Methods and reagents for improving cell therapy by creating novel car targeting mr1 protein

Abstract

The present disclosure relates to CAR constructs targeting the major histocompatibility complex class I-related (MR1) protein (MR1-CAR). The disclosed CAR constructs have an antigen recognition domain comprising an anti-MR1 antibody variable region.

Description

[0001] Related applications

[0002] This application is a patent application for invention and claims priority to U.S. Provisional Application No. 63 / 539,343, filed on September 20, 2023, the entire disclosure of which is incorporated herein by reference.

[0003] Reference to the electronically submitted sequence list

[0004] The sequence list, which was submitted electronically with this application in XML file format, is named 2459S_003WO00.xml, created on September 19, 2024, and is 15,863 bytes in size. Its entire contents are incorporated herein by reference. Technical Field

[0005] This disclosure relates to chimeric antigen receptor (CAR) cells and therapies using such cells. Specifically, this disclosure relates to a novel CAR that targets the major histocompatibility complex class I-associated (MR1) protein. Background Technology

[0006] T-cell therapy has shown great potential in the immunotherapy of diseases, particularly cancer, infectious diseases, and autoimmune diseases. One approach to enhance T-cell therapy is to genetically modify T cells using virus-mediated gene transfer to enhance their activity and / or specificity toward desired target cells. For example, expressing chimeric antigen receptors (CARs) on T cells using lentiviruses and retroviruses has shown great potential in cancer treatment. Autologous T cells expressing chimeric antigen receptors (CAR-T cells) have shown significant efficacy in patients with advanced leukemia, lymphoma, and other malignancies. Since 2017, the U.S. Food and Drug Administration has approved several CAR T-cell therapies. Other methods for genetically modifying immune cells are known, such as, but not limited to, electroporation and nanoparticle-based delivery methods.

[0007] As is well known, a typical CAR structure comprises four domains or regions: an antigen recognition or ligand binding domain, a hinge region (also known as the outer domain), a transmembrane domain, and an intracellular signal transduction / activation domain (also known as the inner domain). The antigen recognition domain is exposed outside the cell and contains the sequence that recognizes the antigen. Typically, the variable region of an antibody (usually a monoclonal antibody) serves as the source of the antigen recognition domain. These antigen recognition domains are often linked together to form a single-chain variable fragment (ScFv), which is itself a chimeric protein with both a light chain (VL) and a heavy chain (VH) of an immunoglobulin. The VL and VH chains are linked together by a linker peptide, which provides flexibility for the VL / VH chain to capture the antigen. Other sources of antigen recognition sites have also been considered, including TNF receptors, innate immune receptors, cytokines, structural proteins, and growth factors.

[0008] The CAR hinge structure (sometimes called the spacer region) is located between the antigen recognition domain and the extracellular membrane expressing the CAR. The hinge region provides flexibility to the CAR, and its optimal length can vary depending on the location of the antigen epitope. Typically, proximal membrane epitopes are equipped with long hinge regions. For distal membrane epitopes of the antigen, a short hinge region may be sufficient.

[0009] The CAR transmembrane domain is located between the hinge region and the intracellular signaling domain. The transmembrane domain stabilizes the entire CAR through its hydrophobic α-helical structure, providing a highly expressed and stable receptor, and may play a role in the immune cell effector function of CAR.

[0010] As is well known, when an antigen binds to a CAR antigen recognition site, the CAR receptor aggregates, leading to the transmission of activation signals. Intracellular signal transduction domains receive the activation signals and transmit them into the cell. Activation of certain types of immune cells, such as T cells, also requires the presence of co-stimulatory domains / molecules.

[0011] As is well known, the T cell receptor (TCR) is a member of the immunoglobulin superfamily. Its structure is that of a membrane-anchored heterodimer protein linked by disulfide bonds, comprising an α-chain and a β-chain. A minority of T cells express alternative TCRs containing γ-chains and δ-chains. Each chain contains a variable (V) domain and a constant (C) domain, both belonging to the immunoglobulin superfamily. The C domain is located proximally on the cell membrane, connected to a transmembrane domain and a short cytoplasmic tail. The V domain is located distally on the cell membrane, forming a structure that binds to the peptide / MHC complex. Macroscopically, the TCR can be likened to a "half-antibody" composed of a single heavy chain and a single light chain, but the heavy chain lacks a crystallizable fragment (Fc). When the TCR V domain binds to the antigenic peptide and MHC, the T cell is activated through signal transduction mediated by various enzymes, co-receptors, specific adaptor molecules, and activated or released transcription factors.

[0012] Chimeric antigen receptor T-cell therapy (CAR-T) is considered highly effective in treating malignancies refractory to conventional therapies. Unfortunately, up to 50% of patients relapse or become refractory, and relapsed / refractory patients have a poor prognosis with conventional chemotherapy (e.g., a complete response rate (CR) of 7% and a median overall survival (OS) of 6.3 months). In contrast, CD19 CAR-T therapy has shown significantly improved prognoses (e.g., 1-year CR >50% and 1-year OS >50%). CAR-T therapy utilizes the patient's own T cells, bypassing major histocompatibility complex limitations, redirecting T cells to specific targets without prior sensitization. The remarkable clinical success of autologous CD19 CAR-T cells in relapsed / refractory non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma highlights the potential of genetically engineered T cells. For aggressive, relapsed, or refractory disease, if still sensitive to chemotherapy, high-dose therapy followed by autologous stem cell salvage is employed.

[0013] However, in the absence of clearly defined tumor-specific antigens, CAR-T therapy for other hematologic malignancies and solid tumors has proven exceptionally challenging. To address the lack of specific targeting antigens for CAR-T therapy, we focused on a recently described antigen called MR1—a major histocompatibility complex class I-related gene protein. MR1 is a non-polymorphic surface protein located on chromosome 1 and highly conserved across many species. For a time, it was considered a pseudogene not expressed because the MR1 protein was undetectable on the surface of many experimental cells. However, it was later discovered that MR1 is expressed only on the surface of infected cells that respond to MAIT T cells. More recently, MR1 has also been found to be expressed on the surface of many tumor cell lines but not in healthy cell lines.

[0014] One drawback of MR1 is its significantly low expression level, making it difficult to target using conventional CARs. To address this need, we designed an MR1-CAR by utilizing the variable region of an anti-MR1 antibody as an antigen recognition domain. Using this approach, we found that this MR1-CAR is highly expressed in immune cells and can be redirected to very effectively kill many tumor cells, regardless of variations in MR1 expression levels. We also demonstrated that this MR1-CAR is effective in vivo against highly aggressive acute myeloid leukemia (AML) mouse models and gastric tumor mouse models. This novel MR1-CAR has clinical potential for treating a variety of tumors for which there are currently no effective treatments. Summary of the Invention

[0015] This document sets forth details of one or more embodiments of the subject matter currently disclosed. Modifications to the embodiments and other embodiments described herein will be apparent to those skilled in the art upon review of the information provided herein. The information provided herein, particularly the specific details of the exemplary embodiments described, is provided primarily for clarity of understanding and should not be construed as unnecessarily limiting. In the event of any conflict, the specification (including definitions) of this document shall prevail.

[0016] In accordance with the purposes and benefits described herein, in one aspect of this disclosure, a novel chimeric antigen receptor (MR1-CAR) targeting major histocompatibility complex class I-associated proteins is disclosed. Among a variety of other uses, the novel CAR can be used in a one-step or multi-step process to modify target cells derived from blood (e.g., peripheral blood mononuclear cells, PBMCs) to specificity for a desired target (e.g., tumor cells). Target cells can be any immune cell, including but not limited to T cells, natural killer (NK) cells, monocytes, and other cells described below.

[0017] This disclosure further relates to the creation of MR1-modified immune cells expressing MR1-CAR, which can directly kill tumor cells derived from a variety of malignant tumors both in vitro and in vivo. MR1-CAR can be used to target any desired tumor because the MR1 protein has been found to be preferentially expressed on the cell surface of a variety of tumor cells compared to normal cells.

[0018] In another aspect, this disclosure describes the creation of a CAR that uses variable light chain (VL) and / or variable heavy chain (VH) domains derived from an MR1 antibody, and these domains can be arranged in any orientation.

[0019] In another aspect, MR1-CARs with conventional structures are described. Conventional MR1-CAR structures or platforms include antigen recognition domains containing VL and VH domains of anti-MR1 antibodies, as well as one or more of the following sequences as described above: adapter sequences, hinge domain sequences, transmembrane domain sequences, intracellular signal transduction domain sequences, and one or more co-stimulatory molecule sequences.

[0020] In another aspect, a TCR-based MR1-CAR structure or platform is described, comprising the VL and VH domains of an anti-MR1 antibody spliced ​​onto the constant region domain of the TCR.

[0021] In one possible implementation, MR1 CAR-T cells are created using PBMCs exposed to immune signals such as CD3 and / or CD28 to activate T cells. MR1 CARs can also be expressed in any other immune cell type, such as B cells, NK cells, natural killer T (NKT) cells, macrophages / monocytes, dendritic cells, neutrophils, basophils, eosinophils, regulatory T (T-reg) cells, etc.

[0022] In one possible implementation, the CAR design can introduce mouse or human TCR-α and TCR-β, wherein each VL domain from TCR-α and TCR-β is replaced by an anti-MR1 VL domain and a VH domain, respectively.

[0023] In one possible implementation, the VL and VH domains of the MR1 antibody for MR1 CAR may contain the amino acid sequence shown below, or any nucleotide sequence encoding a similar protein sequence.

[0024] VL domain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGPVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLAQEDIATYFCQQGNTLPYTFGGGTKLEIK (SEQ ID NO: 1)

[0025] VH domain EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMHWVKKRPELGLEWIGRIDPANGNTNFAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCPRGSGNYYFDYWGQGTTLTVSS (SEQ ID NO: 2)

[0026] In another possible implementation, the differences between the VL and VH domains of the MR1 CAR and the amino acid sequence described above can be integer percentages selected from the following: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15%.

[0027] In another possible implementation, the differences between the VL and VH domains of the MR1 CAR and the amino acid sequence described above can be integer percentages selected from the following ranges: 12-15%, 15-20%, and 20-25%.

[0028] In other possible implementations, the CAR may include additional domains to enable it to perform the desired function on immune cells. Depending on the CAR structure (conventional architecture versus TCR-based architecture), the domains may be selected from one or more of the following: a linker, such as G4S (GGGGSGGGGSGGGGS; SEQ ID NO: 3) or its connecting portion, Whitlow (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 4) or its connecting portion separating the VH and VL domains; a hinge domain that allows optimal spacing between the VL / VH domains and the target antigen; a transmembrane domain (e.g., CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 transmembrane domains); and an intracellular signal transduction domain. As described above, in conventional CAR structures, the intracellular signal transduction domain is used to activate CAR-expressing cells. This may include domains derived from CD3zeta and domains derived from at least one or more other co-stimulatory molecules, including but not limited to: OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137) or their functional variants and MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Other examples of such co-stimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, and IL7R. alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA M, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and ligands that specifically bind to CD83 and CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and cytokine receptors or inhibitory molecules such as PD-1, CLTA-4, TIM-3, LAG-3, and TIGIT (Soler, DC et al., J Neurooncol, Jan 2022; 156(1): 81-96). For the TCR-based MR1-CARs described in this disclosure, certain additional regions are not required. For example, TCR-based MR1-CAR-Ts do not require a linker to provide an antigen recognition domain for binding to the target MR1 antigen.

[0029] In another aspect, this disclosure describes a CAR comprising a variable domain derived from an improved form of an antibody that has been humanized and / or engineered to increase affinity for the MR1 protein.

[0030] While the terminology used herein is believed to be familiar to those skilled in the art, certain definitions are provided to facilitate the explanation of the subject matter disclosed herein.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] Unless otherwise stated, all patents, patent applications, publications, GenBank sequences, databases, websites and other published materials mentioned throughout this publication are incorporated herein by reference in their entirety.

[0033] When referring to URLs or other such identifiers or addresses, it should be understood that such identifiers can change, and specific information on the internet may appear or disappear, but equivalent information can be found by searching the internet. This reference demonstrates the availability and public dissemination of such information.

[0034] As used herein, unless otherwise stated, the abbreviations of any protecting groups, amino acids and other compounds are consistent with their common names, recognized abbreviations or the IUPAC-IUB Committee biochemical nomenclature (see Biochem, (1972) 11(9): 1726-1732).

[0035] Although similar or equivalent methods, apparatuses and materials described herein may be used to implement or test the currently disclosed subject matter, representative methods, apparatuses and materials are described herein.

[0036] In some cases, the nucleotides and peptides disclosed herein are included in publicly available databases such as GENBANK. ® And SWISSPROT. Information included in the databases disclosed herein, including sequences and other information relating to such nucleotides and polypeptides, is expressly incorporated herein by reference. Unless otherwise stated or obvious, references to such public databases are the most recent versions available as of the date of filing of this application.

[0037] Unless otherwise stated, all figures used in the specification and claims to indicate the quantity of components, properties such as reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless indicated to the contrary, any numerical parameters described in this specification and claims are approximate values ​​that may vary depending on the desired properties sought to be obtained from the subject matter of this disclosure.

[0038] Unless otherwise expressly stated, wherever phrases such as “for example,” “such as,” “including,” etc., are used, they should be understood as being followed by the phrase “and not limited to.” Similarly, “example,” “exemplary,” etc., should be understood as non-restrictive. The term “substantially” allows deviations from the descriptive phrase that do not affect the intended purpose. Descriptive terms should be understood as being modified by the term “substantially,” even if the word “substantially” is not explicitly stated. Thus, for example, the phrase “where the lever extends vertically” means “where the lever extends substantially vertically,” as long as the lever does not require a precise vertical arrangement to perform its function.

[0039] The terms “comprising,” “including,” “having,” and “involving” (and similar terms “comprises,” “includes,” “has,” and “involves”) are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of “comprising” under U.S. patent law and is therefore interpreted as an open-ended term meaning “at least the following,” and is also interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b, and c. Wherever the terms “a” or “an” are used, they should be understood as “one or more” unless such an interpretation would be absurd in the context. The terms “comprise,” “have,” “include,” and “contain” (and their variations) are open-ended connecting verbs that allow for the addition of other elements when used in claims.

[0040] Unless the context otherwise requires, when used in conjunction with the term "comprising" in the claims or specification, the use of the words "a" or "an" means one or more.

[0041] As used herein, the term “about” when referring to a value or quantity of mass, weight, time, volume, concentration, or percentage is intended to cover variations from the specified amount by ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, ±0.1% in some embodiments, ±0.01% in some embodiments, and ±0.001% in some embodiments, because such variations are suitable for implementing the disclosed method. The term “or” as used in the claims is used to mean “and / or” unless explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive.

[0042] As used herein, a range can be expressed as "about" a particular value and / or "about" another particular value. It should also be understood that multiple values ​​are disclosed herein, and each value is disclosed herein as "about" that particular value, in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0043] As used herein, "optional" or "optionally" means that the event or situation subsequently described occurs or does not occur, and the description includes both scenarios in which the event or situation occurs and scenarios in which it does not occur. For example, an optional variant section means that the section is a variant or a non-variant.

[0044] As used herein, the term "subject" can refer to a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). Therefore, the "subject" disclosed herein can be a human, a non-human primate, a horse, pig, rabbit, dog, sheep, goat, cattle, cat, guinea pig, rodent, or other animal. This term does not indicate a specific age or sex. Therefore, adult and neonatal subjects, as well as fetuses (whether male or female), are intended to be included. In one respect, a subject is a mammal. A patient refers to a subject suffering from a disease or condition. The term "patient" includes both human and veterinary subjects. In some aspects of the methods disclosed herein, the subject has been diagnosed with a condition of uncontrolled cell proliferation, such as cancer, prior to the administration step.

[0045] As used herein, the term “treatment” refers to the medical management of a patient with the aim of curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or symptom, and causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, pathological condition, or symptom. Furthermore, the term includes palliative treatment, i.e., treatment aimed at relieving symptoms rather than curing a disease, pathological condition, or symptom; preventive treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the development of an associated disease, pathological condition, or symptom; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at improving an associated disease, pathological condition, or symptom. In all respects, the term covers any treatment of a subject, including mammals (e.g., humans), and includes: (i) preventing the development of the disease in a subject who may be susceptible but has not yet been diagnosed with the disease; (ii) suppressing the disease, i.e., preventing its development; or (iii) alleviating the disease, i.e., causing the disease to subside.

[0046] As used herein, the terms “prevent” or “preventing” mean to exclude, avoid, eliminate, prevent, stop, or hinder something from happening, especially through preemptive action. It should be understood that, unless otherwise expressly stated, the use of “reduce,” “suppress,” or “prevent” herein also explicitly discloses the use of the other two terms.

[0047] As used herein, the term "diagnosed" means a condition that has been diagnosed or treated by a technician, such as a physician, and is found to be a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, the term "diagnosed with a condition requiring treatment for uncontrolled cell proliferation" means a condition that has been diagnosed by a technician, such as a physician, and is found to have uncontrolled cell proliferation, such as cancer, which can be treated by a variety of therapeutic agents or methods, including but not limited to the compounds disclosed herein and / or the products of the disclosed methods of preparation.

[0048] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. These methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, intranasal administration, topical administration, intravaginal administration, ocular administration, intraocular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable administration such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In each respect, the preparation can be administered therapeutically; that is, for the treatment of an existing disease or condition. In other respects, the preparation can be administered prophylactically; that is, administered to prevent a disease or condition.

[0049] As used herein, the term “contact” means the binding of a disclosed compound or construct to a target (such as a cell, target protein, or other biological entity) in a manner in which the compound or construct can directly or indirectly affect the activity of the target; directly affecting, for example, through interaction with the target itself; or indirectly affecting, i.e., through interaction with another molecule, cofactor, factor, or protein on which the activity of the target depends.

[0050] As used herein, the terms "effective amount" and "amount effective" refer to an amount sufficient to achieve a desired outcome or have an effect on an undesirable condition. For example, an "effective therapeutic amount" is an amount sufficient to achieve a desired therapeutic outcome or have an effect on an undesirable symptom, but generally insufficient to cause adverse side effects. The specific effective therapeutic dose level for any particular patient will depend on a variety of factors, including the severity of the disease and condition being treated; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration; the route of administration; the excretion rate of the specific compound used; the duration of treatment; the combination with the specific compound used or incidentally used drugs; and similar factors well known in the medical field. For example, it is well known to those skilled in the art to start administration at a dose level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. If necessary, the effective daily dose may be divided into multiple doses for ease of administration. Thus, a single-dose composition may contain such amounts or approximations thereof constituting the daily dose. If any contraindications occur, the dose may be adjusted by an individual physician. Doses may vary and may be administered once or multiple times daily for one or several days. Appropriate dosage guidelines for a given class of medicines can be found in the literature. In other respects, preparations can be administered at an "effective preventive dose"; that is, a dose that effectively prevents disease or ailment.

[0051] As used herein, a “kit” refers to a collection of at least two components that constitute a kit. These components together form a functional unit for a specific purpose. Individual component members may be physically packaged together or packaged separately. For example, a kit containing instructions for use may or may not physically include instructions for use along with other individual component members. Conversely, the instructions for use may be provided as a separate component, or in print or electronic form (available on a computer-readable storage device or downloadable from an internet website), or as a recorded presentation.

[0052] As used herein, “instruction manual” refers to a document describing the materials or methods associated with the kit. These materials may include any combination of the following: background information, a list of components and information on their availability (purchase information, etc.), a brief or detailed protocol for using the kit, troubleshooting, references, technical support, and any other relevant documents. The instruction manual may be provided with the kit or as a separate component, and may be in print or electronic form (available on computer-readable storage devices or downloadable from the internet), or as a recorded presentation. The instruction manual may consist of one or more documents and may include future updates.

[0053] As used herein, the term "therapeutic agent" includes any synthetic or naturally occurring biologically active compound or composition of substances that, when administered to an organism (human or non-human animal), induce the desired pharmacological, immunogenic, and / or physiological effects through local and / or systemic action. Therefore, the term encompasses those compounds or chemicals traditionally considered as drugs, vaccines, and biological agents, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, etc. Examples of therapeutic agents are described in prominent references such as the Merck Index (14th edition), Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and therapeutic agents include, but are not limited to, drugs; vitamins; mineral supplements; substances intended to treat, prevent, diagnose, cure, or alleviate a disease or symptom; substances or prodrugs that affect the structure or function of the body and become biologically active or more active when placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions used in all major therapeutic areas, including but not limited to adjuvants; anti-infectives such as antibiotics and antiviral agents; analgesics and combinations of analgesics, anorexia nervosa, anti-inflammatory agents, antiepileptics, local and general anesthetics, hypnotics, sedatives, antipsychotics, psychotropics, antidepressants, anxiolytics, antagonists, neuronal blocking agents, anticholinergics and cholinergics, antimuscarinic and muscarinic drugs, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritis drugs, antiasthmatics, anticonvulsants, antihistamines, antiemetics, antitumor drugs, antipruritics, antipyretics, anticonvulsants, cardiovascular agents (including calcium channel blockers and beta-blockers) β-receptor agonists and antiarrhythmic drugs, antihypertensive drugs, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic reagents; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressants; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides and their fragments (whether naturally occurring, chemically synthesized or recombinant); and nucleic acid molecules (polymers of two or more nucleotides, whether ribonucleotides (RNA) or deoxyribonucleotides (DNA) (including double-stranded and single-stranded molecules), gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin) and other bioactive macromolecules, such as proteins and enzymes. This reagent can be a bioactive agent used in medicine (including veterinary medicine) and agriculture (e.g., plants) and other fields. The term therapeutic agent also includes, but is not limited to, drugs; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure or alleviate a disease or symptom; or substances that affect the structure or function of the body; or prodrugs that become biologically active or have enhanced activity when placed in a predetermined physiological environment.

[0054] The term "pharmaceutically acceptable" describes a substance that has no undesirable properties in biological or other respects, meaning it will not cause undesirable biological effects at unacceptable levels, nor will it interact with substances in a harmful manner.

[0055] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders intended for reconstitution into sterile injectable solutions or dispersions prior to use.

[0056] Unless otherwise expressly stated, no method described herein should be construed as requiring its steps to be performed in a particular order. Therefore, no inference is made in any respect of the order in which the method claims do not actually state the order of their steps, or where the claims or description do not specifically restrict these steps to a particular order. This applies to any possible non-express basis of interpretation, including: logical problems of the arrangement of steps or operational procedures; simple meanings derived from grammatical organization or punctuation; and the number or type of embodiments described in the description.

[0057] It should be understood that various details of the subject matter may be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing descriptions are for illustrative purposes only, not for limiting purposes. Attached Figure Description

[0058] The subject matter disclosed herein will be better understood in conjunction with the detailed description below, and features, aspects, and advantages beyond those described above will become apparent. This detailed description refers to the following figures, in which:

[0059] Figure 1 A compared the mechanisms of expression on the surface of classical MHC and MR1 cells.

[0060] Figure 1 B shows the expression of MR1 in various tumorigenic and non-tumorigenic cell lines, expressed as the percentage (%) of surface MR1.

[0061] Figure 2 A illustrates the difference between a conventional CAR-T architecture and a TCR-based CAR-T architecture according to this disclosure.

[0062] Figure 2 B illustrates the TCR-based MR1-CAR-T architecture according to this disclosure.

[0063] Figure 2 C graphically illustrates the virus-transduced T cells. Figure 2 B shows the high expression level of TCR-based MR1-CAR-T.

[0064] Figure 2 D shows Figure 2 B shows the high cell expansion of TCR-based MR1-CAR-T cells after transduction.

[0065] Figure 3 A graphically illustrates the effective targeting and killing of various tumor cell lines in vitro by TCR-based MR1-CAR-T according to this disclosure.

[0066] Figure 3 B demonstrates the specificity of the TCR-based MR1-CAR-T for parental AML cell lines compared to AML cell lines lacking b2-microglobulin (KO) expression, according to this disclosure.

[0067] Figure 3 C shows TCR-based MR1-CAR-T-induced degranulation in several cell lines, measured by CD107a expression levels.

[0068] Figure 3 D shows the effects of several cell types on the induction of TCR-based MR1-CAR-T-induced interferon-γ compared to untransduced cells.

[0069] Figure 4 A describes the architecture of a conventional MR1-CAR-T.

[0070] Figure 4 B shows Figure 4 A shows the efficacy of conventional MR1-CAR-T against AML, PC, and GBM cell lines.

[0071] Figure 5 A shows that in an in vivo AML tumor model (Molm13), mice treated with TCR-based MR1-CAR-T cells experienced reduced tumor growth over 4 weeks compared to mice treated with control T-cell therapy and untreated mice.

[0072] Figure 5 B shows that in an in vivo AML tumor model, mice treated with TCR-based MR1-CAR-T therapy according to this disclosure had improved survival rates compared with mice treated with control T-cell therapy and untreated mice.

[0073] Figure 5 C shows that, compared with mice receiving control T-cell therapy and untreated mice, the clearance of CD33+ cells in the blood of mice receiving TCR-based MR1-CAR-T therapy according to this disclosure was improved.

[0074] Figure 6The tumor volume of mice treated with TCR-based MR1-CAR-T according to this disclosure is compared with that of mice treated with vector or untransduced T cells in an in vivo gastric tumor model (SNU-16 implantation) in a graphical manner.

[0075] Figure 7 The killing efficiency of the TCR-based MR1-CAR-T implementation according to this disclosure on Molm13, HL-60, Panc1 and U87jos cell lines is shown graphically.

[0076] Figure 8 A graphically compares the killing efficiency of TCR-based MR1-CAR-T and conventional MR1-CAR-T implementations against the brain tumor cell line (U87).

[0077] Figure 8 A graphically compares the killing efficiency of TCR-based MR1-CAR-T and conventional MR1-CAR-T implementations against the pancreatic tumor cell line (Panc1).

[0078] While this disclosure is readily adaptable to various modifications and alternatives, specific embodiments thereof have been illustrated by way of example in the accompanying drawings and are described in detail below. However, it should be understood that the description of specific embodiments is not intended to limit this disclosure to all modifications, equivalents, and substitutions that cover the spirit and scope of this disclosure as defined by the appended claims. Detailed Implementation

[0079] This document sets forth details of one or more embodiments of the currently disclosed subject matter. Modifications to the embodiments and other embodiments described herein will be apparent to those skilled in the art upon review of the information provided herein. The information provided herein, particularly the specific details of the exemplary embodiments described, is provided primarily for clarity and should not be construed as unnecessarily limiting. In the event of any conflict, the specification (including definitions) of this document shall prevail.

[0080] To address the aforementioned and other drawbacks of existing CAR-T therapies, we demonstrated MR1 expression in several tumor cell lines and designed several novel MR1-CARs, including CARs with a conventional CAR architecture (conventional MR1-CARs) and CARs utilizing the T-cell receptor (TCR) mechanism (TCR-based MR1-CARs). The PBMCs used in this study were derived from healthy donors. For functional evaluation experiments of MR1-CAR-T, T cells were generated from healthy donors and tested in vitro and in vivo against AML and GI cell line models, as detailed below. Materials and Methods DNA construct

[0081] In general, in order to develop the disclosed TCR-based MR1-CAR, the variable region of the TCR is combined with the light and heavy chains (VL, VH) of the MR1 antibody within the framework (Chua WJ et al., J Immunol. 2011 Apr 15; 186(8):4744–4750. doi:10.4049; Liu Y et al., Sci Transl Med. 2021 Mar 24;13(586):eabb5191). Conventional MR1 CAR constructs contain a CD28 transmembrane domain and a CD28 costimulatory domain (Chua WJ et al., J Immunol. 2011 Apr 15; 186(8): 4744–4750; Wang D et al., Sci Transl Med. 2020 Mar 4; 12(533); Soler DC et al., J Neurooncol. 2022 Jan; 156(1):81-96).

[0082] Example 1. In one embodiment referred to as MR4 (see...) Figure 2 B), the TCR-based MR1CAR construct according to this disclosure comprises mouse TCR-α (SEQ ID NO:8) and mouse TCR-β (SEQ ID NO:6), wherein the VL domains of TCR-α and TCR-β are replaced by the VL domain (SEQ ID NO:1) and VH domain (SEQ ID NO:2) of a mouse anti-MR1 antibody, respectively. The construct also comprises a CD8 peptide signaling sequence (SEQ ID NO:5) and a furin P2A self-cleaving peptide separating the TCR-VH and TCR-VL splice sequences. As is well known, the furin site ensures that the polypeptide is cleaved by endogenous cellular proteases, resulting in two independent proteins (TCR-a and TCR-b). P2A is a self-cleaving sequence, further ensuring cleavage between these two distinct proteins. Combining furin and P2A ensures successful cleavage, resulting in two independent chains: TCR-α and TCR-β. It should be understood that the construct can be modified by using human TCR-α and TCR-β sequences and replacing them with the VL domain (SEQ ID NO:1) and VH domain (SEQ ID NO:2) of mouse anti-MR1 antibody or other non-human anti-MR1 antibody to provide a humanized CAR sequence.

[0083] Example 2. In another possible embodiment, referred to as MR3.11, the conventional MR1 CAR construct according to this disclosure comprises a CD8 peptide signaling sequence (SEQ ID NO:5), a VL domain sequence (SEQ ID NO:1), and a VH domain sequence (SEQ ID NO:2). The construct also comprises two linker sequences (SEQ ID NO:3 between VH and VL, and GGG between the CD28 co-stimulatory domain and the CD3 TCR-zeta domain) and an IgG4 hinge sequence (SEQ ID NO:10). The construct further comprises a CD28 transmembrane domain sequence (SEQ ID NO:11) and a CD28 co-stimulatory sequence (SEQ ID NO:12). The construct also comprises a CD3 TCR-zeta chain sequence (SEQ ID NO:13) and may optionally include a V5 tag (SEQ ID NO:9) for confirmation of successful detection of the construct.

[0084] Example 3. In another possible embodiment, referred to as MR3.12, the conventional MR1 CAR construct according to this disclosure comprises a CD8 peptide signaling sequence (SEQ ID NO:5), a VL domain sequence (SEQ ID NO:1), and a VH domain sequence (SEQ ID NO:2). The construct also comprises two linker sequences (SEQ ID NO:3 between VH and VL, and GGG between the 4-1BB co-stimulatory domain and the CD3 TCR-zeta domain) and an IgG4 hinge sequence (SEQ ID NO:10). The construct further comprises a CD4 transmembrane domain sequence (SEQ ID NO:14) and a 4-1BB co-stimulatory sequence (SEQ ID NO:15), and may optionally include a V5 tag (SEQ ID NO:9) for confirmation of successful detection of the construct.

[0085] Example 4. In another possible embodiment, referred to as MR3.13, the conventional MR1 CAR construct according to this disclosure comprises a CD8 peptide signaling sequence (SEQ ID NO:5), a VL domain sequence (SEQ ID NO:1), and a VH domain sequence (SEQ ID NO:2). The construct also comprises two adapter sequences (SEQ ID NO:3 between VH and VL, and GGG between the CD28 co-stimulatory domain and the CD3 TCR-zeta domain) and a CD8 stem sequence (SEQ ID NO:16) provided as a “stem” or spacer, i.e., a very long extracellular adapter in this case, capable of annealing to other expressed CAR-T chains, forming a so-called dimer. Sometimes, a long “stem” is required for a CAR-T, while in other cases, a short “stem” is required. The dimer between the two CAR-Ts (the dimer is essentially two CAR-T chains close to each other) ensures target binding and enhances intracellular activation signaling. The construct also includes a CD8 stem sequence (SEQ ID NO:16), a CD28 transmembrane domain sequence (SEQ ID NO:11), and a CD28 co-stimulatory sequence (SEQ ID NO:12), provided as a short stem or spacer linker sequence. The construct also includes a CD3 TCR-zeta strand sequence (SEQ ID NO:13) and may optionally include a V5 tag (SEQ ID NO:9) for confirmation of successful detection of the construct.

[0086] Example 5. In another possible embodiment, referred to as MR3.14, the conventional MR1 CAR construct according to this disclosure comprises a CD8 peptide signaling sequence (SEQ ID NO:5), a VL domain sequence (SEQ ID NO:1), and a VH domain sequence (SEQ ID NO:2). The construct also comprises two adapter sequences (SEQ ID NO:3 between VH and VL, and GGG between the 4-1BB co-stimulatory domain and the CD3 TCR-zeta domain) and a CD8 stem cell sequence (SEQ ID NO:16). The construct also comprises a CD4 transmembrane domain sequence (SEQ ID NO:14) and a 4-1BB co-stimulatory sequence (SEQ ID NO:15). The construct also comprises a CD3 TCR-zeta chain sequence (SEQ ID NO:13) and may optionally include a V5 tag (SEQ ID NO:9) for confirmation of successful detection of the construct. Lentiviral production

[0087] For lentivirus production, 293T cells (ATCC) were transfected with Trans-TI (Mirus, Madison, Wisconsin). The medium was replaced after 24 hours with 4 mM sodium butyrate, and all medium was collected after 48 hours. Viral particles were filtered through a 0.45 μm filter and purified using a lentivirus concentrator centrifuge (Cellecta, Mountain View, California) or by ultracentrifugation at 98 kg for 2 hours using a Beckman Coulter, following the manufacturer's instructions. The viral particles were resuspended in T cell culture medium and immediately frozen at -80°C until use. CAR-T cell production

[0088] PBMCs were obtained from healthy donor blood samples, purified using a Ficoll gradient, and monocytes were removed by adhering to 10-cm culture dishes at a density of 5 M / mL for 2 hours. T cells were then activated for 48 hours using Trans Act (Miltneyi, Gaithersburg, MD) according to the manufacturer's instructions. Cells were exposed to lentivirus for 24 hours and expanded for 7 days, with the culture medium (RPMI, 10% FBS, and penicillin-streptomycin) changed every 2-3 days. Cytotoxicity assay

[0089] For lactate dehydrogenase (LDH) assay (Takara, San Jose, California), 25 × 10⁻⁶ μg / mL of the enzyme was used. 3 Target cancer cells were seeded in 96-well plates containing 150 μL of culture medium (RPMI, 10% FBS, and penicillin-streptomycin). MR1-CAR-T cells were added at effector cell:target cell (E:T) ratios of 1:1, 5:1, and 10:1, and incubated overnight at 37°C. The next day, LDH analysis was performed with a slight modification: 50 μL of cell supernatant was mixed with 50 μL of HBSS, and 100 μL of the LDH mixture was added. After incubation for 9–11 minutes, the results were read at 450 nm absorbance using a Victor 300X spectrophotometer. The percentage of cell kill was calculated according to the manufacturer's protocol. result MR1 is expressed at low levels in several tumor cell lines, making it difficult to detect.

[0090] MR1 differs from classical MHC in that it is singlet, highly conserved across species, and presents a non-protein metabolite compared to classical MHC, which presents a peptide. Both classical MHC and MR1 require expression of β2 microglobulin to achieve optimal surface expression in cells (see [link to relevant documentation]). Figure 1A). MR1 expression in several tumor and non-tumor cell lines was assessed by flow cytometry (Attune NX, Thermo Fisher Scientific). One million cells were stained with 1–2 μL of MR1 antibody in a 50 μL solution for 15 minutes. Cells were washed three times with 1 mL of washing buffer, resuspended in 100 μL of staining solution, and analyzed by flow cytometry. Figure 1 As shown in B, when quantified as a percentage of surface MR1, MR1 expression is relatively high, but does not reach 100% due to the low presence of antigen on the surface of tumor cells. TCR-based MR1-CAR-T cells are expressed at high levels in T cells.

[0091] Compared to TCR-based CAR-T, the architecture of conventional CAR-T is as follows: Figure 2 As shown in A. To create the disclosed unconventional TCR-based MR1-CAR-T, the variable region of the TCR is replaced with the heavy and light chains of the anti-MR1 antibody (…). Figure 2 B). The mouse constant region of the TCR is used to avoid mismatch with endogenous human TCR. The novel TCR-based MR1-CAR-T cells showed very high expression efficiency in virus-transduced T cells compared to untransduced T cells. Figure 2 C), and was shown to undergo robust amplification after transduction (see C). Figure 2 D). TCR-based MR1-CAR-T cell lines that kill tumor cells are not affected by MR1 expression levels.

[0092] Using calcein-Am and LDH analysis, we tested the killing efficacy of the novel TCR-based MR1-CAR-T cell line from Example 1 against a variety of different tumor cell lines. Figure 3 As shown in Figure A, TCR-based MR1-CAR-T cells can effectively kill various tumor cell lines, including resistant glioblastoma multiforme (GBM) and pancreatic cancer (PC) cell lines. TCR-based MR1-CAR-T cells also exhibit specificity, as AML cell lines lacking b2-microglobulin (b2M—as mentioned above, is essential for MR1 surface expression) are not killed by TCR-based MR1-CAR-T cells compared to parental cell lines. Figure 3 E).

[0093] When exposed to several tumor cell lines, the novel TCR-based MR1-CAR-T also induced degranulation, as measured by CD107a expression (3 μL of CD107-PE antibody [R&D # IC4800P] was incubated overnight with a mixture of effector and target cells. After 24 hours, cells were collected, washed with FACS buffer, and analyzed by flow cytometry using an Attune or Aria-SORP flow cytometer). Figure 3 F), and release INF-γ (using the R&D INF-γ ELISA kit according to the manufacturer's recommended protocol). Figure 3 G). INF-γ is a known cytotoxic factor. TCR-based MR1-CAR-T cells showed superior efficacy compared to conventional MR1-CAR-T cells in vitro.

[0094] We also examined the cytotoxic effects of conventional MR1-CAR-T cells against AML, PC, and GBM cell lines. Figure 3 Compared to TCR-based MR1-CAR-T cells tested in the study, conventional MR1-CAR-T cells were effective, but their efficacy against PC and GBM cell lines was lower. Figure 4 ). Figure 8 AB illustrates a direct comparison of the efficacy of each CAR-T cell type against PC and GBM cell lines. As shown in the figure, while each CAR-T cell type was effective, cells expressing TCR-based MR1-CAR-T were significantly more effective at killing glioblastoma and pancreatic tumor cells than cells expressing conventional MR1-CAR-T. In in vivo AML tumor models, TCR-based MR1-CAR-T therapy demonstrated potent tumor control.

[0095] Next, we evaluated the efficacy of TCR-based MR1-CAR-T in vivo in an AML tumor model. The model used was Molm-13, a known AML tumor model. Molm-13 is a well-known mononuclear cell-derived liquid carcinoma that causes circulatory disease in mice. Molm-13 expresses the luciferase gene, so its presence can be detected by bioluminescence imaging. Untreated mice that received Molm13 cell injections died within 23 days, while mice treated with control T cells (T cells lacking genetic modification but otherwise purified and expanded from the same donor in the same manner as MR1-CAR-T cells) had slightly lower survival rates. Mice injected with Molm13 and treated with TCR-based MR1-CAR-T had 100% survival for 40 days (see [link to relevant documentation]). Figure 5B). Blood samples collected from untreated mice showed the presence of malignant human CD33+ Molm13 cells, while mice treated with TCR-based MR1-CAR-T therapy showed complete clearance of human CD33+ cells (AML cells were undetectable in the blood). Figure 5 C). MR1-CAR-T cells showed activity against MR1 gastric tumor models in vivo.

[0096] We also evaluated the efficacy of TCR-based MR1-CAR-T cells in an in vivo mouse model of gastric tumors, in which mice were injected with SNU-16 cells. Eight million SNU16 cells were subcutaneously injected into each side of the mouse abdomen. Tumor growth was assessed after 11 days, with sizes ranging from 100 to 300 mm. 3 Between [dates missing]. On day 11, 3 million MR1-CAR-T cells were injected into mice via tail injection. Tumor growth was monitored for 41 days. Tumor size was assessed using calipers to provide three-dimensional measurements: height, width, and depth. Figure 6 As shown, tumors grew significantly in mice treated with untransduced T cells compared to mice treated with only the vector (without T cells). However, mice treated with TCR-based MR1-CAR-T therapy were able to control tumor growth for up to 44 days. discuss

[0097] This study demonstrates that, despite the relatively low levels of MR1 surface protein on cell surfaces, the unconventional TCR-based MR1-CAR-T disclosed herein can very effectively target MR1 surface protein present in a variety of tumor cell lines. Therefore, this novel TCR-based MR1-CAR-T can effectively disrupt a variety of tumor cells in vitro. To support our in vitro results, we investigated the potential of TCR-based MR1-CAR-T to inhibit the growth of two tumor types (AML and gastric tumors) using in vivo mouse tumor models. In the in vivo model, TCR-based MR1-CAR-T effectively reduced tumor growth / volume and improved mouse survival. MR1-CARs based on conventional CAR architectures were also shown to effectively target the MR1 cell surface protein on tumor cell lines.

[0098] Therefore, this disclosure provides a novel MR1-CAR that, when expressed by suitable immune cells, can target and kill multiple types of cancerous tumor cells in vivo and in vitro. In fact, those skilled in the art will understand that the disclosed novel MR1-CAR is expected to be effective and therefore usable for treating any type of cancer cell expressing a certain level of cell surface MR1, including but not limited to cells from the following sources: lung cancer, renal cancer, kidney cancer, uterine cancer, colorectal cancer, cervical cancer, colon cancer, gastric cancer, head and neck cancer, and all hematologic malignancies, including lymphoma.

[0099] It should be understood that various details of the currently disclosed subject matter may be altered without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for illustrative purposes only and not for limitation. Based on the foregoing teachings, obvious modifications and variations are possible. All such modifications and variations, when interpreted with due fairness, legality and impartiality, should be covered within the scope of the appended claims.

Claims

1. A major histocompatibility complex class I-related (MR1) chimeric antigen receptor (MR1-CAR) having an antigen recognition domain comprising one or both of an anti-MR1 antibody VL domain and an anti-MR1 antibody VH domain.

2. The MR1-CAR according to claim 1, wherein the VL domain comprises the sequence shown in SEQ ID NO:1, or comprises a sequence having at least 85% identity with SEQ ID NO:

1.

3. The MR1-CAR according to claim 2, wherein the VH domain comprises a sequence having SEQ ID NO:2, or comprises a sequence having at least 85% identity with SEQ ID NO:

2.

4. The MR1-CAR of claim 3, comprising an antigen receptor constant domain sequence derived from a T cell receptor (TCR) sequence to provide a TCR-based MR1-CAR architecture.

5. The MR1-CAR of claim 4, wherein the TCR-based MR1-CAR sequence comprises TCR-α and TCR-β sequences, each of the TCR-α and TCR-β sequences having a variable domain portion respectively replaced by the VL domain of the anti-MR1 antibody and the VH domain of the anti-MR1 antibody to provide an antigen recognition domain.

6. The MR1-CAR of claim 5, wherein the TCR-α sequence comprises SEQ ID NO:8, or comprises a sequence having at least 85% identity with SEQ ID NO:

8.

7. The MR1-CAR of claim 5, wherein the TCR-β sequence comprises SEQ ID NO:6, or comprises a sequence having at least 85% identity with SEQ ID NO:

6.

8. The MR1-CAR of claim 1 further comprises an additional domain selected from: at least one adapter sequence connecting the VL domain and the VH domain, a hinge region, a transmembrane domain, a signal peptide sequence, an IgG4 or CD8 stem sequence, a TCR-zeta sequence, and one or more co-stimulatory domains to provide a conventional MR1-CAR architecture.

9. The MR1-CAR of claim 8, wherein the at least one connector sequence comprises one or more of SEQ ID NO:3, SEQ ID NO:4 and GGG, or comprises at least one connector sequence having at least 85% identity with it.

10. The MR1-CAR according to claim 8, wherein the transmembrane domain is selected from: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154 transmembrane domains.

11. The MR1-CAR of claim 10, wherein the transmembrane domain sequence comprises one or more of SEQ ID NO:11 and SEQ ID NO:14, or comprises a sequence having at least 85% identity with it.

12. The MR1-CAR of claim 8, wherein the one or more co-stimulatory sequences are selected from one or more of the following: CD3-zeta, OX40, CD27, CD28, CD5, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), ICOS (CD278), 4-1BB (CD137) or functional variants thereof, BTLA, Toll ligand receptor, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD 11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and ligands that specifically bind to CD83 and CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, LFA-1, CD2, CD5, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, cytokine receptors or inhibitory molecules, PD-1, CLTA-4, TIM-3, LAG-3, and TIGIT.

13. The MR1-CAR of claim 12, wherein the one or more co-stimulatory sequences comprise one or more of SEQ ID NO:12 and SEQ ID NO:15, or comprise a sequence having at least 85% identity with them.

14. The MR1-CAR of claim 8, wherein the signal peptide sequence comprises SEQ ID NO:5, or comprises a sequence having at least 85% identity with it.

15. The MR1-CAR of claim 8, wherein the IgG4 sequence comprises SEQ ID NO:10, or comprises a sequence having at least 85% identity with it.

16. The MR1-CAR of claim 8, wherein the stem sequence comprises SEQ ID NO:16, or comprises a sequence having at least 85% identity with it.

17. The MR1-CAR of claim 8, wherein the TCR-zeta chain sequence comprises SEQ ID NO:13, or comprises a sequence having at least 85% identity with it.

18. A cytolytic / cytotoxic immune cell modified to express the MR1-CAR of claim 1, wherein the cytolytic immune cell is selected from: T cells, natural killer (NK) cells, and natural killer T cells (NKT).

19. The modified cytolytic / cytotoxic immune cell according to claim 18, wherein the cytolytic immune cell is a T cell or an NK cell.

20. The modified cytolytic / cytotoxic immune cells according to claim 19, wherein the T cells or the NK cells are derived from the peripheral blood mononuclear cell (PBMC) population.

21. A pharmaceutical composition comprising a plurality of modified cytolytic immune cells as described in claim 18, and one or more pharmaceutically or veterinarily acceptable carriers, diluents, adjuvants, and excipients.

22. The pharmaceutical composition of claim 21, formulated for treating a disease or condition characterized by uncontrolled cell proliferation.

23. The pharmaceutical composition of claim 22, wherein the disease or condition is cancer characterized by the expression of MR1 on the surface of cancer cells.

24. The pharmaceutical composition according to claim 23, wherein the cancer is selected from brain cancer, pancreatic cancer, leukemia, gastric cancer, breast cancer, melanoma, prostate cancer, and myeloma.

25. The pharmaceutical composition according to claim 24, wherein the gastric cancer is selected from epithelial carcinoma and gastric adenocarcinoma, and the leukemia is acute myeloid leukemia.

26. A method of treating a disease or condition characterized by uncontrolled cell proliferation, comprising the step of administering an effective therapeutic amount of the pharmaceutical composition of claim 21 to a subject in need of such treatment.

27. The method of claim 26, wherein the disease or symptom is cancer characterized by the expression of MR1 on the surface of cancer cells.

28. The method of claim 27, wherein the cancer is selected from brain cancer, pancreatic cancer, leukemia, gastric cancer, breast cancer, melanoma, prostate cancer, and myeloma.

29. The method of claim 28, wherein the gastric cancer is selected from epithelial carcinoma and gastric adenocarcinoma, and the leukemia is acute myeloid leukemia.

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