target the gut-homing pathway of T-cells, thereby preventing or mitigating the initial inflammatory insult to the gastrointestinal tract following CAR T cell infusion.

[0173] In other embodiments, the at least one agent administered prophylactically is a TNF-α inhibitor. The TNF-α inhibitor acts by neutralizing TNF-α, a key cytokine implicated in T-cell mediated inflammation and apoptosis of intestinal epithelial cells. In a particular embodiment, the TNF-α inhibitor is infliximab. The prophylactic use of a TNF-α inhibitor like infliximab may prevent or reduce the severity of diarrhea by suppressing the inflammatory response before it becomes clinically significant. 32  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0174] In yet another embodiment, the method comprises administering both a gut-selective integrin inhibitor and a TNF-α inhibitor. For example, a subject may receive both vedolizumab and infliximab prophylactically after CAR T cell infusion. This combination may provide a multi-pronged approach, simultaneously blocking T-cell trafficking to the gut and neutralizing a key inflammatory cytokine.

[0175] The inflammatory diarrhea addressed by these prophylactic methods can be characterized by particular pathological features. For example, the diarrhea may be associated with histological findings from a mucosal biopsy, such as diffuse epithelial apoptosis and crypt loss, which are indicative of an immune-mediated enteritis.

[0176] In some embodiments, the methods of the disclosure are directed to treating moderate diarrhea associated with CAR T cell therapy. This method comprises administering dasatinib to the subject, for example, at a dose of 90 mg twice daily (BID). The administration of dasatinib may be performed in combination with a corticosteroid, such as methylprednisolone administered at 1 mg / kg intravenously (IV) BID, until the diarrhea resolves. As detailed in the clinical examples, dasatinib has been administered to patients to manage gastrointestinal toxicity, including in one instance from day 10 to day 13 post-infusion 3 and in another from day 10 to day 22 post-infusion. This intervention may be initiated when a subject presents with moderate diarrhea, defined as 4 to 6 loose or watery stools per 24 hours or a stool volume of 600 to 1200 mL per 24 hours.

[0177] In yet other embodiments, the disclosure provides a method for managing severe or refractory CAR T cell therapy-associated diarrhea that has not responded adequately to other interventions, such as corticosteroids or TNF-α inhibitors. This method comprises administering a Janus Kinase (JAK) inhibitor to the subject. In a specific embodiment, the JAK inhibitor is a JAK1 / 2 inhibitor. In a more specific embodiment, the JAK inhibitor is ruxolitinib. The administration of a JAK inhibitor may be initiated as an escalation of therapy in patients with persistent high-volume diarrhea who have already received corticosteroids, infliximab, and / or ATG.

[0178] In another aspect, the present disclosure provides a kit for use in managing gastrointestinal toxicity, such as diarrhea, in a human subject undergoing CAR T cell therapy. In some embodiments, the kit comprises at least one therapeutic agent and instructions for use. The at least one therapeutic agent may be selected from the group consisting of: vedolizumab, infliximab, budesonide, a corticosteroid, ATG, a JAK inhibitor, and any combination thereof. In some embodiments, the budesonide in the kit may be formulated for oral administration, rectal administration (e.g., as a foam), or both. The instructions for use would direct a healthcare professional to administer the agent(s) according to a method disclosed herein. For instance, the instructions may specify: a prophylactic regimen, such as administering vedolizumab on or about Day 3 and / or infliximab on or about Day 5 post-CAR T cell infusion; a tiered therapeutic algorithm wherein the choice of agent is based on the severity of diarrhea as graded by predefined clinical criteria, such as the MAGIC criteria which consider stool frequency and / or stool volume; specific actions for different grades of diarrhea, such as administering ATG for moderate diarrhea (e.g., 4-6 loose stools / 24 hrs or 600-1200 mL / 24 hrs) or for mild diarrhea that persists for more than 3 days; a warning or instruction 33  Attorney Docket: I071-0123PCT / SDS1.0133PCT   regarding administration timing, such as a directive that infliximab should not be administered within 48 hours after vedolizumab administration.

[0179] The present disclosure also provides, in another aspect, a method of treating inflammatory diarrhea that has already developed in a subject undergoing CAR T therapy. This therapeutic method may comprise several steps. First, a stool output volume of the subject is monitored following the infusion of CAR T cells. Monitoring stool volume, rather than only frequency, provides a more accurate measure of the severity of high-volume, watery diarrhea that can occur with CAR T therapy. Second, based on the monitored stool output volume, a determination is made that the subject has developed grade 3 or higher diarrhea. Finally, in response to this determination, a therapeutically effective amount of a T-cell depleting agent is administered to the subject to control the severe, established diarrhea.

[0180] In some embodiments of this therapeutic method, the determination of grade 3 or higher diarrhea is based on the Mount Sinai Acute GVHD International Consortium (MAGIC) criteria (e.g., <600 mL / 24 hrs, 600–1200 mL / 24 hrs, >1200 mL / 24 hrs). The adoption of the volume-based MAGIC criteria enables a more objective and timely identification of severe diarrhea, potentially allowing for earlier intervention.

[0181] The T-cell depleting agent administered in this method serves to reduce the underlying T-cell mediated immunopathology. In a preferred embodiment, the T-cell depleting agent is antithymocyte globulin (ATG). The use of ATG is particularly contemplated for cases of severe, refractory diarrhea where standard immunosuppressants have failed.

[0182] The inflammatory diarrhea treated by this method may be of a particularly severe nature, characterized by widespread mucosal denudation that resembles acute gastrointestinal graft-versus-host disease (GI-GVHD). This pathological presentation underscores the severity of the immune reaction that necessitates the use of a potent T-cell depleting agent like ATG.

[0183] In some implementations, the therapeutic method for established diarrhea can be combined with the prophylactic strategies. For example, a subject may have first received a prophylactically effective amount of a gut-selective integrin inhibitor and / or a TNF-α inhibitor. If, despite this prophylaxis, the subject proceeds to develop severe diarrhea, the method of monitoring stool volume and administering a T-cell depleting agent can be implemented as a second-line or escalation therapy.

[0184] The present disclosure further contemplates a comprehensive, integrated method for managing a human subject undergoing CAR T therapy, combining both prophylactic and therapeutic elements in a layered strategy. This management method comprises several steps performed in sequence. First, after an infusion of CAR T cells, a prophylactically effective amount of a first therapeutic agent is administered to the subject. This first agent is selected from a gut-selective integrin inhibitor and a TNF-α inhibitor. Second, the subject's stool output volume is monitored subsequent to the administration of the first therapeutic agent. Third, an onset of grade 3 or higher inflammatory diarrhea is identified based on the monitored stool output volume (e.g., <600 mL / 24 hrs, 600–1200 mL / 24 hrs, >1200 mL / 24 hrs). Finally, in response to identifying the onset of severe diarrhea, a therapeutically effective amount of a second, different therapeutic agent is administered to the subject, wherein the second therapeutic agent is a T-cell depleting agent. This integrated 34  Attorney Docket: I071-0123PCT / SDS1.0133PCT   approach allows for early, targeted prevention, followed by systematic monitoring and a clear plan for escalating to a more potent therapy if needed.

[0185] In various embodiments of this comprehensive management method, the first therapeutic agent may be an α4β7 integrin inhibitor, such as vedolizumab. In other embodiments, the first therapeutic agent is a TNF-α inhibitor, such as infliximab. The second therapeutic agent, administered upon escalation, may be ATG. The identification of the need for escalation, that is, the onset of grade 3 or higher diarrhea, can be performed using the MAGIC grading system to provide an objective, volume-based trigger for the second therapeutic intervention. This layered management protocol represents a novel and non-obvious strategy derived from the clinical reinterpretation of patient outcomes.

[0186] In embodiments, the disclosure provides a composition comprising vedolizumab, infliximab, and budesonide, for use in a method of reducing an incidence or severity of a CAR T-cell therapy-associated gastrointestinal toxicity in a human subject. The present disclosure also relates to the use of a composition comprising vedolizumab, infliximab, and budesonide in the manufacture of a medicament for the prophylaxis of gastrointestinal toxicity in a subject receiving a CAR T cell therapy. In these embodiments, the therapeutic agents are intended for administration in a specific sequence and time frame relative to the infusion of the CAR T cells to prophylactically manage potential toxicities. For example, the vedolizumab may be for administration on day 3 post-infusion of the CAR T cells, the infliximab for administration on day 5 post-infusion, and the budesonide for administration for a duration of time, such as from day 5 to day 12 post-infusion of the CAR T cells.

[0187] The present disclosure further provides for therapeutic agents for use in treating CAR T cell therapy-associated gastrointestinal toxicity in specific, defined patient subgroups, wherein the selection of the patient for treatment is based on an assessment of their clinical symptoms.

[0188] In embodiments, antithymocyte globulin is provided for use in a method of treating a CAR T cell therapy-associated gastrointestinal toxicity. The use of antithymocyte globulin may also be described in the context of manufacturing a medicament for treating this toxicity. The treatment is directed to a human subject or patient subgroup selected for treatment on the basis that the subject exhibits a particular severity of diarrhea. For example, the subject may be selected based on having steroid-refractory diarrhea and a stool output volume of greater than 600 mL per 24 hours.

[0189] This patient subgroup can be further stratified. In one implementation, the antithymocyte globulin is for use in a subject who exhibits a stool output volume of between 600 mL and 1200 mL per 24 hours. For this subgroup, the antithymocyte globulin may be for administration at a specific dose, for example, a dose of 0.75 mg / kg. In another implementation, the antithymocyte globulin is for use in a subject exhibiting a more severe toxicity, characterized by a stool output volume of greater than 1200 mL per 24 hours. In such cases of severe toxicity, the antithymocyte globulin may be for administration on at least two consecutive days to achieve sufficient T-cell depletion. These uses are contemplated for toxicities arising from CAR T cell therapies targeting antigens such as GUCY2C or CEA.

[0190] In another embodiment, a different therapeutic agent is provided for a different patient subgroup. For example, methylprednisolone is provided for use in a method of treating CAR T cell therapy-associated 35  Attorney Docket: I071-0123PCT / SDS1.0133PCT   gastrointestinal toxicity in a human subject selected for treatment on the basis that the subject exhibits a stool output volume of less than 600 mL per 24 hours, and wherein the toxicity persists for more than 3 days. This embodiment describes an intervention for a lower-grade but persistent toxicity.

[0191] In a further aspect, the disclosure provides a kit for the prophylactic treatment of a CAR T cell therapy-associated gastrointestinal toxicity. Such a kit may facilitate the proper administration of the combination therapies described herein. In embodiments, the kit comprises a first pharmaceutical composition comprising vedolizumab; a second pharmaceutical composition comprising infliximab; and a third pharmaceutical composition comprising budesonide. The kit further comprises instructions for the administration of these compositions. The instructions may specify the sequential administration of the first, second, and third pharmaceutical compositions in a period relative to an infusion of CAR T cells. In embodiments of the kit, the instructions for use specify a precise administration schedule. For example, the instructions may specify the administration of the first pharmaceutical composition (comprising vedolizumab) on day 3 post-infusion of the CAR T cells, and the administration of the second pharmaceutical composition (comprising infliximab) and the third pharmaceutical composition (comprising budesonide) beginning on day 5 post-infusion. This provides the practitioner with a clear protocol for implementing the prophylactic regimen.

[0192] Embodiments of the present disclosure are directed to methods of managing CAR T cell therapy- associated diarrhea in a human subject undergoing CAR T cell therapy for colorectal cancer (CRC), wherein the CAR T cells target a CRC-associated antigen.

[0193] In an exemplary embodiment, the method comprises administering a prophylactic regimen to the subject post-infusion of the CAR T cells. The prophylactic regimen, in various aspects, comprises vedolizumab, infliximab, or prophylactic budesonide. The method further comprises monitoring the subject for the development and severity of diarrhea based on predefined clinical criteria, including stool frequency or stool volume, after the CAR T cell therapy. This monitoring is important for early detection and management of CAR T cell therapy-associated diarrhea.

[0194] In some embodiments, the CAR T cells administered to the subject target a CRC-associated antigen. The CRC-associated antigen may be, for example, GCC or CEA. Other CRC-associated antigens known in the art may also be targeted by the CAR T cells. Clinical experience has shown that CAR T cell therapies targeting antigens expressed on normal intestinal tissue, such as GCC and CEA, can lead to significant on-target, off-tumor gastrointestinal toxicities, including severe diarrhea and colitis.

[0195] The term "CRC-associated antigen" refers to an antigen that is preferentially or abundantly expressed on colorectal cancer cells compared to normal cells, and which can be targeted by CAR T cell therapy. The restricted or elevated expression of such antigens on colorectal cancer cells makes them potential targets for such therapies. Examples of CRC-associated antigens include, but are not limited to, GCC, CEA, Epithelial Cell Adhesion Molecule (EpCAM), Mesothelin (MSLN), Human Epidermal Growth Factor Receptor 2 (HER2), Mucin-1 (MUC1), Doublecortin-like Kinase 1 (DCLK1), CD133, B7-H3 (CD276), Tumor-Associated Glycoprotein 72 (TAG-72), and NKG2D ligands (NKG2DL). In specific embodiments described herein, the CRC-associated antigen may comprise GCC or CEA. 36  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0196] The monitoring of the subject for the development and severity of diarrhea involves predefined clinical criteria. In certain embodiments, these predefined clinical criteria for monitoring the subject comprise the daily collection of a complete blood count with differential (CBC w / diff). This can help in assessing the overall health status of the subject and identify signs of inflammation or infection. The criteria may also include an assessment to rule out an infectious cause of the diarrhea. It is important to distinguish between CAR T cell therapy-associated diarrhea and diarrhea caused by infectious agents, as the management strategies may differ. Furthermore, the monitoring can include the monitoring of intake and output volumes to assess the subject's hydration status and the severity of fluid loss due to diarrhea. The term "complete blood count with differential (CBC w / diff)" refers to a blood test used to evaluate overall health and detect a wide range of disorders, including anemia, infection, and leukemia.1 It measures several components and features of2 the blood, including red blood cells, white blood cells (with a breakdown of the different types), hemoglobin, hematocrit, and platelets.

[0197] Details of the prophylactic regimen can vary. In one embodiment, where the prophylactic regimen comprises vedolizumab, the vedolizumab is administered to the subject on or about Day 3 post-infusion of the CAR T cells. Administration around this time point is intended to preempt or mitigate the onset of diarrhea.

[0198] In another embodiment, the prophylactic regimen comprises infliximab. In such cases, or where infliximab is used in combination with another prophylactic agent as described herein (e.g., vedolizumab), the infliximab may be administered to the subject on or about Day 5 post-infusion of the CAR T cells.

[0199] In yet another embodiment, the prophylactic regimen comprises prophylactic budesonide. This prophylactic budesonide may be administered from on or about Day 5 to on or about Day 12 post-infusion of the CAR T cells. This extended administration period aims to provide sustained protection against diarrhea during a common window of onset.

[0200] The methods disclosed herein also encompass therapeutic interventions if diarrhea occurs despite the prophylactic regimen. In some embodiments, if diarrhea occurs in the subject on or after Day 5 post- infusion of the CAR T cells and meets the predefined clinical criteria based on its severity, the method further comprises administering at least one therapeutic agent to the subject. This administration follows a tiered therapeutic algorithm, which is based on the severity of the diarrhea as determined by the predefined clinical criteria. The tiered therapeutic algorithm, in some aspects, comprises administration of a corticosteroid or administration of ATG.

[0201] The term “tiered therapeutic algorithm,” as used herein, refers to a structured, stepwise approach to treatment wherein the selection and administration of therapeutic interventions are based on the severity of a clinical condition as determined by predefined criteria. In the context of the present disclosure, the tiers typically correspond to different grades of diarrhea severity (e.g., mild, moderate, severe), and each tier is associated with a specific, recommended therapeutic action or set of actions, such as the administration of a corticosteroid or antithymocyte globulin (ATG), often in a manner of escalating intensity that corresponds to increasing clinical severity. 37  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0202] The tiered therapeutic algorithm provides specific interventions based on the severity of diarrhea, which can be classified, for example, as mild, moderate, or severe.

[0203] In an embodiment addressing mild diarrhea, if mild diarrhea occurs on or after Day 5 post- infusion of the CAR T cells, wherein mild diarrhea is defined as 2 to 3 loose or watery stools per 24 hours or less than 600 mL of stool volume per 24 hours, then administering the at least one therapeutic agent comprises administering methylprednisolone. Methylprednisolone is a corticosteroid aimed at reducing inflammation contributing to the diarrhea.

[0204] Should mild diarrhea persist despite initial treatment, further intervention may be necessary. In a specific embodiment, the method further comprises administering ATG if the mild diarrhea persists for more than 3 days despite administration of the methylprednisolone. ATG provides a broader immunosuppressive effect.

[0205] For cases of moderate diarrhea, in some embodiments, if moderate diarrhea occurs on or after Day 5 post-infusion of the CAR T cells, wherein moderate diarrhea is defined as 4 to 6 loose or watery stools per 24 hours or 600 mL to 1200 mL of stool volume per 24 hours, then administering the at least one therapeutic agent comprises administering ATG. The use of ATG for moderate diarrhea reflects a step-up in therapeutic intensity.

[0206] In situations where the subject has received or is scheduled to receive infliximab as part of their prophylactic regimen, a specific consideration for ATG administration may apply. In an embodiment, if the prophylactic regimen comprises infliximab, and if the subject has received the infliximab as part of the prophylactic regimen or is scheduled to receive the infliximab on the same day as the onset of the moderate diarrhea, then the administration of the ATG is delayed for 24 hours. This delay allows for an assessment of the subject's response to the infliximab before escalating to ATG therapy.

[0207] For instances of severe diarrhea, a different therapeutic approach may be implemented. In some embodiments, if severe diarrhea occurs on or after Day 5 post-infusion of the CAR T cells, wherein severe diarrhea is defined as 7 or more loose or watery stools per 24 hours, or greater than 1200 mL of stool volume per 24 hours, or any milder diarrhea not controlled by other therapeutic agents, then administering the at least one therapeutic agent comprises administering therapeutic budesonide. This therapeutic budesonide, in certain aspects, comprises orally administered budesonide and rectally administered budesonide foam, providing both systemic and local anti-inflammatory effects.

[0208] The duration of therapeutic budesonide administration can be specified. In a particular embodiment, the therapeutic budesonide is administered until on or about Day 19 post-infusion of the CAR T cells or for at least 7 days after the severe diarrhea is controlled, whichever is later. This ensures that treatment is continued until the diarrhea is resolved and for a period thereafter to prevent recurrence.

[0209] Further management strategies for diarrhea occurring at later time points are also contemplated. In an embodiment, the method further comprises administering a second dose of infliximab for management of diarrhea if the diarrhea occurs on or after Day 12 post-infusion of the CAR T cells. This may be particularly relevant if the subject previously responded to infliximab or if a delayed onset or recurrence of diarrhea occurs. 38  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0210] Following the administration of potent immunosuppressive agents such as ATG, subjects may be at an increased risk of infections. Accordingly, in some embodiments where ATG is administered (e.g., for persistent mild diarrhea or for moderate diarrhea), the method further comprises administering at least one agent selected from the group consisting of an antifungal agent, an antibacterial agent, and an antiviral agent, after administration of the ATG. This prophylactic measure aims to prevent opportunistic infections.

[0211] The methods described herein provide a comprehensive approach to managing CAR T cell therapy-associated diarrhea, encompassing prophylactic strategies and a tiered, severity-based therapeutic algorithm to address diarrhea if it occurs. The specific timings, agents, and criteria are designed to optimize patient care and outcomes during CAR T cell therapy for CRC.

[0212] The present disclosure further relates to methods for predicting, grading, managing, and overseeing gastrointestinal (GI) toxicity, particularly diarrhea, in human patients receiving GCC-targeted CAR T therapy for conditions such as metastatic colorectal cancer. These methods aim to improve patient outcomes and the tolerability of such advanced cancer treatments by integrating risk assessment, real-time monitoring, tiered prophylactic and therapeutic interventions, and structured safety oversight.

[0213] In some embodiments, a method for managing GI toxicity in a patient receiving GCC-targeted CAR T therapy includes monitoring the patient for the development of GI symptoms, such as diarrhea, subsequent to the infusion of CAR T cells. If diarrhea develops, its severity is graded. This grading can be based on criteria such as the MAGIC (Mount Sinai Acute GVHD International Consortium) criteria, which may consider a combination of stool frequency (number of bowel movements per 24-hour period) and stool volume (measured in milliliters per 24-hour period). For instance, diarrhea may be classified as moderate or severe when the measured stool volume is equal to or exceeds a predefined threshold, or when the combined assessment of frequency and volume meets criteria for MAGIC grade 2 or higher. Stool volume may be measured using a dedicated stool collection and measurement system, potentially a sensor- integrated system configured to log volume and frequency data continuously or at frequent intervals. Such a sensor system for stool volume measurement might employ a fluid-level sensor calibrated for a range, for example, from 100 mL to 2000 mL, with a clinically acceptable accuracy, such as within approximately ±5%. Stool frequency may be recorded using a motion sensor system or a pressure sensor system integrated into the patient's toilet or commode.

[0214] The term "MAGIC criteria" refers to a standardized, multi-center grading system originally established for assessing the severity of acute graft-versus-host disease (GVHD), with a particular focus on grading gastrointestinal (GI) GVHD based on diarrhea stool output volume or, when precise volume measurement is not feasible, stool frequency as a surrogate. The original MAGIC criteria provide specific quantitative thresholds for adults to stage diarrhea severity, for example: Stage 0 for stool output <500 mL / day; Stage 1 for 500-999 mL / day; Stage 2 for 1,000-1,500 mL / day; and Stage 3 for >1,500 mL / day, with Stage 4 characterized by severe symptoms such as intense abdominal pain or grossly bloody stools. For the purposes of the present disclosure, the fundamental principles of the MAGIC criteria, specifically the grading of diarrhea severity based on quantified stool volume or stool frequency, are utilized. In some clinical protocols or contexts relevant to the embodiments described herein, specific thresholds adapted 39  Attorney Docket: I071-0123PCT / SDS1.0133PCT   from the original MAGIC criteria may be employed. For instance, an adaptation, may define thresholds for adults such as less than 600 mL / 24h for mild diarrhea, 600 mL to 1200 mL / 24h for moderate diarrhea, and greater than 1200 mL / 24h for severe diarrhea. Accordingly, when specific definitions for diarrhea severity (e.g., mild, moderate, severe) are provided in the embodiments of this specification, they are based on such principles and may reflect these or other similar adaptations, and should be understood as applying the MAGIC principles with specific, potentially modified, quantitative criteria.

[0215] The MAGIC criteria used for grading may be supplemented with baseline-adjusted thresholds specific to the patient's pre-treatment bowel habits for a more individualized assessment. In some applications, diarrhea may be classified as severe specifically when the measured stool volume exceeds 1200 mL per 24 hours, irrespective of stool frequency. Alternatively, the MAGIC grading system may define diarrhea severity based on stool output with specific thresholds, such as: Grade 1 corresponding to a stool volume of less than 500 mL per 24 hours; Grade 2 corresponding to a stool volume between 500 mL and 1000 mL per 24 hours; Grade 3 corresponding to a stool volume between 1000 mL and 1500 mL per 24 hours; and Grade 4 corresponding to a stool volume exceeding 1500 mL per 24 hours. A modified grading tier, for instance, a Grade 4+, may be defined for extreme volume diarrhea, such as that exceeding 2000 mL / day. The MAGIC grading may be applied independently of patient-reported symptoms, incorporating such quantitative stool volume thresholds as the primary criterion. The grading may be applied at regular intervals, for example, every 8 hours, particularly during the first 7 days post-CAR T infusion.

[0216] Based on this classification, a therapeutic intervention, such as the administration of Anti- Thymocyte Globulin (ATG), may be initiated. For example, ATG administration may be initiated for persistent grade 2 or higher diarrhea that has not responded adequately to other interventions like corticosteroids or infliximab, ideally before symptoms progress to grade 4 severity. ATG may be administered intravenously, for example, at a dosing regimen of 0.75 milligrams per kilogram (mg / kg) of patient body weight per day, potentially for up to four consecutive days, with the total cumulative dosing for a treatment course not exceeding a certain limit, such as 3 mg / kg. The administration of ATG is adjusted based on the patient's response, including observed reductions in stool volume and stool frequency. ATG administration may be discontinued earlier than planned if a satisfactory clinical response is observed, such as if the patient's stool volume falls below a threshold like 600 mL per 24 hours within 48 hours of the initial ATG dosing. In some instances, early discontinuation of ATG may be warranted if stool volume reduces to below 600 mL / 24 hours and additionally, if associated biomarkers like Interleukin-6 (IL-6) levels decline by a significant margin (e.g., more than 30%) within 48 hours of starting ATG. ATG dosing may also be paused or modified if certain hematological parameters are met, such as an Absolute Neutrophil Count (ANC) falling below 500 / µL or a platelet count falling below 30,000 / µL. Pharmacokinetic (PK) monitoring for ATG may be employed to maintain therapeutic serum levels, for example, between 100– 400 micrograms / mL.

[0217] The management of GI toxicity can be temporally stratified. A period spanning approximately Day 4 through Day 10 post-CAR T cell infusion can be designated as a primary window of toxicity, during 40  Attorney Docket: I071-0123PCT / SDS1.0133PCT   which symptom onset is considered more likely. A subsequent period, for example, from approximately Day 11 through Day 30 post-infusion, may comprise a secondary phase focusing on recovery, tapering of corticosteroids, and identification of late-onset or recrudescent GI inflammation. Diarrhea occurring very early, for instance, before Day 5 post-infusion, may often be attributable to other causes. Day 9 post- infusion may emerge as an inflection point for onset or intensification of CAR T cell-associated toxicities.

[0218] Further embodiments provide comprehensive methods for predicting, grading, and managing GI toxicity by integrating predictive biomarker analysis with a detailed, multi-parameter system for grading diarrhea severity, guiding a tiered treatment escalation protocol. Subsequent to CAR T cell infusion, a biological sample (e.g., blood, serum, plasma) is obtained and analyzed to measure concentrations of a panel of cytokines. This panel may include IL-6, Interleukin-10 (IL-10), Interferon-gamma (IFN-γ), Interleukin-2 (IL-2), Interleukin-12 (IL-12), and Tumor Necrosis Factor-alpha (TNF-α), measured using techniques like multiplex immunoassay panels. Based on predefined thresholds for these cytokines, the patient's risk of developing CAR T-induced diarrhea is classified (e.g., low-risk, moderate-risk, high-risk).

[0219] Diarrhea severity is graded using a dual-parameter system considering both stool frequency (e.g., number of loose / watery bowel movements per 24 hours, potentially graded per National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE)) and stool volume (mL per 24-hour period, potentially measured per MAGIC criteria). For example, severe diarrhea may be classified if stool volume exceeds 1200 mL per 24 hours or stool frequency exceeds 7 bowel movements per 24 hours. Based on the risk classification and diarrhea severity grade, a treatment escalation protocol is initiated. This may involve administering vedolizumab to moderate-risk patients with a certain diarrhea grade, or ATG to high-risk patients with a similar or higher grade. Treatment intensity is dynamically adjusted based on real-time or frequently updated changes in cytokine levels, stool frequency, and stool volume, allowing for personalized therapy escalation or de-escalation, including tapering of immunosuppressives during a "cool-down" period.

[0220] Infection risk management is also incorporated, particularly with ATG use. Infection risk can be determined based on levels of cytokines like IL-6 and TNF-α. Based on an assigned infection risk category (e.g., low, moderate, high), tiered antimicrobial prophylaxis may be initiated: no prophylaxis for low-risk; single-agent antimicrobial (e.g., broad-spectrum antibacterial) for moderate-risk; and a combination of antibacterial, antifungal, and antiviral agents for high-risk. This prophylaxis may be initiated prior to ATG or other systemic immunosuppressive therapy in high-infection-risk patients. For patients receiving ATG who also show an increased infection risk based on elevated IL-6 and TNF-α levels exceeding predefined thresholds, prophylactic antimicrobial therapy (e.g., levofloxacin, fluconazole, acyclovir) may be initiated.

[0221] Cytokine data and stool monitoring data can be integrated into a Clinical Decision Support System (CDSS), which may process data, apply risk classification and grading rules, and deliver automated treatment recommendations, potentially via an Electronic Medical Record (EMR) interface. The stool volume sensor may comprise a fluid-level detection system integrated into a collection container, measuring volume with high accuracy (e.g., ±5%). Stool frequency can be determined using pressure or motion sensor arrays. This automated system may comprise a cloud-connected data processor for longitudinal trend 41  Attorney Docket: I071-0123PCT / SDS1.0133PCT   analysis. Such monitoring may occur for at least 14 to 21 days post-infusion, with features like automatic recalibration. The system may trigger alerts, for instance, if IL-6 or TNF-α levels increase by more than 50% within 24 hours, prompting stool volume reassessment.

[0222] Machine learning (ML) models (e.g., random forest classifier, neural network) trained on historical data (cytokine patterns, stool trends, outcomes) may be used to predict the likelihood of severe GI toxicity. Risk classification thresholds for cytokines may be personalized based on baseline patient characteristics (e.g., prior chemotherapy, age, Body Mass Index (BMI), baseline cytokine levels). The CDSS may prioritize recommendations based on toxicity classification, biomarker trends, and comorbidity- adjusted risk assessment.

[0223] Further embodiments focus on predicting and managing GI toxicity through early risk identification via biomarker analysis and fever monitoring, enabling preemptive interventions even before observable clinical symptoms of GI toxicity, or before they become severe. A biological sample is obtained post-CAR T infusion to measure cytokines (e.g., IL-6, IL-10, IFN-γ, IL-2, IL-12, TNF-α), which are compared to predefined thresholds. Concurrently, body temperature is monitored for fever (e.g., ≥38°C). A patient is determined at high risk if at least one cytokine exceeds its threshold and a fever event is detected. Upon high-risk determination, a preemptive clinical intervention is initiated. This intervention may comprise administering vedolizumab (e.g., 300 mg intravenously, potentially on or after Day 8 post- infusion or within 24 hours of a qualifying fever), especially if IL-6 and IL-10 exceed specific thresholds. Alternatively, the intervention may be corticosteroid therapy (e.g., Methylprednisolone, Budesonide) or anti-TNF therapy (e.g., Infliximab, Etanercept, potentially administered at least 48 hours after any prior vedolizumab and within 24 hours of high-risk determination). Fluid resuscitation (e.g., intravenous lactated Ringer's solution or normal saline) may also be part of the preemptive intervention.

[0224] Predefined cytokine thresholds for this predictive approach may be tailored to a GI-specific risk model. Examples include IL-6 between 20-50 picograms per milliliter (pg / mL), IL-10 between 5-15 pg / mL, TNF-α between 10-25 pg / mL, and IFN-γ between 15-35 pg / mL. These thresholds may be dynamically adjusted based on baseline biomarker levels or patient-specific factors (BMI, autoimmune disorders, prior chemotherapy). A real-time clinical alert may be generated for high-risk patients, deliverable via EMR or mobile device, potentially including intervention recommendations and escalation to a rapid response team. Fever monitoring may involve continuous tracking (wearable / implantable sensor), and other vital signs (heart rate variability, blood pressure, oxygen saturation) may be integrated to refine risk prediction. A diarrhea grading system (stool frequency / volume) can be used adjunctively. ML models may be applied to the high-risk determination process. This preemptive approach can be integrated into an AI-driven CDSS.

[0225] Methods for optimizing treatment escalation integrate biomarker-based risk assessment with clinical observations. Cytokine concentrations (e.g., IL-6, IL-10, IFN-γ, IL-2, IL-12, TNF-α) are measured from biological samples post-CAR T infusion and compared to predefined thresholds to classify GI toxicity risk (low, moderate, high). A treatment escalation protocol is initiated based on this risk. For example, moderate-risk patients might receive vedolizumab (e.g., 300 mg IV) on Day 5 post-infusion, conditional on absence of significant fever (e.g., ≥38°C) in the first 4 days. High-risk patients might receive ATG (e.g., 42  Attorney Docket: I071-0123PCT / SDS1.0133PCT   0.75 mg / kg IV) on or after Day 4 post-infusion, contingent on meeting predefined clinical criteria for severe immune toxicity. Treatment is dynamically adjusted based on real-time cytokine trends and clinical symptoms. High-risk patients may receive combination therapy (e.g., infliximab and corticosteroids), with infliximab administered no earlier than 48 hours after any prior vedolizumab and after meeting criteria for GI toxicity progression. Treatment intensity may be tapered if cytokine levels rapidly return to near- baseline (e.g., within 48 hours of intervention).

[0226] Risk classification can include stool volume / frequency data. Cytokine levels may be monitored frequently (e.g., daily for at least 7 days). If vedolizumab is given on Day 5, additional cytokine monitoring at Day 7 may assess response. ATG might be considered earlier (e.g., Day 3), with adjustment around Day 6 based on biomarkers. A sequential strategy might use vedolizumab first-line; if cytokine levels do not decrease significantly (e.g., within 48 hours), infliximab and corticosteroids may be second-line. Preemptive vedolizumab for moderate / high risk may be accompanied by prophylactic antibiotics. Vedolizumab may be combined with dual-form (oral / rectal) budesonide as a prophylactic or early therapeutic intervention, potentially triggered by early fever post-infusion, even without concurrently elevated cytokines.

[0227] Specific cytokine thresholds can be defined: e.g., moderate-risk if IL-6 is 20-50 pg / mL; high-risk if IL-6 exceeds 50 pg / mL, especially with increased TNF-α. These may be adjusted based on pre-CAR T baseline levels. Biomarker kinetics (e.g., increasing IL-6 over 24 hours plus fever / GI symptoms) may trigger earlier / more aggressive ATG. Secondary indicators like C-reactive protein (CRP) levels greater than 20 mg / L or sustained stool volume exceeding 1000 mL / 24 hours (for >24h) may support escalation to ATG (no earlier than Day 4). Continuous heart rate variability (HRV) monitoring (significant decrease, e.g., >20%) or high fever (e.g., >39°C within 72 hours) may trigger escalation. Rapid bedside cytokine testing (results within 30 minutes) or wearable biosensors for continuous monitoring can facilitate timely decisions. Protocols may be adapted for specific populations (e.g., patients with pre-existing autoimmune disorders might receive an IL-23 inhibitor instead of vedolizumab). High-risk patients (even before Day 3) may receive prophylactic corticosteroids. Dosing may be adjusted for BMI (e.g., BMI ≥30 kg / m²) or renal function.

[0228] Methods for predicting and stratifying GI toxicity risk and severity utilize cytokine measurements (e.g., IL-6, IL-10, IFN-γ, TNF-α) compared to predefined thresholds to determine a GI toxicity risk score. This score stratifies risk into categories (e.g., Low: cytokines below thresholds; Moderate: at least one cytokine exceeds moderate threshold; High: two or more cytokines exceed high thresholds). The risk score is correlated with measured stool volume (and optionally frequency) to refine overall toxicity severity classification. Thresholds may be dynamically adjusted based on patient baseline levels or aggregated population data (updated periodically). Risk scores may be recalculated frequently (e.g., daily for at least 7 days). Cytokine measurements may occur at multiple time points (e.g., Day 3, 7, 14). Illustrative thresholds: IL-6 between 20-50 pg / mL, IL-10 between 5-15 pg / mL. The risk score can be a weighted index, with cytokines like IL-6 and TNF-α having greater weight. A "very high risk" category may exist if multiple key cytokines (IL-6, TNF-α, IFN-γ) simultaneously exceed high thresholds. Stool volume may be quantified 43  Attorney Docket: I071-0123PCT / SDS1.0133PCT   using automated sensor-equipped devices, monitored daily (e.g., Day 0-12), integrated with real-time grading (e.g., MAGIC) and alerts. High risk may be classified if stool volume exceeds 1000 mL / 24 hours and at least two cytokines exceed high-risk thresholds. Personalized treatment recommendations (e.g., Vedolizumab for moderate-risk; Infliximab / corticosteroids for high-risk; ATG / combination therapy for very high-risk) can be delivered via EMR-integrated CDSS. Cytokines may be measured by multiplex immunoassay. Data may be analyzed by a cloud-based system. ML models trained on retrospective data can refine the risk score, identifying complex interactions. The risk score can be further adjusted by patient- specific clinical factors (age, liver function, pre-existing GI disorders).

[0229] A system for grading diarrhea severity in immunotherapy patients can comprise a data collection module (for stool frequency per 24 hours, manual or automated), a grading engine (processing frequency to classify severity, e.g., mild: ≤4 / 24h; moderate: 5-6 / 24h; severe: ≥7 / 24h), and a classification output module (providing severity level to healthcare professionals). The system may include a sensor-based stool monitoring device (detecting frequency via infrared / pressure sensors; volume via smart toilet insert / fluid collection with weight / volumetric sensors, accuracy e.g., ±10%). A wearable sensor platform might log bowel activity. A clinical decision support (CDS) interface may present treatment recommendations based on severity, potentially integrated with EMR. An alert system can notify providers of moderate / severe diarrhea, with guidance. Grading logic can incorporate patient baseline bowel habits (if no chronic GI conditions) or be adjusted for weight, hydration, age. The grading engine may use ML algorithms (decision tree, random forest, neural network) trained on historical data. The output module can visualize severity trends. A biomarker monitoring interface may receive GI-related cytokine data (IL-6, TNF-α) as supportive context, with optional escalation advisories if thresholds are exceeded (not sole criteria for intervention).

[0230] Methods for dynamically adjusting treatment sequencing correlate measured biomarker levels (e.g., IL-6, IL-10, IFN-γ, TNF-α, CRP) with GI toxicity parameters (stool volume mL / 24h, stool frequency / 24h) to classify toxicity status into escalation levels (e.g., Mild: biomarkers below thresholds AND stool volume <600 mL / 24h; Moderate: at least one biomarker exceeds threshold OR stool volume 600–1200 mL / 24h; Severe: two or more biomarkers exceed thresholds OR stool volume >1200 mL / 24h). Treatment recommendations are generated: e.g., Moderate toxicity: Vedolizumab (300 mg IV) or Infliximab (5 mg / kg IV). Sequentially for moderate: vedolizumab first; if no significant biomarker improvement within 72 hours, escalate to infliximab (after ≥48h post-vedolizumab). Severe toxicity: combination therapy (ATG, corticosteroids e.g., Methylprednisolone / Budesonide, Dasatinib). Sequentially for severe: corticosteroids first; if IL-6 remains elevated after 48 hours, escalate to ATG (0.75 mg / kg IV). Biomarker thresholds may be adjusted based on pre-treatment baselines. Longitudinal trends (e.g., progressive increase in IL-6, IL-10, TNF-α over 48h, or change ≥25% over 48h) may trigger earlier escalation. Severe toxicity classification can incorporate a biomarker-based sepsis risk factor (e.g., IL-6 >100 pg / mL + CRP >50 mg / L triggers immediate combination therapy). Patient-reported symptom severity (stool consistency, pain, nausea) and physiological parameters (HRV drop >30%, hypotension systolic <90 mmHg) can refine stratification. Dehydration (serum sodium <135 mEq / L, BUN >25 mg / dL) may trigger IV fluids. High-risk patients (early cytokine trends) may receive preemptive IL-6 inhibitor (Tocilizumab) 44  Attorney Docket: I071-0123PCT / SDS1.0133PCT   and TNF inhibitor (Etanercept). Steroid-refractory toxicity may be treated with dasatinib. AI / ML models can predict response to therapies or refine classification. Treatment de-escalation (tapering steroids, discontinuing biologics) may occur if IL-6 / TNF-α decline significantly (e.g., >50% from peak within 48h). Dosing intervals for Vedolizumab / Infliximab may be modified based on rate of cytokine decline (e.g., >30% / day supports extending intervals). De-escalation to supportive care if cytokines at baseline for ≥72h and symptoms resolved (stool ≤500 mL / 24h).

[0231] Methods for treating toxicities and reducing infection risk involve obtaining a biological sample post-CAR T infusion but prior to immunosuppressive therapy to measure IL-6 and TNF-α. These are compared to predefined infection risk thresholds to assign risk (low, moderate, high). Prophylactic antimicrobial treatment is administered: e.g., Low-risk: no initial therapy / close monitoring; Moderate-risk: single-agent (levofloxacin, potentially upon moderate infection indicators); High-risk: combination (levofloxacin, fluconazole, acyclovir, potentially upon high-grade GI toxicity or confirmed infection). Subsequently, an immunosuppressive agent (prednisone, methylprednisolone, ATG, infliximab, vedolizumab) is administered. Prophylactic antimicrobials are dynamically modified based on subsequent IL-6 / TNF-α changes (e.g., escalation if levels rise / remain elevated >48h post-immunosuppression). Cytokine thresholds (e.g., IL-6 >50 pg / mL, TNF-α >10 pg / mL) may prompt intensified monitoring without necessarily triggering prophylaxis if no other risks. Cytokines may be measured by rapid multiplex immunoassay (results ~2h) or point-of-care (results ~60min). High infection risk may be based on cytokine elevation plus clinical GI toxicity. Risk stratification may be updated regularly (e.g., every 24h for ≥7 days post-immunosuppression), with significant cytokine increase (e.g., >30%) triggering risk escalation. Antimicrobial agents (e.g., levofloxacin; fluconazole / micafungin; acyclovir / valganciclovir) can be chosen based on local antibiograms. Antifungal prophylaxis may be for high-risk neutropenic patients (ANC <500 / µL). Additional biomarkers (IL-1β, IL-8, procalcitonin, CRP, ANC) can refine infection risk scores. CDSS can generate recommendations. ML models can refine risk classification. Thresholds may be personalized (age, baseline immune status, renal function, infection history).

[0232] Methods for selecting human subjects for GCC-targeting CAR T cell therapy involve multi-step evaluation. Medical history confirms metastatic colorectal cancer refractory to prior standard therapies. GCC expression in a tumor sample is detected (e.g., Immunohistochemistry (IHC) on FFPE tissue with anti-GCC monoclonal antibody, quantified by tumor proportion score; or RT-qPCR for GCC mRNA). Subjects are evaluated to exclude significant baseline GI toxicity (stool frequency / consistency per MAGIC / CTCAE; e.g., exclude if baseline diarrhea >Grade 0 or ≥Grade 1). Systemic inflammatory risk is screened (CRP and serum ferritin below predetermined thresholds, e.g., CRP <21 mg / L, ferritin <2x ULN). Organ function is assessed: liver (AST, ALT, total bilirubin), renal (serum creatinine, creatinine clearance), hematologic (hemoglobin, ANC, platelet count). ECOG performance status 0 or 1 may be required. Absence of active significant viral infections (HIV, HBV, HCV) is confirmed. Selection may be based on GCC expression on tumor cells and minimal / no significant GCC expression in critical systemic non- malignant tissues. Liver function assessment may exclude cirrhosis / portal hypertension. Prior treatment history may require surgical resection of primary tumor and ≥1 line of systemic chemotherapy. Hepatic 45  Attorney Docket: I071-0123PCT / SDS1.0133PCT   tumor burden may be limited (e.g., ≤7 metastases, no lesion >3 cm, determined by CT / MRI). Patients with cholecystectomy history may be excluded from dose escalation phases but potentially eligible for dose expansion, pending safety review; if CAR T cells co-express pro-inflammatory cytokines (IL-6, IL-12, IFN-γ), cholecystectomy history may be an exclusion. A subject might be selected if all criteria met: GCC+ tumor, Grade 0 baseline diarrhea, low hepatic tumor burden, and low inflammatory markers. Leukapheresis scheduled after criteria met (e.g., within 7 days of last assessment). Post-infusion, prophylactic vedolizumab (300 mg IV) may be initiated within 24h of first fever (≥38°C), or on Day 8 post-CAR T, or within 24h of fever (≥38°C) in first 4 days. Daily stool monitoring for ~14 days follows prophylactic vedolizumab. Optional endoscopic biopsies (Week 4, Month 12) for GI mucosal pathology (multiplex immunofluorescence for CD8, FOXP3, PD-L1, CD3; histopathology for epithelial disruption, edema, lymphocytic infiltration; PCR / IHC for CAR transgene / persistence).

[0233] Pre-treatment screening may occur within ~28 days pre-leukapheresis and ~7 days pre- lymphodepletion. Neurological function (ICE score ≥10) and cardiac function (LVEF ≥50%) are assessed. Liver metastases occupying >30% hepatic volume may be exclusionary. Lab criteria: ANC ≥1,000 / µL, platelets ≥75,000 / µL, Hb ≥9 g / dL; CrCl ≥50 mL / min; AST / ALT ≤2.5x ULN, total bilirubin ≤1.5x ULN. No active / latent infections (procalcitonin, viral PCR). Brain MRI to rule out CNS malignancy. Cytokine profiling pre-infusion (IL-6, IL-10, IL-22, IL-17, IFN-γ, TNF-α); elevations (IL-6 >50 pg / mL, IFN-γ >500 pg / mL) may prompt prophylactic vedolizumab pre- or early post-CAR T, or exclusion. Post-infusion, focused GI toxicity assessment begins ~Day 5 (stool, biomarkers, fever), with MAGIC grading. Thresholds for concern: stool volume >600 mL / day or diarrhea persistence >5 days. IL-6 >50 pg / mL and IL-10 >15 pg / mL may prompt intensive surveillance.

[0234] Multivariate toxicity risk scores may be calculated pre-infusion using biomarkers (IL-6, IL-8, G- CSF, LDH), age, and CAR T dose. Each parameter (e.g., IL-6 ≥4.05 pg / mL, IL-8 ≥8.00 pg / mL, G-CSF ≥0.75 ng / mL, LDH ≥220.41 U / L, age ≥47 years, CAR T dose ≥1.72×10⁶ cells / kg) can be assigned points. A cumulative score (e.g., 0-14) classifies DLT risk (e.g., ≥9 is high-risk), guiding protocols. GI-specific scores (IL-6, IL-10, ferritin, CRP, neutrophils, albumin) classify diarrhea risk (low, moderate, high), guiding prophylaxis (vedolizumab, budesonide). High-risk patients may need inflammatory marker reduction before proceeding.

[0235] Early prophylactic / preemptive interventions can be based on cytokine trajectory. IL-6 >50 pg / mL or IFN-γ >500 pg / mL within 72h post-CAR T (absent diarrhea) may trigger preemptive vedolizumab (300 mg IV). If IL-6 or IL-22 rise despite vedolizumab, with fever or CRP >30 mg / L, dasatinib (e.g., 90 mg BID) may be given. The biological confinement of GCC (apical intestinal epithelia, shielded by tight junctions) may localize on-target toxicity to the gut, supporting gut-directed therapies. ML models can assign real-time diarrhea toxicity risk scores based on daily cytokines, stool output, fever, baseline labs, guiding early interventions (vedolizumab, budesonide, advanced stool monitoring). Patient-Reported Outcomes (PROs) via diaries (Day 5 to ≥Day 28 or resolution) track stool consistency (Bristol Stool Scale), urgency, pain, bloating, frequency. PROs can supplement objective measures; significant urgency / cramping might classify Grade 2 diarrhea even if volume <600 mL. Structured de-escalation 46  Attorney Docket: I071-0123PCT / SDS1.0133PCT   protocols for diarrhea resolution (stool frequency <4 / 24h and volume <600 mL / day for ≥3 consecutive days) include discontinuing NPO, reintroducing bland diet, tapering TPN, and tapering immunosuppressants (corticosteroids, vedolizumab, infliximab, ATG) per schedule based on symptom resolution and declining inflammatory markers (IL-6, CRP). CDSS may alert for de-escalation and recommend cytokine re-testing.

[0236] Gastroenterology specialist consultation may be triggered by severe diarrhea (e.g., ≥7 bowel movements / 24h or stool output >1200 mL / 24h), guiding diagnostics (imaging, endoscopy) and therapy. For severe GI complications (suspected ileus, ischemia, necrosis; signs: reduced / absent bowel sounds, distension, hypotension, paradoxical absence of stool output with high inflammatory markers), interventions include NPO, TPN. If CT scan (contrast) shows severe bowel injury (mucosal edema, wall thickening, pneumatosis intestinalis), escalate to potent immunosuppression (e.g., ATG 1.5 mg / kg initial, then q48h up to 4 doses). Colonoscopy with biopsies may assess mucosal injury and rule out infectious colitis (C. difficile, CMV). Endoscopy may be reserved for prolonged symptoms (>7 days) or inadequate response to initial therapies. Extended follow-up for late-onset / persistent diarrhea (>14 days or unresolved by Day 28) involves a chronic diarrhea surveillance program (e.g., through Month 4+), with monthly outpatient evaluations (stool parameters, symptoms, questionnaires, stool logs) and cytokine monitoring (IL-6, IL-10, IFN-γ, TNF-α, IL-22). Persistent elevation of IL-10 / IL-22 may indicate ongoing mucosal inflammation. Stepwise de-escalation of gut-directed agents (budesonide, infliximab) after sustained resolution (e.g., 3 monthly assessments) and biomarker normalization.

[0237] To differentiate concurrent CRS and diarrhea: if systemic CRS symptoms (fever, hypotension) and high systemic IL-6 (>100 pg / mL) and IL-10 (>15 pg / mL), presume CRS-driven diarrhea; treat CRS (tocilizumab ± corticosteroids). If diarrhea persists / worsens >48h post-CRS therapy, initiate gut-specific treatments (vedolizumab, infliximab). Sustained IL-22 / IL-17 despite IL-6 resolution may confirm gut- localized inflammation. Fever-based preemptive vedolizumab: if first fever ≥38.0°C between Day 0-4, administer vedolizumab (300 mg IV) within 24h. If fever on / after Day 5, vedolizumab within 24h. If no fever by Day 7, prophylactic vedolizumab on Day 8. Exploratory stool biomarker analysis during diarrhea (daily for up to 7 days for moderate / severe): microbiome, viral PCR, multiplex cytokines (IL-22, IL-17) correlated with blood levels.

[0238] Dietary interventions: for low-grade diarrhea, restricted bland diet (low fiber, non-spicy, non- fatty). For stool volume >600 mL / day or persistence >72h, NPO and IV fluids. For volume >1000 mL / day or persistence >5 days, TPN. NPO until stool <400 mL / day stable for ≥72h. Reintroduction of oral feeding after high-grade diarrhea for >5 days may be delayed until 10 days without recurrence. Early pharmacological management for Grade 1 diarrhea: oral budesonide (e.g., 9 mg total daily) ± rectal budesonide foam; subcutaneous / IV octreotide (100–150 mcg TID (three times daily)) ± loperamide / diphenoxylate-atropine. dasatinib (90-100 mg BID) for severe / refractory GI toxicity despite corticosteroids / infliximab; tapered upon resolution. Post-cholecystectomy patients may receive preemptive cholestyramine (4g BID, starting Day 5 or first loose stool, up to 14 days). Antimicrobial prophylaxis escalation: if IL-6 >100 pg / mL or CRP >50 mg / L, empiric broad-spectrum antibiotics, antifungals, 47  Attorney Docket: I071-0123PCT / SDS1.0133PCT   antivirals, with microbiological investigations. Evaluate for mucosal necrosis / ischemia if very high-volume (>1200 mL / day) or high-frequency (≥7 stools / day) diarrhea (exam, labs, CT scan). If hemodynamic instability or signs of necrosis / ischemia, aggressive immunosuppression with ATG (1.5 mg / kg initial, then q48h up to 4 doses). Sensor-based stool collection (pressure-sensitive pads, catheter-connected fecal containment with volumetric sensors, accuracy ±10%) for real-time / near real-time volume, data to dashboard / EMR.

[0239] Structured safety oversight for GI adverse events may involve real-time monitoring by an internal Safety Review Team (SRT) and external Data Safety Monitoring Committee (DSMC). Safety triggers (e.g., Grade 3 diarrhea unresolved within 7 days, any Grade 4 GI event, ICU admission for diarrhea) may pause trial enrollment or escalate oversight. Investigator discretion for additional diagnostics or therapy escalation is documented. Comprehensive documentation (eCRFs / CRFs) of interventions, assessments, deviations is maintained, ensuring data integrity and subject privacy. Each treatment escalation event may be logged for regulatory review and quality analysis. The grading and treatment sequence may be implemented in prospective clinical trials or validated using real-world patient registries (e.g., ≥100 cases).

[0240] The features described are not intended to be mutually exclusive and can be combined or modified. References to particular grading criteria, thresholds, or dosages are illustrative, and pharmaceutically acceptable variants and equivalents are encompassed. The overall approach may adapt best practices from related fields, tailored to the unique toxicity profile of these advanced cellular therapies.

[0241] The present disclosure, in further aspects, details the broad applicability of the disclosed methods for managing gastrointestinal (GI) toxicity, particularly diarrhea, to a range of human patients receiving various immunotherapies beyond a single type or target. While many specific embodiments herein focus on CAR T therapy, particularly for conditions like colorectal cancer (CRC), the underlying principles and strategies for GI toxicity management are relevant to other immunotherapeutic modalities that can induce similar adverse events.

[0242] In one embodiment, the methods are directed toward patients undergoing CAR T cell therapy targeting tumor-associated antigens. Examples of such antigens include, but are not limited to, GCC, CEA, Natural Killer Group 2D (NKG2D) ligands, or other CRC-relevant markers. A known risk in such therapies is the potential for on-target, off-tumor toxicity, which can arise if the target antigen, while predominantly expressed on tumor cells, also has low-level expression on normal GI epithelial cells. Clinical experience with CAR T products targeting antigens like GCC or CEA has shown that such therapies can induce diarrhea and immune-mediated colitis, sometimes necessitating interventions such as corticosteroids, anti- Tumor Necrosis Factor (anti-TNF) agents, or, in severe cases, surgical intervention. Consequently, the disclosed methods encompass both prophylactic measures (administered before toxicity onset) and responsive management steps (administered once toxicity develops). These steps include vigilant GI symptom monitoring, early initiation of immunosuppressive interventions, and strategies for dose adjustment or interruption of the immunotherapy if required.

[0243] In another embodiment, the GI toxicity management methods are applied to patients receiving other classes of immunotherapy. These may include, but are not limited to, immune checkpoint inhibitors 48  Attorney Docket: I071-0123PCT / SDS1.0133PCT   (ICIs), such as antibodies targeting Programmed Death-1 (PD-1), Programmed Death-Ligand 1 (PD-L1), or Cytotoxic T-Lymphocyte-Associated Protein14 (CTLA-4). Other applicable therapies include bispecific T-cell engagers (BiTEs), for example, those designed to simultaneously bind a tumor antigen like CEA and an activating receptor on T-cells like CD3 (e.g., CEA × CD3 BiTEs), thereby redirecting T-cells to attack tumor cells. Monoclonal antibodies with inherent immune-activating properties, such as anti-Epidermal Growth Factor Receptor (anti-EGFR) antibodies like cetuximab (which can mediate antibody-dependent cell-mediated cytotoxicity), also fall within this scope. Each of these therapeutic modalities has been clinically associated with GI toxicity, although the precise mechanisms and severity profiles can vary. For instance, checkpoint blockade can result in immune-related colitis (irColitis) through a generalized or nonspecific activation of T-cells within the gut mucosa. In contrast, T-cell engagers and some antibody- drug conjugates may trigger on-target cytotoxicity against normal GI cells expressing the target antigen at low levels, or they may induce cytokine release that disrupts GI mucosal integrity.

[0244] It has now been recognized by the inventors that although these diverse immunotherapy classes differ in their molecular architecture and primary immunologic targets, the downstream pathophysiological mechanisms that contribute to GI toxicity—particularly diarrhea and colitis—are often overlapping. In many cases, these mechanisms converge on common inflammatory pathways within the GI tract. These shared mechanisms include, for example: disruption of epithelial barrier integrity: This can be caused by cytokine-mediated apoptosis of epithelial cells (e.g., driven by TNF-α or Interferon-gamma (IFN-γ) signaling) or by direct damage from infiltrating immune cells; aberrant activation of cytotoxic lymphocytes: Immune cells, such as cytotoxic T-lymphocytes or Natural Killer (NK) cells, can become activated within the intestinal lamina propria, leading to damage of the mucosal epithelium; chemokine-driven trafficking of effector T-cells into the gut mucosa: Specific chemokine gradients and adhesion molecule interactions (e.g., involving the α4β7 integrin and its ligand MAdCAM-1) can promote the recruitment of inflammatory T-cells to the GI tract. This mechanism has been observed in both CAR T-associated GI toxicity and in checkpoint inhibitor–induced colitis; local release of pro-inflammatory cytokines: Elevated levels of cytokines such as Interleukin-6 (IL-6), TNF-α, and others within the gut microenvironment can cause secretory diarrhea and contribute to mucosal edema. This is particularly relevant in the context of systemic inflammatory responses like Cytokine Release Syndrome (CRS), which can have significant GI manifestations; and on-target antigen expression in normal GI tissues: If the immunotherapy target antigen (e.g., CEA or GCC) is also expressed, even at low levels, on normal GI crypt cells or other epithelial components, this can lead to "collateral damage" as the immunotherapy engages these cells.

[0245] Given these shared mechanisms of GI injury, the inventors have determined that the GI toxicity management methods disclosed herein, initially described in the context of CAR T therapy, may be beneficially adapted and applied to other immunotherapies that carry a risk of similar adverse events. As such, the broadly applicable methods may comprise the administration of various immunosuppressive or immunomodulatory agents (e.g., corticosteroids such as prednisone or budesonide; anti-TNF agents like infliximab; or gut-selective agents like vedolizumab). The methods may also involve instituting dose interruption, dose reduction, or step-up dosing regimens for the primary immunotherapy to manage toxicity. 49  Attorney Docket: I071-0123PCT / SDS1.0133PCT   Furthermore, premedication protocols (e.g., with dexamethasone prior to immunotherapy infusion) and specific diagnostic workflows (e.g., stool cultures, endoscopy with biopsy, imaging) may be employed to accurately distinguish immune-mediated diarrhea from infectious or drug-induced etiologies, thereby guiding appropriate therapy.

[0246] The utility of certain GI toxicity mitigation strategies across multiple immunotherapy platforms further supports this concept of generalized applicability. For instance, vedolizumab, which acts as a gut- specific α4β7 integrin blocker to prevent lymphocyte trafficking to the intestine, has demonstrated clinical utility in managing both CAR T cell-induced GI toxicity and checkpoint inhibitor–associated colitis. Similarly, strategies like step-up dosing (gradually increasing the dose of the immunotherapy) and corticosteroid prophylaxis have been employed successfully to reduce the incidence or severity of early- onset diarrhea and cytokine release associated with some bispecific antibody therapies. These clinical findings underscore the value of applying common management principles to GI toxicities arising from different types of immune-engaging treatments.

[0247] Accordingly, in some embodiments, the methods for managing GI toxicity described herein are not limited to a specific immunotherapy modality (e.g., only CAR T cells) or a single antigen target (e.g., only GCC). Rather, they encompass general strategies applicable to reducing or managing GI toxicity that results from various underlying causes, including systemic immune activation with secondary gut effects, localized mucosal immune responses, or direct on-target epithelial injury by immunotherapeutic agents. The inventors contemplate that these methods are applicable to any immunotherapy agent, or combination thereof, wherein GI toxicity—including but not limited to diarrhea, colitis, enteritis, or mucosal ulceration—may arise as a treatment-associated adverse event. This broad applicability allows for a more unified and potentially standardized approach to managing a common and often dose-limiting side effect of a growing class of cancer therapies.

[0248] In some embodiments, the present disclosure provides methods for the prophylactic management of diarrhea associated with a therapy comprising Chimeric Antigen Receptor T-cells (CAR T cells) targeting a colorectal cancer-associated antigen (CRC-associated antigen). These methods are particularly relevant for a human patient receiving such CAR T cell therapy for the treatment of relapsed or refractory metastatic colorectal cancer (CRC). The goal of such prophylactic management is to reduce the incidence, duration, or severity of diarrhea potentially linked to the CAR T cell therapy. In certain aspects, this prophylactic management may also reduce the initial onset of diarrhea associated with the therapy.

[0249] According to certain embodiments, the method of prophylactic management comprises administering Vedolizumab to the patient at approximately Day 3 post infusion of the CAR T cells. In other embodiments, the method comprises administering Infliximab to the patient at approximately Day 5 post infusion of the CAR T cells. In yet other embodiments, the method comprises administering Budesonide to the patient for a duration, for example, from approximately Day 5 to approximately Day 12 post infusion of the CAR T cells.

[0250] It is contemplated that these prophylactic administrations can be performed individually or in combination. For example, an embodiment may involve administering only Vedolizumab at Day 3 post 50  Attorney Docket: I071-0123PCT / SDS1.0133PCT   CAR T cell infusion. Another embodiment may involve administering only Infliximab at Day 5 post CAR T cell infusion. A further embodiment may involve administering only Budesonide from Day 5 to Day 12 post CAR T cell infusion. In other exemplary embodiments, combinations of these agents are administered. For instance, a patient may receive Vedolizumab at Day 3 and Infliximab at Day 5. Alternatively, a patient may receive Vedolizumab at Day 3 and Budesonide from Day 5 to Day 12. In another combination, a patient may receive Infliximab at Day 5 and Budesonide from Day 5 to Day 12. In a comprehensive approach, an embodiment includes administering Vedolizumab at Day 3, administering Infliximab at Day 5, and also administering Budesonide from Day 5 to Day 12 post CAR T cell infusion. The specific CRC- associated antigen targeted by the CAR T cells can vary. In a particular embodiment, this antigen is GCC. These prophylactic methods may also incorporate the step of monitoring the patient for the development of any signs or symptoms of diarrhea.

[0251] In other embodiments, the present disclosure provides methods for managing diarrhea that may arise in a human patient undergoing CAR T cell therapy targeting a CRC-associated antigen for relapsed or refractory metastatic CRC. These methods involve monitoring the patient and implementing interventions based on the severity of the diarrhea. The management strategy often focuses on diarrhea occurring subsequent to Day 4 post infusion of CAR T cells.

[0252] According to these embodiments, the method comprises monitoring the patient for symptoms of diarrhea, particularly after Day 4 post CAR T cell infusion. If diarrhea occurs, it is graded using established criteria, such as the MAGIC criteria. The MAGIC criteria typically consider stool frequency and stool volume, irrespective of the patient's baseline bowel habits, to assess acute changes. Based on the grade determined by the MAGIC criteria, a therapeutic intervention is implemented. In some instances, as part of patient management, a patient may be excluded from initiating the CAR T cell therapy if they exhibit Grade 1 or greater diarrhea by MAGIC criteria at baseline. Should diarrhea develop during the course of therapy, the monitoring may be intensified to include strict monitoring of the patient’s intake and output volumes to guide fluid and electrolyte management. The therapeutic intervention itself can be selected from a tiered therapeutic algorithm, where the choice of treatment is dictated by the MAGIC criteria grade of the diarrhea, allowing for a graduated response to increasing severity. The CAR T cells involved in these methods may target any suitable CRC-associated antigen.

[0253] In further embodiments, the present disclosure provides specific methods for treating an identified level of diarrhea, such as moderate diarrhea, associated with CAR T cell therapy targeting a CRC-associated antigen in a human patient. These methods are typically applied when diarrhea occurs subsequent to Day 4 post infusion of the CAR T cells and the patient is undergoing treatment for relapsed or refractory metastatic CRC.

[0254] According to these embodiments, the method involves identifying that the patient has moderate diarrhea. Moderate diarrhea, for the purpose of these embodiments, can be defined as experiencing 4 to 6 loose or watery stools per 24 hours or producing 600 to 1200 milliliters (mL) of stool per 24 hours, according to the MAGIC criteria. Once moderate diarrhea is identified, the method comprises administering 51  Attorney Docket: I071-0123PCT / SDS1.0133PCT   ATG to the patient. An exemplary dose for ATG administration is 0.75 milligrams per kilogram (mg / kg) of the patient's body weight, administered as a single intravenous infusion.

[0255] In situations where a patient identified with moderate diarrhea has received or is scheduled to receive infliximab on the same day, an embodiment of the method may include delaying the administration of ATG for approximately 24 hours. This delay allows for assessment of the patient's response to infliximab before proceeding with ATG. Additionally, following the administration of ATG, it may be clinically indicated to administer antifungal, antibacterial, or antiviral agents to the patient to mitigate risks of infection due to immunosuppression. In specific applications of this treatment method, the CRC-associated antigen targeted by the CAR T cells is GCC, although other CRC-associated antigens may also be relevant.

[0256] It will be understood by a person of ordinary skill in the art, such as a treating physician, that the specific doses and administration schedules for the therapeutic agents described herein, whether administered alone or in combination, are exemplary and can be adjusted. Such adjustments may be made based on standard clinical practice and a variety of patient-specific factors, including but not limited to, the patient's body weight, the severity and trajectory of the clinical toxicity as graded by established criteria, the patient's response to previous interventions, and the status of their hematologic and other organ functions. The management protocols described herein provide a framework that allows for investigator discretion to tailor therapy to individual patient presentations, thereby optimizing the balance between managing toxicity and preserving the efficacy of the primary CAR T therapy. For instance, in embodiments where multiple therapeutic agents are administered in combination, specific dosing and safety monitoring protocols are employed. As an illustrative example, when Anti-Thymocyte Globulin (ATG) is administered in combination with corticosteroids for severe or refractory diarrhea, an exemplary dose of ATG is 0.75 mg / kg / day intravenously, and an exemplary dose of methylprednisolone is 1 mg / kg administered intravenously twice daily. During such combination therapy, patients are closely monitored with, for example, daily complete blood count (CBC) with differential, with particular attention given to Absolute Neutrophil Count (ANC) and platelet counts. As described elsewhere herein, ATG dosing may be adjusted, held, or reduced based on these hematologic parameters, such as when the platelet count falls below a certain threshold. Furthermore, due to the heightened risk of infection from combined immunosuppression, prophylactic anti-infective agents may be initiated, as also described herein.

[0257] To the extent any descriptions in the present disclosure are phrased as alternatives or options, such alternatives may be combined unless such combination is expressly excluded. The disclosure should be construed as covering such permutations and combinations of features, steps, or elements as would be apparent to one skilled in the art based on the teachings provided herein.

[0258] The present disclosure is further described by reference to the following and examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following exemplary embodiments and examples but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. EXAMPLES 52  Attorney Docket: I071-0123PCT / SDS1.0133PCT   Example 1: Prophylactic Regimen for Prevention of CAR T Cell-Associated Gastrointestinal Toxicity

[0259] This example describes a prophylactic drug regimen to be administered to subjects undergoing CAR T cell therapy. The primary objective of this regimen is the prevention or reduction in severity of CAR T cell-associated gastrointestinal toxicity, with a particular focus on diarrhea. This prophylactic approach is intended for subjects with relapsed or refractory metastatic colorectal cancer who are receiving infusion of CAR T cells as part of a therapeutic regimen.

[0260] Subjects eligible for this prophylactic regimen are those diagnosed with relapsed or refractory metastatic colorectal cancer who are scheduled to receive an infusion of CAR T cells. The prophylactic agents are administered according to a specific schedule relative to the CAR T cell infusion.

[0261] The prophylactic regimen includes vedolizumab, administered as a 300 mg intravenous (IV) infusion on Day 3 post-CAR T infusion. Additionally, infliximab is administered at a dose of 5 mg / kg IV on Day 5 post-CAR T infusion. Budesonide is also part of the prophylactic protocol; it is administered as 9 mg tablets orally once daily, concurrently with budesonide rectal foam, 2 mg per metered dose, administered twice daily. The administration of both forms of budesonide commences on Day 5 post-CAR T infusion and continues through Day 12 post-CAR T infusion.

[0262] Throughout the period of CAR T cell therapy and prophylactic administration, subjects undergo routine monitoring. This includes a daily complete blood count (CBC) with differential. Investigations are conducted as needed to rule out infectious etiologies if diarrhea develops. Intake and output volumes are also closely monitored.

[0263] The administration of this multi-agent prophylactic regimen is expected to lead to a reduction in the incidence, overall duration, and / or severity of diarrhea associated with CAR T cell therapy in treated subjects. By proactively addressing potential gastrointestinal inflammation, this regimen aims to improve the subjects' tolerance of the CAR T cell therapy.

[0264] This prophylactic strategy provides a method for mitigating gastrointestinal toxicity, a common and potentially dose-limiting adverse event observed with CAR T cell therapies. The combination of vedolizumab, infliximab, and budesonide targets multiple pathways implicated in gastrointestinal inflammation. Successful implementation of this prophylactic protocol is anticipated to enhance the safety and tolerability profile of CAR T cell treatment, potentially allowing for more consistent therapy administration and improved patient quality of life. This regimen is to be incorporated into the clinical study protocol for ongoing evaluation. Example 2: Management Protocol for CAR T Cell-Associated Diarrhea

[0265] This example details a comprehensive management protocol for treating subjects who develop diarrhea on or after Day 5 following an infusion of CAR T cells. This protocol is specifically designed for subjects with relapsed or refractory metastatic colorectal cancer undergoing CAR T cell therapy. It is important to note that any diarrhea occurring from Day 0 to Day 4 post-CAR T cells infusion is not considered related to CAR T cells and is to be managed according to standard institutional guidelines. The 53  Attorney Docket: I071-0123PCT / SDS1.0133PCT   primary objective of the herein described protocol is to effectively manage CAR T cell-associated diarrhea, thereby reducing its duration and overall severity.

[0266] The patient population for this management protocol comprises subjects with relapsed or refractory metastatic colorectal cancer who have received CAR T cell infusion and subsequently develop diarrhea on or after Day 5 post-infusion.

[0267] Upon development of diarrhea meeting these criteria, initial management steps are implemented. These include ensuring the completion of any ongoing prophylactic regimen as detailed in Example 1. Supportive care measures, including hydration, appropriate diet modification, and the use of antidiarrheal agents such as loperamide, are initiated. Clinical monitoring involves closely tracking intake and output volumes, daily collection of CBC with differential, and investigations to rule out infectious causes for the diarrhea.

[0268] The treatment interventions are escalated based on the severity of diarrhea, categorized by the number of loose or watery stools per 24 hours or the total volume of diarrheal output per 24 hours.

[0269] For diarrhea characterized by 2 to 3 loose or watery stools per 24 hours or less than 600 mL per 24 hours: Budesonide is administered at a dose of 9 mg orally once daily, supplemented with budesonide rectal foam (2 mg per metered dose) twice daily. This treatment continues until Day 19 post-infusion or for 7 days after the diarrhea is fully under control, whichever period is longer. Controlled diarrhea or diarrhea under control refers to a state in which diarrhea symptoms have improved to a mild or manageable level— specifically such that the diarrhea is responsive to current tier interventions (e.g., loperamide, budesonide), and no escalation to systemic corticosteroids or biologics (like infliximab) is needed. Fully Under Control (diarrhea) refers to a state in which diarrhea has resolved to the point that stool frequency and volume have returned to baseline or normal for the patient, no antidiarrheal or immunosuppressive treatment is further ongoing or required, and the patient can transition to routine follow-up. Methylprednisolone is administered at 1 mg / kg IV twice daily (BID) until complete resolution of diarrhea is achieved. A second dose of infliximab, at 5 mg / kg (with a maximum of two doses per patient), may be administered if symptoms occur on or after Day 12 post-infusion; however, infliximab is not to be administered within 48 hours after vedolizumab administration. If this grade of diarrhea persists for more than 3 days, ATG is administered at a dose of 0.75 mg / kg IV once on the fourth day of persistent diarrhea. Should there be no improvement in diarrhea 24 hours after this initial ATG dose, ATG is administered every 48 hours thereafter until the diarrhea improves or stops, for a maximum of 4 doses of ATG. If diarrhea does not improve or stop after 4 doses of ATG, other immunomodulatory antibodies, including but not limited to those targeting CD52 or CD25, may also be considered. If ATG is used, concomitant consideration of antibacterial, antifungal, and antiviral agents is recommended. In the event of recurrent diarrhea of this severity, ATG 0.75 mg / kg IV is immediately administered once, with subsequent doses as previously described if no improvement is observed.

[0270] For diarrhea characterized by 4 to 6 loose or watery stools per 24 hours, or a volume of 600 to 1200 mL per 24 hours: Prophylaxis (if ongoing), hydration, diet modification, and antidiarrheals are continued. Budesonide is administered at 9 mg orally once daily along with rectal foam (2 mg per metered 54  Attorney Docket: I071-0123PCT / SDS1.0133PCT   dose) twice daily, until Day 19 post-infusion or for 7 days after diarrhea is fully controlled, whichever is later. Dasatinib 90 mg BID is administered in combination with methylprednisolone 1 mg / kg IV BID until complete resolution of diarrhea. A second dose of infliximab (5 mg / kg, maximum two doses per patient) may be administered if symptoms occur on or after Day 12 post-infusion, avoiding administration within 48 hours of vedolizumab. If diarrhea does not improve after three days, the patient is kept Nothing by Mouth (NPO). If diarrhea persists after 3 days of NPO status, Total Parenteral Nutrition (TPN) is initiated and continued until diarrhea improves to equal to or less than 3 loose or watery stools per 24 hours and less than 600 mL per 24 hours. ATG is administered at a dose of 0.75 mg / kg IV once. If the patient has received or is scheduled to receive infliximab on the same day, ATG administration is held for 24 hours to assess the response to infliximab; if there is no improvement, ATG is then administered. Careful monitoring of ATG administration is advised when the White Blood Cell (WBC) count is less than 2,000 / μL and / or the platelet count is less than 50,000 / μL. If there is no improvement in diarrhea 48 hours after using ATG, ATG is administered every 48 hours until the number of diarrhea episodes is less than 4 and the diarrhea volume is less than 600 mL, for a maximum of 4 doses of ATG. If after 4 doses of ATG, diarrhea episodes are 4 or more, or the diarrhea volume is 600 mL or more, other immunomodulatory antibodies, including but not limited to those targeting CD52 or CD25, may also be considered. Concomitant antibacterial, antifungal, and antiviral agents are considered if ATG is used. For recurrent diarrhea of this severity, ATG 0.75 mg / kg IV is immediately administered once, with subsequent doses as previously described if no improvement is seen.

[0271] For diarrhea characterized by 7 or more loose or watery stools per 24 hours, or a volume greater than 1200 mL per 24 hours: A Gastrointestinal (GI) consultation is obtained, and abdominal Computed Tomography (CT) scan, colonoscopy, and / or other evaluations are performed per the investigator’s discretion. Budesonide 9 mg is administered orally once daily with rectal foam (2 mg per metered dose) twice daily, until Day 19 post-infusion or for 7 days after diarrhea is fully controlled, whichever is later. ATG is administered at 0.75 mg / kg IV for 2 consecutive days. If, on the day after this initial ATG administration, the number of diarrhea episodes is greater than 3, or the diarrhea volume is greater than 600 mL, ATG 0.75 mg / kg IV is continued once daily, for a maximum of 4 total days of ATG administration. If the number of diarrhea episodes drops to 3 or less, and the diarrhea volume is 600 mL or less, ATG is stopped. If after 4 days of ATG administration, diarrhea episodes remain greater than 3, or the diarrhea volume is greater than 600 mL, other immunomodulatory antibodies, including but not limited to those targeting CD52 or CD25, may also be considered. Dose reduction of ATG and careful monitoring are considered for patients with platelet counts of 50,000-75,000 / μL or WBC counts of 2,000-3000 / μL. Methylprednisolone 1 mg / kg IV BID is administered until complete resolution of diarrhea. A second dose of infliximab (5 mg / kg, maximum two doses per patient) may be administered if symptoms occur on or after Day 12 post-infusion, but administration should be avoided within 48 hours of administering vedolizumab. The patient is kept NPO, and TPN is initiated until resolution of diarrhea to less than 4 loose or watery stools per 24 hours and less than 600 mL per 24 hours. If there are 7 or more loose or watery stools or greater than 1200 mL of diarrhea per 24 hours for more than 5 days, the normal diet is not resumed 55  Attorney Docket: I071-0123PCT / SDS1.0133PCT   until 10 days after the diarrhea has stopped. Concomitant antibacterial, antifungal, and antiviral agents are considered if ATG is used.

[0272] For life-threatening gastrointestinal events, such as perforation, ischemia, necrosis, bleeding, toxic megacolon, or hemodynamic collapse: The patient is admitted to an Intensive Care Unit (ICU). A GI consultation is obtained, and abdominal CT scan, colonoscopy, and other evaluations are performed per the discretion of the investigator. ATG is immediately administered at a dose of 1.5 mg / kg IV daily for 3 days, or ATG 0.75 mg / kg IV daily for 4 days, depending on the platelet count. This is administered in addition to methylprednisolone IV, which is continued until loose or watery stools decrease to less than 4 episodes and less than 600 mL per 24 hours. Dose reduction of ATG and careful monitoring are considered for patients with platelet counts of 50,000-75,000 / μL or WBC counts of 2,000-3000 / μL. If after 4 days of ATG administration, diarrhea episodes are greater than 6, or the diarrhea volume is greater than 1200 mL, other immunomodulatory antibodies, including but not limited to those targeting CD52 or CD25, may also be considered. Methylprednisolone is administered at 1 mg / kg IV BID until complete resolution. A second dose of infliximab (5 mg / kg, maximum two doses per patient) may be administered if symptoms occur on or after Day 12 post-infusion, avoiding administration within 48 hours of vedolizumab. The patient is kept NPO, and TPN is initiated until resolution of the life-threatening event. For associated diarrhea, TPN is continued until improvement of diarrhea to less than 4 loose or watery stools per 24 hours. If there are 7 or more loose or watery stools or greater than 1200 mL of diarrhea per 24 hours for more than 5 days, the normal diet is not resumed until 10 days after the diarrhea has stopped. Concomitant antibacterial, antifungal, and antiviral agents are considered to treat potential infectious causes of the life-threatening event. In certain embodiments, antibodies targeting CD52 or CD25 are considered as alternatives to ATG.

[0273] The implementation of this tiered and severity-graded management protocol is expected to lead to the effective control and resolution of CAR T cell-associated diarrhea. This approach is anticipated to minimize complications arising from diarrhea, reduce its duration and severity, and thereby improve overall patient outcomes and tolerability of the CAR T cell therapy.

[0274] This detailed management algorithm provides clinicians with clear, actionable guidance for the treatment of CAR T cell-associated diarrhea, tailored to the clinical severity of the presentation. The protocol incorporates a stepwise escalation of therapeutic interventions, including corticosteroids, immunomodulatory agents like infliximab and ATG, and supportive measures such as NPO status and TPN. This structured approach aims to achieve rapid symptom control and prevent the development of severe gastrointestinal morbidity. By standardizing the management of this adverse event, the protocol contributes to the overall safety, feasibility, and therapeutic window of CAR T cell therapy for patients with relapsed or refractory metastatic colorectal cancer. These updated management recommendations are intended for incorporation into future amendments of the clinical trial protocol. Example 3: Clinical Study of CAR T cells in Patients with Metastatic Colorectal Cancer

[0275] This example describes a phase 1 clinical study conducted to evaluate the safety and preliminary efficacy of an investigational CAR T cell therapy, CAR T cells, in patients with mCRC. The study also 56  Attorney Docket: I071-0123PCT / SDS1.0133PCT   provided data on the incidence and management of treatment-related adverse events, including diarrhea, which is relevant to the management protocols of the present invention.

[0276] A phase 1, open-label, nonrandomized clinical study was performed in China. The study was conducted in accordance with the Declaration of Helsinki and received approval from the relevant institutional review board. All participants provided written informed consent prior to enrollment.

[0277] Adult patients with histologically confirmed mCRC that was refractory to standard lines of therapy were enrolled in the study. Eligibility criteria required participants to have tumors expressing Guanylyl Cyclase C (GCC), as determined by immunohistochemistry of archival tumor tissue or a recent biopsy. Patients were also required to have adequate organ function and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0278] Enrolled patients underwent leukapheresis for the collection of autologous T-cells. Following lymphodepleting chemotherapy, which consisted of fludarabine and cyclophosphamide, participants received a single infusion of CAR T cells. This CAR T cell product consisted of autologous T cells transduced with lentiviral vectors to express CARs targeting both CD19 and GCC (GCC19CAR product). Notably, the CD19 CAR component included subpopulations of cells engineered to express immunomodulatory cytokines. Two dose levels of CAR T cell were evaluated: 1 × 10⁶ cells / kg (dose level 1) and 2 × 10⁶ cells / kg (dose level 2). Further details regarding the CAR T cell product, including aspects of CAR T cell manufacturing, specific sequences, and techniques to make and use the product, are described in various publications, including PCT Publication Nos: WO2018126369, WO2018064921, WO2020106843, WO2020146743, and the peer-reviewed publication by Chen N., et al., JAMA Oncology, 2024, all of which are incorporated herein by reference in their entirety.

[0279] Further comprehensive details regarding this clinical study of the CAR T cell product, including aspects of CAR T cell manufacturing, specific dose levels administered, complete safety and efficacy outcomes, observed cytokine dynamics, specific sequences related to the CAR constructs, and correlative biomarker analyses, were reported in the peer-reviewed publication by Chen N., et al., “Phase 1 Evaluation of CAR T cells in Metastatic Colorectal Cancer”, JAMA Oncology, published online September 19, 2024 (doi:10.1001 / jamaoncol.2024.3891). The contents of this publication are hereby incorporated by reference in their entirety.

[0280] Clinical safety, including the incidence and severity of CRS, neurotoxicity, and gastrointestinal adverse events such as diarrhea, was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Clinical response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and metabolic response was assessed by Positron Emission Tomography (PET) imaging using PET Response Criteria in Solid Tumors (PERCIST).

[0281] Fifteen patients were treated in the study. The median age of the participants was 44 years, with a range of 33 to 61 years. The CAR T cell treatment was generally well tolerated by the study participants. The most common adverse events reported were CRS, which was observed in 93% of patients, and diarrhea, also observed in 93% of patients. These adverse events, including instances of diarrhea, were generally 57  Attorney Docket: I071-0123PCT / SDS1.0133PCT   manageable with standard supportive care measures. Such measures included the use of tocilizumab (for CRS), corticosteroids, and / or antidiarrheal agents. A subset of patients who experienced grade 3 or higher adverse events required an escalation of care; this included the administration of infliximab or dasatinib for the management of these more severe events.

[0282] Objective clinical activity was observed in the treated patient population. Six out of the fifteen patients (40%) achieved a partial response, and an additional five patients demonstrated stable disease following the infusion of CAR T cells. Analysis of progression-free survival (PFS) indicated that the median PFS was greater in the higher-dose cohort (dose level 2) compared to the lower-dose cohort (dose level 1), reported as 6.0 months versus 1.9 months, respectively. At the time of data cutoff for the study, the median overall survival (OS) across both dose cohorts was 22.8 months.

[0283] This phase 1 clinical study demonstrated that the CAR T cell therapy product was generally well tolerated and exhibited promising anti-tumor activity in patients with heavily pretreated mCRC. Diarrhea was identified as a common adverse event, occurring in 93% of the treated patients. The observation that this diarrhea was manageable with standard supportive care, and with escalation to specific agents such as infliximab or dasatinib for more severe cases, highlights the clinical setting in which such adverse events occur and underscores the utility of defined and systematic management protocols, such as those detailed in Example 1 and Example 2 of the present specification. The effective management of CAR T therapy- related adverse events, including diarrhea, is important for optimizing the overall therapeutic benefit and patient experience with CAR T cells and similar cell therapies. Example 4: Clinical Evaluation of CAR T cells in Metastatic Colorectal Cancer in a U.S. Population and Evolution of Diarrhea Management Strategies

[0284] This example describes a multicenter, open-label, Phase 1 clinical study conducted in the United States (U.S.) to further evaluate the safety, feasibility, and preliminary efficacy of the Guanylyl Cyclase C (GCC)-directed CAR T cell product, CAR T cells, in adult patients with advanced mCRC. This study was designed for patients who had progressed following at least two prior lines of systemic therapy. A significant aspect of this example is the detailed presentation of diarrhea experienced by several study participants, the management approaches actually employed, the rationale behind these approaches, and the development and contemplation of revised and improved management protocols based on these clinical experiences and observations.

[0285] The study was a multicenter, open-label, Phase 1 clinical trial conducted in the U.S. Adult patients diagnosed with advanced mCRC who had shown disease progression after at least two previous lines of systemic therapy were eligible for enrollment. The CAR T cell product was prepared and characterized as generally described in Example 3. Further specific details regarding the clinical study of the CAR T cell product, including aspects of CAR T cell manufacturing, specific dose levels administered, comprehensive safety and efficacy outcomes, cytokine dynamics, genetic sequences related to the CAR constructs, and correlative biomarker analyses, are described in Example 3 and the publications incorporated by reference therein. The study included careful assessment and grading of adverse events, including diarrhea, and documentation of the management strategies utilized. 58  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0286] The following patient case studies illustrate the clinical course of diarrhea observed during the U.S. Phase 1 study, the actual management interventions undertaken, and the contemplated revisions to management strategies based on these experiences. These descriptions are supported by clinical trial timelines and hypothetical plan timelines for management revisions as indicated in the study documentation (e.g., FIG. 1A, 1B for patient 104-1031; FIG.2A, 2B for patient 101-1052; FIG.3A, 3B for patient 102- 1005; FIG. 4A, 4B for patient 104-1013; FIG. 5A, 5B for patient 104-1003; FIG. 6 for patient 101-1110; FIG.7A, 7B for patient 101-1114; and FIG.8A, 8B for patient 104-1039).

[0287] Hereinafter, references to a “contemplated revised management strategy,” “modified treatment plan,” or similar phrases, particularly in the context of figures depicting potential clinical courses (e.g., FIG. 1B, 2B, 3B, 4B, 5B, 7B, and 8B), generally refer to a layered, proactive, and criteria-guided approach to managing gastrointestinal toxicity. This strategic framework is distinguished from regimens that may have been actually administered in early clinical experiences by its emphasis on earlier and more targeted interventions, often initiated prophylactically or preemptively based on early clinical signs such as fever, or on predefined biomarker thresholds, rather than waiting for the establishment of high-grade diarrhea.

[0288] Key components of such a revised strategy often include the early introduction of gut-targeted immunomodulatory agents, such as vedolizumab and / or dual-form (oral and rectal) budesonide, to establish a protective mucosal barrier and manage local inflammation. The strategy further includes a structured escalation pathway, wherein subsequent interventions with agents like infliximab, systemic corticosteroids, dasatinib, or potent immunosuppressants like ATG are guided by the severity and duration of symptoms, often graded using objective criteria such as the MAGIC criteria. The overarching goals of these contemplated revised strategies are to shorten the duration and reduce the severity of diarrhea, prevent long- term intestinal inflammation and damage, shorten patient recovery time, and preserve the therapeutic efficacy of the CAR T cell therapy by minimizing or delaying the need for broad systemic immunosuppression. The specific agents and timings shown in each figure depicting a revised plan (e.g., FIG.1B, 2B, etc.) represent non-limiting examples of how this general strategic framework can be applied to different clinical scenarios.

[0289] Patient 104-1031 experienced an onset of diarrhea on day 5 post- infusion of CAR T cells, which progressed to grade 2 on the same day and persisted at grade 2 until day 7. The diarrhea rapidly resolved to grade 0 by day 8. However, a recurrence of grade 1 diarrhea was observed around day 15, which, after a brief remission between day 17 and day 19, returned as grade 1 diarrhea and continued until day 26. Although these later symptoms were mild, their persistence suggested ongoing intestinal inflammation. Treatments administered in relation to the diarrhea included dexamethasone (days 1 to 4), methylprednisolone (days 6-7, with an additional 1g administered on day 11 as a pulse), and loperamide, which was used for symptomatic relief during the initial peak of symptoms (days 5-7). Budesonide was initiated on day 6 and continued until day 7. A single dose of infliximab was administered on day 6, after which the initial symptoms rapidly subsided. Dasatinib was also administered, starting on day 6 and continuing for two days. No additional treatment was given for the later-onset grade 1 diarrhea. 59  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0290] Patient 101-1052 developed gastrointestinal toxicity following CAR T cell infusion, with symptoms starting around day 13 and requiring clinical management until day 27. Under the original management protocol, treatment with corticosteroids and dasatinib was initiated on day 13 when diarrhea appeared. From day 14, dexamethasone (4mg), methylprednisolone (2mg / kg / day), loperamide, and one dose of infliximab were administered. This treatment regimen continued until day 18, by which time the diarrhea had improved to grade 1. The patient maintained grade 1 diarrhea until day 24 and achieved complete resolution on day 25. This was followed by a tapering course of prednisone (70-100mg / day) until day 27. The diarrhea rapidly progressed to grade 2 on day 13. A fixed dose of 480mg of infliximab was administered on day 14, along with loperamide (days 14-26) and octreotide (days 15-16). The diarrhea improved to grade 1 by day 18 and resolved completely on day 25.

[0291] A contemplated revised management strategy for cases similar to Patient 101-1052 involves earlier and more targeted interventions. This revised approach includes the administration of vedolizumab (300mg) on day 8, combined with a 3-day course of budesonide (9mg orally plus rectal foam). If grade 2 diarrhea still occurs around day 13, infliximab at a dose of 5mg / kg body weight is administered. During the peak of diarrhea (e.g., days 15-17), the patient is kept Nothing by Mouth (NPO) and receives Total Parenteral Nutrition (TPN). Upon improvement of symptoms to grade 1 (e.g., by day 18), a second course of budesonide is initiated to promote mucosal repair. This revised protocol, by incorporating vedolizumab, budesonide (oral / rectal), potentially ATG, and nutritional support (NPO / TPN), aims to create a layered intervention strategy. It is expected that this revised strategy may offer advantages over the original protocol, such as earlier prevention of diarrhea by establishing a protective barrier, earlier management of emergent diarrhea which is anticipated to shorten its duration, reduce the risk of long-term intestinal inflammation and damage, shorten intestinal recovery time, and by controlling diarrhea sooner, potentially shorten the duration of immunosuppression, thereby avoiding negative impacts on therapeutic efficacy.

[0292] Patient 102-1005 experienced severe gastrointestinal toxicity after CAR T cell infusion. Diarrhea appeared on day 8, rapidly progressed to grade 3 on the same day, and persisted at this grade until day 12. The diarrhea improved to grade 2 on day 13 and further to grade 1 by day 18. The management strategy involved multiple agents. Infliximab was initiated on day 10. Methylprednisolone (2mg / kg / day) was started on day 11 and continued until day 22. Dasatinib was administered long-term, from day 10 to day 22. Loperamide was initiated on day 10 and used throughout the course. During the peak of symptoms (day 11), diphenoxylate-atropine, octreotide, and cholestyramine were added. Due to gastrointestinal intolerance and / or absorption issues, TPN was started on day 26, indicating interruption of enteral nutrition.

[0293] A contemplated revised management strategy for patients similar to 102-1005, especially those who do not present with fever, involves initiating a diarrhea prevention protocol on day 8, consisting of vedolizumab 300mg and budesonide (9mg oral plus rectal foam). Concurrently, methylprednisolone (2mg / kg / day) is administered and continued, which is expected to slow the progression of diarrhea. If diarrhea still progresses to grade 3, ATG is administered for 4 consecutive days, and the patient is kept NPO with TPN support to prevent further intestinal damage. If infliximab is required, it is to be administered 48 hours after vedolizumab, per guidelines. The anticipated advantages of this revised 60  Attorney Docket: I071-0123PCT / SDS1.0133PCT   protocol include reducing the severity and duration of diarrhea, preventing recurrence, and minimizing cumulative intestinal damage through early and sustained immunomodulation and optimized supportive care.

[0294] Patient 104-1013 experienced fever and gastrointestinal symptoms following CAR T cell infusion, with clinical management extending to 30 days post-infusion. The patient developed low-grade fever on day 5, which peaked at 39°C on day 7 and gradually returned to normal by day 10. Diarrhea symptoms appeared later, starting on day 11, reaching a grade 2 peak on day 16, and completely resolving by day 22 without recurrence. Treatment measures included tocilizumab on day 6 for management of CRS. For diarrhea control, since it started at grade 1 and did not progress rapidly, loperamide (days 11-21) was used in combination with diphenoxylate-atropine (days 14-24). A single dose of infliximab was administered on day 16, the peak day of symptoms.

[0295] The contemplated revised strategy for similar cases involves initiating a diarrhea prevention protocol on the second day after fever onset, consisting of a single dose of vedolizumab 300mg plus budesonide (9mg oral plus rectal foam) for three days. If grade 1 diarrhea appears on day 11, a single dose of infliximab is administered in conjunction with budesonide (standard antidiarrheals like loperamide and diphenoxylate-atropine are used as needed). If diarrhea were to persist for 5 days without resolution and progress to grade 2 by day 16 (as in the original course), then ATG combined with dasatinib would be used for treatment. This revised approach emphasizes earlier prevention and management, with timely escalation of treatment if diarrhea worsens, and is expected to shorten symptom duration, reduce severity, and prevent progression through combined systemic and gut-targeted immunosuppression.

[0296] Patient 104-1003 experienced a brief period of high fever after cell infusion, followed by mild but persistent diarrhea. Early fever started on day 5, peaked at 39°C between days 5-8, and resolved by day 9. Diarrhea symptoms appeared on day 18, remained at grade 1 throughout without progression, and persisted until day 28. Treatment measures included four doses of tocilizumab to manage CRS, with dexamethasone added on days 8-9 (primarily for neurotoxicity symptoms, which also indirectly alleviated CRS). Loperamide was used prophylactically and for management of diarrhea from day 10 to day 30. Since the diarrhea remained at grade 1, treatment measures were not intensified.

[0297] To reduce the risk of long-term or subclinical gastrointestinal inflammation, a contemplated revised strategy for similar cases involves an early, layered intervention. This includes gut-targeted immunomodulation: on the second day after fever onset, a single dose of vedolizumab (300mg) plus budesonide (9mg oral plus rectal foam) is administered for 3 days to prevent diarrhea (prophylactic loperamide use is not altered). For biologics optimization, if grade 1 diarrhea appears on day 18, infliximab (5mg / kg) is administered directly instead of waiting for progression. Mucosal protection is enhanced by administering budesonide (9mg oral plus rectal foam) throughout the period of grade 1 diarrhea (e.g., days 18-28). Immunosuppression is strengthened if grade 1 diarrhea persists for 5 days despite treatment (e.g., by day 23), at which point IV ATG (0.75mg / kg) is administered to control T-cell mediated damage. This revised approach changes the strategy of primarily observing low-grade diarrhea to managing it early to control inflammation, prevent progression to higher grades, and avoid long-term intestinal damage. 61  Attorney Docket: I071-0123PCT / SDS1.0133PCT

[0298] Patient 101-1110, a 49-year-old male with metastatic rectal cancer, exhibited a biphasic pattern of diarrhea and significant immune-related gastrointestinal toxicity following CAR T cell infusion. The patient was clinically stable for the first 10 days post-infusion. On day 10, grade 2 diarrhea, based on Common Terminology Criteria for Adverse Events (CTCAE) frequency, developed but was self-limiting and resolved by day 14. A more severe secondary episode began on day 17, progressing to grade 3 diarrhea by day 22 and persisting until day 31. During this period, stool volume increased dramatically, reaching a peak of nearly 6 liters per day on day 30, indicative of severe mucosal inflammation and barrier dysfunction, despite relatively stable stool frequency. The diarrhea trajectory showed two peaks: the first from day 10- 13, and the second from day 17 to beyond day 30.

[0299] Multiple immunosuppressive and supportive therapies were employed: methylprednisolone (1 mg / kg BID) was started concurrently with symptom onset on day 10; dasatinib (100 mg BID) was administered from day 10 to day 13; vedolizumab, an integrin blocker targeting the gut, was used on day 6 to prevent immune-mediated epithelial damage; infliximab at 5 mg / kg was initiated on day 10 for the grade 2 diarrhea; budesonide (days 14-39) provided local gastrointestinal anti-inflammatory effects; and octreotide (days 23-31) was used to reduce intestinal fluid secretion. A second dose of infliximab (5 mg / kg) was administered on day 18 due to increasing stool volume. Despite these interventions, high-volume diarrhea continued, leading to the initiation of ATG therapy on day 26 (total dose of 50 mg over 4 days, approximately 0.8 mg / kg). On day 31, ruxolitinib, a JAK1 / 2 inhibitor, was added as a steroid-sparing immunomodulator. Stool volume decreased after ATG treatment, and diarrhea resolved to grade 0 by day 34. The patient resumed oral intake on day 38 and had formed stools by day 39.

[0300] However, the recovery period was complicated by a transfer to the ICU and intubation for respiratory failure on day 31; although briefly extubated on day 32, re-intubation was necessary the same day. Candida dubliniensis was identified in sputum culture on day 34, and fungal bloodstream infection was confirmed on day 46. These events occurred in the context of high-dose corticosteroids, multiple biological agents, and sustained immunosuppression. The patient ultimately succumbed to sepsis. Autopsy confirmed immune-mediated intestinal damage.

[0301] Based on this case, a significantly revised management protocol was contemplated. A critical change is the adoption of MAGIC standards, which are based on stool volume, to replace CTCAE grading based on frequency, for diarrhea assessment. According to MAGIC criteria, this patient would have met criteria for grade 3 diarrhea on days 17-18, potentially prompting ATG initiation 9 days earlier. The new contemplated protocol suggests that in similar future cases, if stool volume exceeds 1 liter per day for 2 consecutive days, ATG should be administered on the third day at a dose of 0.75 mg / kg, in conjunction with other agents like vedolizumab and infliximab to achieve immunomodulation and mucosal protection. Dasatinib was retained in the revised protocol (days 9-22). Ruxolitinib was not included, possibly reflecting uncertainty about its delayed use. The new strategy also incorporates expanded infection prophylaxis: for moderate to high-volume diarrhea, concurrent initiation of antibacterial, antifungal, and antiviral prophylaxis is recommended starting on the third day of sustained diarrhea. Budesonide and octreotide are still utilized but with an emphasis on earlier initiation and continuation into the recovery phase. Monitoring 62  Attorney Docket: I071-0123PCT / SDS1.0133PCT   was also revised to include earlier use of rectal collection devices (e.g., Flexiseal) for accurate stool volume measurement, rather than subjective assessment, to guide treatment decisions; this was introduced on day 29 in the original management but is recommended much earlier in the revised plan.

[0302] The introduction of ATG into the CAR T cell clinical trial for diarrhea management represented a departure from then-current standard practices, as guidelines from ASCO / NCCN / SITC primarily recommended corticosteroids and infliximab, without mentioning ATG for CAR T-related diarrhea. The decision was prompted by the observation that diarrhea post-CAR T cell infusion closely resembled acute Gastrointestinal Graft-versus-Host Disease (GI-GVHD), sharing onset timing (4-8 days post-infusion), presentation (high-volume watery stool), and cytokine profiles (elevated Interleukin-6 (IL-6), Interferon- gamma (IFN-γ), Interleukin-10 (IL-10), Tumor Necrosis Factor-alpha (TNF-α)).

[0303] The prospectively modified protocol therefore incorporated MAGIC criteria for grading, listed ATG as a third-line immunosuppressant for ≥grade 2 diarrhea unresponsive to 72 hours of corticosteroids plus infliximab, or for ≥grade 3 diarrhea unresponsive to 4 days of combined therapy. Earlier ATG use was considered for high-risk patients (e.g., prior colectomy, rapidly worsening stool volume). ATG dosing was adjusted for body weight and platelet count: 1.5 mg / kg / day for 3 days if platelets >50×10⁹ / L, and 0.75 mg / kg / day for 4 days if platelets <50×10⁹ / L.

[0304] Subsequent patients were managed under aspects of the evolving revised protocols. Patient 101- 1114 experienced mild, self-limiting immune-mediated diarrhea following CAR T infusion. Diarrhea appeared on day 9 and resolved by day 11 without recurrence. Treatment measures included methylprednisolone (1 mg / kg BID, days 9-12), dasatinib (days 3-13, transitioning from 100 mg QD to BID), infliximab (5 mg / kg on day 9), budesonide (days 9-12), and vedolizumab (300 mg on day 8) administered as pre-symptomatic prophylaxis.

[0305] It was contemplated that if protocols were strictly followed as anticipated, the duration of dasatinib and budesonide administration could potentially be shortened in such cases. This reflects a more proactive management strategy aiming to prevent symptom escalation by administering key immunomodulatory agents earlier. While the original management for this patient led to rapid resolution, the revised thinking suggested even earlier intervention for high-risk patients or those needing early inflammation control.

[0306] In the case of Patient 101-1110, on Day 26, colonoscopy and mucosal biopsy were performed due to persistent grade 3 diarrhea with daily stool volumes exceeding 5 liters. Endoscopic findings revealed widespread mucosal sloughing. Histological analysis showed diffuse epithelial apoptosis, crypt loss, lamina propria inflammation, and mucosal denudation. The pathological pattern was assessed by the clinical team as resembling GI-GVHD, a phenomenon not previously described in autologous CAR T settings. Based on these findings and the failure of standard immunosuppressants to control symptoms, ATG was introduced on Day 26. The patient exhibited a sustained decrease in stool volume after initiation of ATG, with resolution of diarrhea by Day 34.

[0307] This unexpected observation led to the incorporation of mucosal pathology as a key factor in therapeutic decision-making. In addition, the pattern of high-volume watery stool—despite normal stool 63  Attorney Docket: I071-0123PCT / SDS1.0133PCT   frequency—highlighted the inadequacy of CTCAE frequency-based diarrhea grading in such cases. As a result, the MAGIC criteria, which are based on stool volume, were adopted into the revised management protocol. Under the revised grading scheme, Patient 101-1110 would have qualified for grade 3 diarrhea by Day 17, potentially enabling earlier ATG administration.

[0308] Separately, Patient 102-1005 presented with early-onset grade 3 diarrhea on Day 8 post-infusion, without preceding fever. This case revealed that severe inflammatory diarrhea could arise rapidly and without classical CRS prodromes. Although the patient responded to infliximab and corticosteroids, the initial progression was abrupt and required multiple secondary agents. Post hoc analysis suggested that earlier intervention with vedolizumab—targeting gut-selective integrin α4β7—might have prevented lymphocyte trafficking to the gut mucosa and delayed or mitigated disease progression. This insight, combined with the experience in Patient 101-1110, led to a revised layered prophylactic and treatment model: vedolizumab and infliximab were incorporated as prophylactic measures to suppress gut-directed immune activation, and therapeutic escalation was guided in part by the adoption of the MAGIC grading system, which, unlike frequency-based criteria alone, emphasizes stool volume as an indicator of disease burden.

[0309] The formulation of this regimen was not based on any prior CAR T treatment algorithm or guideline. ATG had not previously been proposed for CAR T–associated diarrhea. Vedolizumab and infliximab, although individually known agents, had never been employed in the specific sequence or indications defined above. The rationale and structure of the protocol emerged solely from direct clinical data, endoscopic and histological findings, and retrospective cross-patient analysis. As such, the approach described herein represents a novel and non-obvious strategy derived from mechanistic reinterpretation of actual patient outcomes.

[0310] Patient 104-1039 experienced a brief and distinct episode of non-immune-mediated diarrhea after CAR T cell infusion. Grade 1 diarrhea occurred on day 1 post-infusion, at a time when CAR T cells had not yet started to expand and there were no systemic signs of inflammation, suggesting it was likely an incidental increase in bowel movements rather than immune-related diarrhea. The diarrhea resolved, and no recurrence was observed during the 30-day monitoring period. Management during this episode included early use of budesonide, starting on day 1 when diarrhea appeared and continuing until day 11. Vedolizumab was administered on day 8 as a prophylactic measure against inflammation-mediated diarrhea. The early use of these drugs indicated an intent to control both mucosal surface and systemic gut-specific inflammation. The absence of recurrent inflammatory diarrhea during the observation period suggested the intervention was effective in suppressing immune-mediated gastrointestinal symptoms.

[0311] A contemplated revised treatment strategy for such presentations includes the combined use of infliximab, methylprednisolone, budesonide, and vedolizumab within a compact treatment window to manage early or potentially steroid-refractory inflammation. Systemic corticosteroids like methylprednisolone would be used with budesonide to provide broad immunosuppression via both systemic and local pathways. Vedolizumab would remain a component of the prophylactic strategy, potentially administered earlier or in multiple doses to maintain gut-selective immunomodulation. This intensified 64  Attorney Docket: I071-0123PCT / SDS1.0133PCT   protocol reflects a proactive management approach aimed at shortening symptom duration and preventing recurrence, even for mild, self-limiting diarrhea, by ensuring early, layered use of biologics and corticosteroids to achieve gastrointestinal stability and reduce the risk of further immune-driven toxicity, especially in cases where immune activation could progress rapidly.

[0312] In the U.S. Phase 1 trial, CAR T cells was evaluated across two dose levels (1×10⁶ CAR T cells / kg and 2×10⁶ CAR T cells / kg), with a total of nine patients enrolled across these levels by the time of this analysis; follow-up was planned to continue until March 2025. Despite the occurrence of immune-mediated adverse events, including diarrhea, the results indicated clinically meaningful anti-tumor activity, with responses including both partial remissions and complete remissions observed among the participants.

[0313] Elements of the revised diarrhea management strategies were applied in the treatment of subsequent patients, yielding positive outcomes. For example, Patient 104-1039, who received vedolizumab on day 8 and budesonide starting on day 1, experienced rapid resolution of grade 1 diarrhea within three days, without the need for systemic corticosteroids or treatment escalation. Patient 101-1114 received vedolizumab on day 8, and upon developing diarrhea on day 9, was treated with infliximab and budesonide (continued until day 10); symptoms completely resolved by day 11. These cases provided evidence that early intervention with gut-selective therapeutic agents could prevent progression to more severe inflammation and obviate the need for broader immunosuppressive measures. Indeed, patients who were subsequently managed with an approach involving early combination of vedolizumab, budesonide, and infliximab had their conditions controlled without requiring the use of ATG, indirectly validating the efficacy of this escalated and refined management strategy. This evolution in management, particularly the incorporation of ATG based on a re-evaluation of the immunopathology as GI-GVHD-like, challenged traditional perceptions of CAR T related diarrhea and represented an innovative application of patient- derived data.

[0314] The U.S. Phase 1 clinical study of CAR T cells provided further evidence of its anti-tumor activity in advanced mCRC and allowed for a detailed characterization of its safety profile, where diarrhea was a commonly observed adverse event. The in-depth analysis of individual patient experiences with diarrhea, including their actual clinical courses and the responses to various interventions, facilitated an iterative process for developing and contemplating refined, multi-layered diarrhea management strategies. These strategies encompass both prophylactic measures and active treatment algorithms that emphasize earlier introduction of gut-targeted therapies (such as vedolizumab and budesonide) and timely, criteria-guided escalation to systemic agents (like infliximab and ATG, the latter based on revised severity grading and understanding of underlying pathology).

[0315] The contemplated (improved protocols) derived from these clinical observations represent scientifically reasoned approaches expected to enhance the management of CAR T cell-associated diarrhea by reducing its severity, duration, and associated complications. Importantly, the initial application of elements from these revised strategies in managing subsequent patients demonstrated success in preventing severe diarrhea and, in some instances, avoiding the need for more intensive immunosuppressive agents like ATG, thereby validating the value of this adaptive and data-driven approach to toxicity management. 65  Attorney Docket: I071-0123PCT / SDS1.0133PCT   Effective control of such adverse events is crucial for improving the therapeutic index of CAR T cells and similar CAR T cell therapies. Example 5: Unexpected Occurrence of Severe Diarrhea as an Adverse Event in GCC-Targeted CAR T Cell Therapy

[0316] This example describes the prevailing scientific and clinical understanding prior to the initiation and subsequent data analysis of the CAR T cells clinical trial (the results of which are detailed in part in Examples 3 and 4) concerning the anticipated gastrointestinal toxicity profile of GCC-targeted therapies. The primary objective of this example is to elucidate why the frequent and sometimes severe diarrhea observed in subjects treated with CAR T cells was an unexpected clinical finding, thereby underscoring the novelty and non-obviousness of the methods developed for its management.

[0317] Before the CAR T cells clinical trial was conducted, the prevailing scientific and clinical expectation was that severe gastrointestinal toxicity, particularly severe diarrhea, would not be a prominent adverse event associated with GCC-targeted CAR T cell therapy in patients with colorectal cancer. This expectation was largely based on the known anatomical and molecular characteristics of GCC expression. GCC is understood to be expressed almost exclusively on the apical, luminal surface of intestinal epithelial cells. In healthy intestinal tissue, these epithelial cells form a barrier maintained by structurally intact tight junctions, which preserve epithelial polarity and are considered to restrict the access of systemically administered therapeutic agents, including immune effector cells like CAR T cells and large biologic molecules such as antibodies, to the apically-expressed GCC.

[0318] Based on this understanding, it was widely believed in the field that the apical confinement of GCC would effectively prevent CAR T cells or other therapeutic antibodies from engaging the target antigen in healthy intestinal tissues. Consequently, the biological consensus posited that targeting GCC in solid tumors, where epithelial integrity and junctional complexes might be compromised or disrupted, would permit tumor-selective engagement by the therapeutic agent while sparing normal intestinal tissues from significant on-target, off-tumor toxicity. This rationale had underpinned the development of previous GCC-directed therapeutic modalities, including vaccines, antibody-drug conjugates (ADCs), and bispecific T-cell engagers. Notably, clinical studies involving these prior GCC-targeted agents did not report high rates of severe diarrhea. The observed safety profile of these earlier agents further reinforced the prevailing assumption that GCC-targeted therapeutics, including CAR T cells, would not cause significant intestinal toxicity leading to severe diarrhea.

[0319] The role of inflammation-induced disruption of tight junctions, a mechanism now understood to be significantly driven by cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF- α), and interleukin-6 (IL-6), was not fully appreciated in the context of GCC-targeted CAR T cell therapy during earlier preclinical evaluations. These inflammatory mediators, which can be robustly produced upon CAR T cell activation and engagement with target cells, have the capacity to degrade epithelial tight junction integrity. Such degradation can, in turn, allow effector T cells to infiltrate the intestinal mucosa and gain access to GCC-expressing epithelial surfaces that would ordinarily remain immunologically privileged or inaccessible due to the intact epithelial barrier. However, these complex cytokine-driven 66  Attorney Docket: I071-0123PCT / SDS1.0133PCT   dynamics and their potential clinical consequences were often absent or significantly underrepresented in prior animal models, which generally lacked the resolution or physiological similarity to human subjects to accurately capture these specific changes and predict their impact on gastrointestinal toxicity.

[0320] Contrary to the prior expectations, the CAR T cells clinical studies (detailed in Examples 3 and 4) revealed that diarrhea was a frequent and, in some cases, severe adverse event experienced by treated patients. The emergence of such gastrointestinal toxicity represented a substantial and unexpected deviation from the anticipated safety profile that was based on the previously understood apical sequestration of GCC behind functional tight junctions. For the purposes of the present disclosure, unless specified otherwise or clearly indicated by the context, any reference to 'a CAR T cells clinical trial,' 'the CAR T cells clinical trial,' 'clinical trial evaluation of CAR T cells,' or similar phrases concerning clinical trials of CAR T cells, shall refer to a clinical trial utilizing or evaluating 'GCC19CAR product', as defined in Examples 3 and 4.

[0321] The occurrence of frequent and sometimes severe diarrhea as a significant adverse event in the CAR T cells clinical trial was unexpected, considering the prior understanding of GCC biology, the behavior of other GCC-targeted therapeutic agents, and the limitations of preclinical models. This unexpected clinical finding highlights the novelty and non-obviousness of developing specific, and often intensive, management protocols, such as those described in Examples 1, 2, and the evolved strategies discussed in Example 4, which are designed to effectively address this previously underappreciated toxicity associated with GCC-targeted CAR T cell therapy. The clinical observations from the CAR T cells program suggest a mechanism involving CAR T cell activation, subsequent release of inflammatory cytokines, ensuing disruption of intestinal epithelial tight junctions, and consequent on-target, off-tumor effects in the intestine. This underscores a previously underestimated risk for CAR T cell therapies directed against antigens that exhibit polarized expression on epithelial surfaces, particularly when utilizing effector cells capable of amplifying local inflammatory responses. Example 6: Rationale and Development of a Balanced Immunomodulatory Strategy for Managing Severe CAR T Cell-Associated Diarrhea

[0322] This example describes the clinical considerations, challenges, and rationale that guided the development of the therapeutic algorithm for managing severe, and particularly steroid-refractory, diarrhea observed during the CAR T cells clinical trial. The primary objective is to detail the complex process of balancing the critical need to mitigate this serious adverse event (AE) with the equally important goal of preserving the anti-tumor efficacy of the GCC-targeting CAR T cell product. This example explains how direct clinical observations led to a novel, indication-specific management framework. For the purposes of the descriptions herein, the terms “protocol,” “treatment protocol,” and “treatment plan” refer interchangeably to a structured therapeutic approach designed to manage gastrointestinal adverse events following CAR T cell infusion, encompassing both actual clinical regimens administered and proposed or modified versions based on real-time clinical observations and trial outcomes.

[0323] During the course of the CAR T cells clinical trial, severe gastrointestinal toxicity, notably diarrhea that proved refractory to initial steroid treatment, emerged as a significant clinical issue. This presented a challenging clinical dilemma: how to effectively suppress the pathogenic immune activity 67  Attorney Docket: I071-0123PCT / SDS1.0133PCT   presumed to be driving the toxicity without concurrently impairing the therapeutic function of the CAR T cells, which itself is dependent on T cell activity. The management strategy therefore had to be responsive to these emerging adverse events, focusing on protecting patient safety while attempting to maintain the efficacy of the CAR T cell therapy.

[0324] The management strategy developed incorporated several pharmacologic agents known for their immunomodulatory or immunosuppressive effects, including methylprednisolone, infliximab, dasatinib, ATG, and broad-spectrum antibiotics. The use of these agents in the context of a T cell-dependent immunotherapy like CAR T therapy was approached with caution due to their known mechanistic risks of compromising CAR T cell activity.

[0325] Specifically, methylprednisolone, a systemic corticosteroid, was known to potentially attenuate CAR T cell expansion and persistence, particularly if administered early in the treatment course. Infliximab, an antibody targeting TNF-α, while effective for managing immune-mediated colitis, carried a risk of dampening T cell signaling pathways mediated by TNF-α. Dasatinib, characterized in some contexts as a pharmacologic “off switch” for CAR T cells, can reversibly inhibit CAR signaling, which might suppress therapeutic activity during its administration. ATG, traditionally used in transplant conditioning and for GVHD, causes broad lymphocyte depletion, thereby posing a direct threat to CAR T cell numbers and viability. Furthermore, certain empirical antibiotics, particularly those that significantly disrupt the gut microbiota composition, had been associated in other settings with impaired CAR T cell responses, potentially due to altered microbial-immune interactions.

[0326] Despite the known immunological effects and potential risks of these drugs, their incorporation into the diarrhea management protocol was not predicated on theoretical models alone. Instead, each decision to include an immunomodulator, as well as the specific sequence, dose, and conditions under which each agent was introduced, was guided by an iterative process based on real-time clinical observations, patient-level efficacy data, and safety data gathered directly during the CAR T cells clinical trial. This approach allowed for the dynamic optimization of the management plan to achieve control of the diarrhea while attempting to minimize any negative impact on the underlying CAR T cell activity.

[0327] The therapeutic algorithm that evolved from this process represented a carefully calibrated balance between achieving necessary immune suppression to control gastrointestinal toxicity and preserving CAR T cell function. This algorithm included strategies such as the early use of corticosteroids, followed by the introduction of infliximab for patients who did not respond adequately to steroids, escalation to ATG in refractory cases, and the incorporation of gut-specific anti-inflammatory agents such as vedolizumab and budesonide.

[0328] The application of this specific combination and sequence of immunomodulatory agents within a management framework for high-grade gastrointestinal toxicity arising from GCC-targeted CAR T therapy in solid tumors was distinctive. Although these individual drugs may have been used in other immunotherapy contexts or for other inflammatory conditions, their precise impact on CAR T cells function—and particularly their strategic sequencing and integration into a protocol for this specific type of severe diarrhea—was defined through direct clinical evidence obtained from the CAR T cells trial, rather 68  Attorney Docket: I071-0123PCT / SDS1.0133PCT   than being simply extrapolated from unrelated therapeutic areas. The development and implementation of this management framework was a direct and inventive response to an unexpected and severe toxicity profile observed in the trial. It involved incorporating existing pharmacologic agents into a novel, indication-specific algorithm designed to support both patient safety and the continued efficacy of the CAR T therapy for solid tumors.

[0329] The development of the comprehensive diarrhea management protocol for the CAR T cell therapy involved navigating significant clinical challenges, primarily balancing the need to control severe, treatment-emergent gastrointestinal toxicity against the risk of known immunomodulatory interventions impairing CAR T cell efficacy. The resulting multi-agent, sequenced therapeutic algorithm, which was iteratively refined through direct clinical experience and patient outcomes within the trial, represents a novel and non-obvious approach to managing this specific and severe adverse event. This strategic framework, born from the necessity to address an unexpected toxicity profile, was crucial for improving the overall risk-benefit profile of the CAR T cell therapy and enabling its continued development. Example 7: Early and Prioritized Use of Infliximab for Management of CAR T Cell-Associated Diarrhea: A Deviation from Conventional Guidelines

[0330] This example describes a key and distinctive component of the adaptive toxicity management strategy that was developed and employed in the CAR T cells clinical trial for addressing severe and persistent diarrhea: the early and often prioritized administration of infliximab. The objective of this example is to detail this specific therapeutic approach, explain its deviation from then-conventional treatment guidelines for immune-mediated gastrointestinal toxicities, present the underlying clinical rationale derived from the trial, and highlight its contribution to managing severe diarrhea while attempting to preserve the efficacy of the CAR T cell therapy.

[0331] Prior to and during the period of the CAR T cells clinical trial, major oncology and immunotherapy guidelines, such as those from the National Comprehensive Cancer Network (NCCN), American Society of Clinical Oncology (ASCO),1 and European Society for Medical Oncology (ESMO), generally recommended a specific sequence for immunosuppressive intervention in cases of immune- mediated diarrhea or colitis. Systemic corticosteroids, for example methylprednisolone, were typically recommended as the primary, first-line treatment. Infliximab, a Tumor Necrosis Factor-alpha (TNF-α) blocking agent, was generally positioned strictly as a second-line intervention, to be employed only in cases where systemic corticosteroids had failed to achieve adequate symptom control after a period of 48 to 72 hours of treatment. This conservative sequencing was rooted in several considerations, including concerns that the early or more aggressive use of biologic agents like infliximab might heighten the risk of infection, unnecessarily blunt systemic immune responses (including potential anti-tumor responses), or obscure the determination of whether the underlying inflammation was indeed steroid-responsive.

[0332] The clinical trial protocol for CAR T cells explicitly deviated from this conventional paradigm for the management of CAR T cell therapy-associated diarrhea. According to the CAR T cell protocol implemented in the trial, a deliberate inversion of the conventional immunosuppressive sequencing was adopted. Specifically, patients experiencing Grade 1 or Grade 2 diarrhea that persisted beyond five days 69  Attorney Docket: I071-0123PCT / SDS1.0133PCT   despite supportive therapy were administered infliximab at a dose of 5 mg / kg intravenously as an early intervention. If symptoms of diarrhea continued beyond Day 6 of their course, corticosteroids, typically intravenous methylprednisolone at a dose of 1 mg / kg twice daily, were subsequently introduced. This model prioritized early biological intervention with infliximab, aiming to prevent symptom progression, while deferring the use of systemic corticosteroids unless deemed clinically necessary.

[0333] Even in cases of Grade 2 or Grade 3 diarrhea, infliximab was frequently administered as the initial immunomodulatory agent, unless there was a clear clinical indication for immediate systemic immunosuppression with corticosteroids. In instances of severe (Grade 3 or Grade 4) diarrhea, infliximab and corticosteroids were often initiated concurrently; however, the protocol maintained infliximab as a preferred agent for achieving early control of the inflammatory process.

[0334] This strategic approach involving the early use of infliximab was supported by emerging clinical observations during the CAR T cells trial. The clinical rationale for this deviation arose from observations suggesting that the early inflammatory signaling in these patients appeared to be driven by TNF-α– dominant cytokine patterns. It was hypothesized that rapid TNF-α blockade with infliximab could potentially contain mucosal damage more efficiently and rapidly than could be achieved with the generally slower immunosuppressive kinetics of corticosteroids.

[0335] An additional and critical consideration was the potential to preserve CAR T cell efficacy. Early infliximab use was thought to allow for more selective immune modulation, potentially avoiding or reducing the broad immunosuppression of CAR T cell function that is known to be associated with systemic corticosteroid administration. This aspect was of particular importance in the context of treating solid tumors with CAR T cells, where achieving robust and persistent CAR T cell expansion and effector function can be more challenging than in hematologic malignancies. The approach was documented in a companion publication in JAMA Oncology, which reported on the treatment of 11 patients with infliximab, including cases where the agent was used in parallel with, or even before, the administration of corticosteroids.

[0336] The decision to prioritize and implement the early use of infliximab in the management of CAR T cell-associated diarrhea was not intuitive at the time and represented a meaningful divergence from established clinical standards and guidelines for managing immune-mediated gastrointestinal toxicities. Prior to the experience gained in the CAR T cell study, no widely published protocols or immunotherapy guidelines endorsed the use of infliximab before corticosteroids in this specific clinical scenario. The findings and the resultant protocol modifications from the CAR T cell trial therefore represented a significant deviation from then-current clinical norms, a deviation that was justified by the real-time clinical data and mechanistic insights that emerged during the study and which were not available in the existing literature. This early and prioritized application of infliximab within a CAR T therapy framework—where the preservation of engineered T-cell activity is of vital importance—constituted a non-obvious innovation in adverse event management and challenged long-standing assumptions regarding the sequencing of immunosuppressive therapies for such toxicities.

[0337] The implementation of an early and prioritized infliximab administration strategy within the CAR T cell clinical trial for the management of severe diarrhea, often preceding or used in place of systemic 70  Attorney Docket: I071-0123PCT / SDS1.0133PCT   corticosteroids, marked a deliberate and clinically significant departure from conventional treatment guidelines. This innovative approach was developed reactively in response to the specific challenges and unexpected toxicities encountered during the CAR T cell therapy of solid tumors. The rationale was based on emerging evidence from the trial suggesting a TNF-α driven mechanism and aimed to achieve rapid control of gastrointestinal inflammation while minimizing the potentially detrimental effects of broad and early immunosuppression (e.g., from corticosteroids) on CAR T cell efficacy. This strategy, validated by clinical experience within the trial, highlights a non-obvious advancement in managing CAR T cell therapy- related toxicities, particularly in a setting where preserving effector T cell function is paramount. Example 8: Introduction of ATG and Revised Grading Criteria for Managing Severe, Refractory CAR T Cell-Associated Diarrhea

[0338] This example describes crucial refinements made to the diarrhea management protocol within the CAR T cell clinical program. These refinements included the incorporation of ATG as an escalation therapy and the updating of diarrhea grading criteria based on clinical experience with particularly severe cases. The objective is to detail the clinical impetus for these modifications, primarily arising from observations in patient 101-1110 (whose case is also discussed in Example 4), and to explain how these changes were intended to address previously identified gaps in the management of high-risk, refractory gastrointestinal toxicity associated with CAR T cell therapy.

[0339] While the adverse event management protocol utilized in the initial phases of the CAR T cell clinical development (derived from the Chinese clinical trial and aspects of which were published in JAMA Oncology) demonstrated substantial success in controlling most instances of immune-mediated diarrhea, it proved insufficient in preventing a severe clinical outcome in patient 101-1110. As detailed in Example 4, this patient developed high-volume, persistent diarrhea that progressed and became refractory despite the implementation of the standard therapeutic measures available at that time, which included corticosteroids, infliximab, dasatinib, and comprehensive supportive care. The severity and refractoriness of the gastrointestinal toxicity in this specific case revealed a critical gap in the existing protocol’s capacity to effectively suppress the underlying inflammatory cascade in certain high-risk clinical scenarios.

[0340] In direct response to the experience with patient 101-1110 and other clinical observations, the diarrhea management protocol was further refined for patients enrolled in the subsequent U.S.-based CAR T cell trial. A key modification was the introduction of ATG as an available escalation therapy for patients who presented with severe diarrhea that was refractory to both corticosteroid and infliximab treatments.

[0341] Concurrently, the grading and monitoring system for diarrhea was updated. This update involved the incorporation of volume-based scoring criteria, such as those provided by the MAGIC system, to allow for a more accurate and objective assessment of diarrhea severity. This was considered particularly important for patients who might have an elevated baseline bowel movement frequency, where frequency- based grading alone might not fully capture the clinical severity of the diarrheal episode.

[0342] These significant modifications to the treatment protocol were directly informed by the detailed analysis of the clinical trajectory of patient 101-1110. It was observed that the delayed intervention with highly potent immunosuppression, such as ATG, in this patient contributed to prolonged fluid overload and 71  Attorney Docket: I071-0123PCT / SDS1.0133PCT   eventually necessitated ICU-level care. Consequently, the rationale for incorporating ATG into the protocol, and specifically for considering its use earlier in the treatment course for subsequent high-risk patients, was to more effectively prevent refractory cytokine-driven volume loss and mitigate the risk of systemic instability.

[0343] The decision to refine the protocol by including ATG and revising grading criteria reflected a proactive effort to close identified safety gaps in the management of CAR T cell–related gastrointestinal toxicity. The overarching goal of these modifications was to manage this severe adverse event more effectively, thereby enhancing patient safety, while simultaneously striving to preserve the therapeutic efficacy of the CAR T cell product.

[0344] The clinical experience with patient 101-1110, who suffered from severe and refractory diarrhea, served as a critical learning point and underscored the necessity for further evolution of the diarrhea management strategy for CAR T cell therapy. The subsequent incorporation of ATG as an escalation option for refractory cases and the adoption of volume-based diarrhea grading criteria (e.g., MAGIC system) in the U.S. clinical trial protocol represented important, data-driven refinements. These changes were designed to provide a more robust and timely therapeutic intervention for patients identified as being at high risk of developing severe, uncontrolled gastrointestinal toxicity. By addressing this specific treatment gap, these modifications aimed to improve overall patient safety and support the continued development and viability of the CAR T cell cell therapy. This iterative development and adaptation of the management protocol, based on challenging clinical cases, highlights an inventive approach to addressing unexpected and severe adverse events encountered in novel immunotherapy regimens. Example 10: Early Intervention with Combined Oral and Rectal Budesonide Based on Systemic Inflammatory Signs or Early Gastrointestinal Symptoms for Management of Potential CAR T Cell- Associated Gastrointestinal Toxicity

[0345] This example describes a novel component of the gastrointestinal toxicity management strategy that was developed and implemented in the CAR T cell clinical trial: the early administration of a combined oral and rectal budesonide regimen. The objective is to detail this specific therapeutic intervention, emphasize its novelty by contrasting it with previously reported uses of budesonide in similar clinical contexts, explain the clinical algorithm that guided its initiation based on early systemic signs of inflammation or initial gastrointestinal symptoms, and underscore its effectively prophylactic nature aimed at preventing the development or escalation of clinically significant diarrhea.

[0346] Prior to the approach taken in the CAR T cell trial, the combined use of oral and rectal budesonide, particularly for the management of Grade 1 diarrhea or as an intervention triggered by early systemic inflammation in patients receiving CAR T cell therapy, had not been reported in the published scientific literature. Budesonide, a glucocorticoid characterized by its low systemic bioavailability and predominantly local action within the gut, had been occasionally referenced in the management of immune-related colitis, for instance, in settings such as checkpoint inhibitor therapy. However, such documented applications typically involved the use of oral budesonide alone and were generally reactive, meaning treatment was initiated after the onset of more established symptoms, rather than in an early-phase or prophylactic capacity. 72  Attorney Docket: I071-0123PCT / SDS1.0133PCT   Importantly, no previous studies or treatment protocols had documented the simultaneous use of both oral budesonide and rectal budesonide foam, nor its application at the earliest stages of gastrointestinal distress, such as upon the presentation of Grade 1 symptoms, or based on antecedent systemic signs like fever.

[0347] In the CAR T cell clinical trial, a dual-form budesonide regimen was implemented with the aim of addressing low-grade diarrhea promptly if it arose, and critically, to prevent its progression to higher grades that would likely necessitate systemic immunosuppression with its attendant risks. The treatment protocol specified the administration of a 9 mg oral budesonide capsule taken daily, with concurrent administration of rectal budesonide foam.

[0348] The initiation of this dual budesonide administration was explicitly linked by the protocol to the first appearance of systemic inflammation (manifested as fever) or early gastrointestinal symptoms. Specifically, the regimen was initiated on Day 5 following CAR T cell infusion if the patient developed a Grade 1 or higher fever at any point between Days 0 and 4 post-infusion. If fever appeared on or after Day 5, the dual budesonide regimen was started within 24 hours of the fever's onset. In patients who did not develop fever, or if fever arose after Day 7, the treatment was initiated on Day 8. This approach positioned the budesonide intervention as an early response strategy, triggered by initial signs rather than being a strictly preemptive prophylactic measure administered to all patients irrespective of early signs.

[0349] This early intervention strategy with combined oral and rectal budesonide differed fundamentally from prior immunotherapy toxicity management algorithms. Conventional approaches generally escalated to specific anti-inflammatory therapy only after diarrhea reached Grade 2 or a higher level of severity. Moreover, the combination of two localized delivery routes—oral for broader small and large bowel coverage and rectal for targeted distal colon and rectal delivery—represented an innovative step aimed at maximizing anti-inflammatory effects throughout the entire lower gastrointestinal tract. A key rationale for this approach was to achieve this enhanced local coverage without imposing the significant immunosuppressive risks and systemic side effects typically associated with systemic corticosteroids. The development and refinement of this novel protocol component were driven by direct clinical observations during the CAR T cell trial and were specifically tailored to address the unique pattern and early evolution of diarrhea observed in the context of GCC-targeting CAR T cell therapy.

[0350] The budesonide regimen, as implemented, was determined to be effectively prophylactic in nature. Although its initiation was triggered by early systemic signs such as low-grade fever or very early gastrointestinal symptoms, this often occurred prior to the development of clinically significant or escalating diarrhea. Therefore, this early intervention strategy represented a novel and data-driven approach aimed at preventing the escalation of gastrointestinal toxicity. The combined use of oral and rectal budesonide in this manner, particularly at such an early stage of potential toxicity development in CAR T cell therapy recipients, was not found in existing literature and constituted a non-obvious therapeutic approach.

[0351] The early and combined administration of oral and rectal budesonide, triggered by initial systemic signs of inflammation (such as fever) or the very first appearance of low-grade gastrointestinal symptoms, as developed and implemented in the CAR T cell clinical trial, constituted a novel and non-obvious strategy 73  Attorney Docket: I071-0123PCT / SDS1.0133PCT   for the management of potential CAR T cell therapy-associated gastrointestinal toxicity. This approach differed significantly from previous clinical practices by intervening at a very early stage—often prophylactically against the subsequent development or escalation of diarrhea—and by utilizing dual local delivery routes to maximize gut-targeted anti-inflammatory effects while minimizing systemic immunosuppressive exposure. This refined method, developed through direct clinical experience during the trial, was designed to improve the gastrointestinal safety profile and overall tolerability of CAR T cell therapy.

[0352] All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entireties as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. 74

Claims

Attorney Docket: I071-0123PCT / SDS1.0133PCT   CLAIMS 1. A method of managing chimeric antigen receptor T (CAR T) cell therapy-associated diarrhea in a human subject undergoing the CAR T cell therapy for colorectal cancer (CRC), wherein CAR T cells of the CAR T cell therapy target a CRC-associated antigen, the method comprising: administering a prophylactic regimen to the human subject post-infusion of the CAR T cells, wherein the prophylactic regimen comprises administering vedolizumab (VDZ), infliximab (IFX), and / or budesonide (BUD); and monitoring the subject for development and severity of diarrhea based on predefined clinical criteria including stool frequency or stool volume, after the CAR T cell therapy.

2. The method of claim 1, wherein the predefined clinical criteria further comprise a complete blood count with differential (CBC w / diff), a presence or absence of an infectious cause, and intake and output volumes.

3. The method of claim 1 or 2, wherein the CRC-associated antigen is Guanylate Cyclase C (GCC) or Carcinoembryonic Antigen (CEA).

4. The method of any one of claims 1-3, wherein the prophylactic regimen comprises administering VDZ on or about Day 3 post-infusion of the CAR T cells.

5. The method of any one of claims 1-3, wherein the prophylactic regimen comprises administering IFX on or about Day 5 post-infusion of the CAR T cells.

6. The method of any one of claims 1-3, wherein the prophylactic regimen comprises administering BUD from on or about Day 5 to on or about Day 12 post-infusion of the CAR T cells.

7. The method of any one of claims 1-3, further comprising administering at least one therapeutic agent to the subject if diarrhea occurs on or after Day 5 post-infusion of the CAR T cells and the diarrhea meets the predefined clinical criteria for severity.

8. The method of claim 7, wherein the at least one therapeutic agent is methylprednisolone (MP), wherein MP is administered if the diarrhea is mild, and wherein mild diarrhea is defined as 2 to 3 loose or watery stools per 24 hours or less than 600 mL of stool volume per 24 hours.

9. The method of claim 8, further comprising administering antithymocyte globulin (ATG) if the mild diarrhea persists for more than 3 days despite the administration of the MP.

10. The method of claim 7, wherein the at least one therapeutic agent is ATG, wherein ATG is administered if the diarrhea is moderate, and wherein moderate diarrhea is defined as 4 to 6 loose or watery stools per 24 hours or 600 mL to 1200 mL of stool volume per 24 hours.

11. The method of claim 10, wherein if the prophylactic regimen comprises IFX and if the subject has received or is scheduled to receive IFX on a same day as an onset of the moderate diarrhea, administration of the ATG is delayed for 24 hours to assess response to IFX.

12. The method of claim 7, wherein the at least one therapeutic agent is therapeutic BUD, wherein BUD is administered if the diarrhea is severe, and wherein the severe diarrhea is defined as: 7 or more loose or watery stools per 24 hours; or greater than 1200 mL of stool volume per 24 hours. 75  Attorney Docket: I071-0123PCT / SDS1.0133PCT   13. The method of claim 12, wherein the therapeutic BUD is administered orally and / or rectally, wherein the rectally administered BUD is in a form of BUD foam and is administered until on or about Day 19 post-infusion of the CAR T cells or for at least 7 days or after the severe diarrhea is under control, whichever is later.

14. The method of claim 7, further comprising administering a second dose of IFX if the diarrhea occurs on or after Day 12 post-infusion of the CAR T cells.

15. The method of any one of claims 9 or 10, further comprising administering at least one agent subsequent to the administration of the ATG, wherein the at least one agent is selected from the group consisting of an antifungal agent, an antibacterial agent, and an antiviral agent.

16. The method of any one of claims 1-3, wherein the prophylactic regimen comprises administering both VDZ and IFX, and wherein the IFX is administered at least 48 hours after the VDZ is administered.

17. The method of claim 14, wherein if the subject has previously received VDZ, the second dose of IFX is administered at least 48 hours after administration of the VDZ. 76

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