WO2025064828A1 - Methods and reagents to improve cell therapy with the creation of novel car-targeting mr1 protein - Google Patents

Abstract

The present disclosure is directed to CAR constructs (MR1-CAR) that target Major Histocompatibility complex, class I-related (MR1) protein. The disclosed CAR constructs have an antigen recognition domain including the variable regions of an anti-MRl antibody.

Description

METHODS AND REAGENTS TO IMPROVE CELL THERAPY WITH THE CREATION OF NOVEL CAR-TARGETING MR1 PROTEIN

RELATED APPLICATIONS

This is a utility patent application claiming the benefit of priority in U.S. Provisional Application Serial No. 63/539,343 filed September 20, 2023, the entire disclosure of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] A sequence listing electronically submitted with the present application as an XML file named 2459S_003WO00.xml, created on 9-19-2024 and having a size of 15863 bytes, is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0003] This disclosure relates to Chimeric Antigen Receptor (CAR) cells and to therapies employing such cells. In particular, the disclosure is directed to a novel CAR that targets Major Histocompatibility complex, class I-related (MR1) protein.

BACKGROUND

[0004] T cell therapy has shown enormous potential in the immunotherapeutic treatment of diseases, particularly cancer, infectious diseases and autoimmune diseases. One method to enhance T cell therapy is to genetically modify T cells using viral mediated gene transfer to enhance their activity and/or specificity to the desired target cells. For example, the expression of chimeric antigen receptors (CARs) on T cells using lentivirus and retrovirus has shown enormous potential in cancer therapy. Autologous T cells that express a chimeric antigen receptor (CAR-T cells) have exhibited significant efficacy in patients with advanced leukemia, lymphomas, and other malignancies. A number of CAR T-cell therapies have been approved by the U.S. Food and Drug Administration since 2017. Other methods of genetically modifying immune cells are known, for example and without intending any limitation electroporation methods and nanoparticle delivery-based methods.

[0005] As is known, a conventional CAR structure comprises four domains or regions, in particular an antigen recognition or ligand binding domain, a hinge region (also referred to as an ectodomain), a transmembrane domain, and an intracellular signaling/activation domain (also referred to as an endodomain). The antigen recognition domain is exposed to the exterior of a cell and comprises sequences which recognize antigens. Conventionally, the variable region of an antibody (typically a monoclonal antibody) is used as a source for the antigen recognition domain. The antigen recognition domain is conventionally linked together as a single chain of variable fragments (ScFv) which is itself a chimeric protein having light (VL) and heavy (VH) chains of immunoglobulins. The VL and VH chains are linked together by linker peptides which provide flexibility for capture of antigen by the VL/VH chains. Other sources of antigen recognition sites have been considered, including TNF receptors, innate immune receptors, cytokines, structure proteins, and growth factors.

[0006] The CAR hinge structure (sometimes called a spacer) is positioned between the antigen recognition domain and the outer membrane of a cell expressing the CAR. The hinge region provides flexibility to the CAR, and the optimal length of the CAR hinge region may vary according to the position of an antigen epitope. Conventionally, long hinge regions are provided for membrane-proximal epitopes. Short hinge regions may suffice for membrane-distal epitopes of an antigen.

[0007] The CAR transmembrane domain is positioned between the hinge region and the intracellular signaling domain. The transmembrane domain stabilized the complete CAR through a hydrophobic alpha-helix structure providing a highly expressed and stable receptor, and may play a role in CAR-immune cell effector function. [0008] As is known, when antigen binds to the CAR antigen recognition site, CAR receptors cluster together resulting in transmission of an activation signal. The intracellular signaling domain receives the activation signal and transmits it to the cell interior. Activation of certain types of immune cells such as T-cells also require the presence of co-stimulatory domains/molecules.

[0009] The T-cell receptor (TCR) structure is as is known a member of the immunoglobulin superfamily, and is a disulfide-linked membrane-anchored heterodimeric protein comprising an alpha and a beta chain. A minority of T cells express an alternate TCR comprising gamma and delta chains. Each chain comprises a variable (V) domain and a constant (C) domain, both of the immunoglobulin superfamily. The C domain is proximal to the cell membrane, and connected to a transmembrane domain and a short cytoplasmic tail. The V domain is distal to the cell membrane, and forms the structure binding to a peptide/MHC complex. The TCR can at a high level be analogized to a “half-antibody” consisting of a single heavy chain and a single light chain, except that the heavy chain lacks the crystallizable fraction (Fc). When the TCR V domain engages with antigenic peptide and MHC, the T-cell is activated via signal transduction mediated by various enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0010] Chimeric Antigen Receptor T cells (CAR-T) are acknowledged as very effective in treating malignancies refractory to conventional therapies. Unfortunately, up to 50% of patients relapse or become refractory and the outcomes of the relapsed/refractory patients are poor with traditional chemotherapy (ex. 7% complete response rate (CR) and 6.3 month median overall survival (OS). CD 19 CAR-T therapies in contrast have demonstrated significantly improved outcomes (ex. >50% CR and >50% OS at 1 year) in these patients. CAR-T therapy harnesses the patient’s own T-cells and bypasses the major histocompatibility complex restriction to redirect the T-cells towards specific targets without needed priming. The potential of genetically engineered T cells is highlighted by the remarkable clinical success of autologous CD19 CAR-T cells in relapsed/refractory Non-Hodgkin lymphomas (NHL) and acute lymphoid leukemia (ALL), and mantle cell lymphomas. Aggressive, relapsed, or refractory disease is treated with high dose therapy followed by autologous stem cell rescue if the disease is still chemotherapy sensitive. [0011] However, implementation of CAR-T therapy for other blood malignancies and solid tumors without clear tumor-specific antigens has proven very challenging. In order to address the lack of specific antigens to target using CAR-T, we have focused on a recently described antigen called MR1 - Major histocompatibility complex class I-related gene protein. MR1 is a non- polymorphic surface protein, located in chromosome 1 and highly conserved across many species. For some time it was thought to be a pseudogene without expression, since the MR1 protein could not be detected in the surface of many cells tested. However, MR1 was subsequently found to be expressed solely in the surface of infected cells that react to MAIT T- cells. More recently MR1 was also found to be expressed in the surface of many tumor cell lines but not healthy ones.

[0012] One of the disadvantages of MR1 is its apparent low expression level which makes it difficult for targeting using conventional CAR. To address this need, we have engineered an MR1-CAR using the variable regions of an anti-MRl antibody as the antigen recognition domain. Using this approach, we have found that this MR1-CAR expresses at high levels in immune cells and can redirect them to kill many tumor cells very efficiently regardless of varying levels of MR1 expression. We have also shown that this MR1-CAR is effective in vivo against a very aggressive Acute Myeloid Leukemia (AML) murine model as well as against a gastric tumor murine model. The described novel MR1-CAR finds utility in clinical use to treat a wide array of tumors for which treatments are not yet available.

SUMMARY

[0013] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0014] In accordance with the purposes and benefits described herein, in one aspect of the disclosure novel Major histocompatibility complex class I-related protein-targeting Chimeric Antigen Receptors (MR1-CAR) are disclosed. The novel CARs find utility in, among multiple other uses, a single-step or multi-step process for engineering cells of interest derived from blood, such as Peripheral Blood Mononuclear Cells (PBMC), for specificity to desired targets such as tumor cells. The cells of interest can be any immune cells including but not limited to T cells, natural killer (NK) cells, monocytes, and others as will be described.

[0015] The present disclosure further relates to the creation of MR1 -modified immune cells expressing MR1-CAR that can directly kill tumor cells derived from a broad range of malignancies in vitro as well as in vivo. The MR1-CAR can be used to target any desired tumor as MR1 protein has been found to be expressed preferentially on the cell surface of a wide variety of tumor cells as compared to normal cells.

[0016] In another aspect, the present disclosure describes creation of a CAR using one or both of the variable light (VL) and variable heavy (VH) domains derived from an MR1 antibody in any orientation.

[0017] In yet another aspect, an MR1-CAR having a traditional structure is described. The traditional MR1-CAR structure or platform includes an antigen recognition domain comprising VL and VH domains of an anti-MRl antibody, and further include one or more of a linker sequence, a hinge domain sequence, a transmembrane domain sequence, an intracellular signaling domain sequence, and one or more costimulatory molecule sequences as described above.

[0018] In still yet another aspect, a TCR-based MR1-CAR structure or platform is described, comprising VL and VH domains of an anti-MRl antibody spliced onto the TCR constant domain regions.

[0019] In one possible embodiment, MR1 CAR-T cells are created using PBMCs exposed to immune signals such as CD3 and/or CD28 to activate T cells. The MR1 CAR can also be expressed into 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, and others.

[0020] In one possible embodiment, a CAR design can incorporate a mouse or human TCR- alpha and TCR-beta, wherein each VL domain from TCR-alpha and TCR-beta is substituted by the VL and VH domains of anti-MRl respectively.

[0021] In one possible embodiment, the VL and VH domains of MR 1 antibody used for the MR1 CAR can comprise amino acid sequences shown below, or any nucleotide sequence encoding a similar protein sequence. [0022] VL domain

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGPVKLLIYYTSRLHSG

VPSRFSGSGSGTDYSLTINNLAQEDIATYFCQQGNTLPYTFGGGTKLEIK (SEQ ID

NO: 1)

[0023] VH domain

EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMHWVKKRPELGLEWIGRIDPANG

NTNFAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCPRGSGNYYFDYWGQGTT

LTVSS (SEQ ID NO: 2)

[0024] In another possible embodiment, the VL and VH domains used for the MR1 CAR may differ from the above amino acid sequences by an integer selected from the group consisting of: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15%.

[0025] In yet another possible embodiment, the VL and VH domains used for the MR1 CAR may differ from the above amino acid sequences by a range of integers selected from the group of ranges consisting of: 12-15%, 15-20%, and 20-25%.

[0026] In still yet other possible embodiments, the described CAR may include additional domains to enable its desired function on immune cells. According to the structure of the CAR (traditional architecture versus TCR-based architecture), the domains may be selected from one or more of: a linker such as G4S (GGGGSGGGGSGGGGS; SEQ ID NO: 3) or a linking portion thereof, or Whitlow (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 4) or a linking portion thereof separating the VH and VL domains; a hinge domain to allow optimal spacing of the VL/VH domains to the target antigen; a transmembrane domain (e.g., CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 transmembrane domains); and intracellular signaling domains. As noted above, in the traditional CAR structure an intracellular signaling domain is utilized to activate the CAR-expressing cells. This may include domains derived from CD3zeta as well as domains from at least 1 or more additional co-stimulatory molecules including but not limited to: 0X40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137) or functional variant thereof and MHC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, 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, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, and a ligand that specifically binds with CD83 and CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, B7-H3, and Cytokine receptor or inhibitory molecules as PD-1, CLTA-4, TIM-3, LAG-3 and TIGIT. (Soler, DC et al., J Neurooncol. 2022, Jan; 156(1 ): 81 -96). Certain of the additional regions are not required for the described TCR-based MR1-CAR of the present disclosure. For example, the TCR-based MR1-CAR-T does not require a linker to provide the antigen recognition domain that binds target MR1 antigen.

[0027] In another aspect, the present disclosure describes a CAR comprising variable domains derived from an improved version of the antibody that has been humanized and/or engineered for an increased affinity to MR1 protein.

[0028] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0030] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0031] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0032] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9): 1726-1732).

[0033] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0034] In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases, such as GENBANK® and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the fding date of this Application.

[0035] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, any numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0036] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly “an example,” “exemplary” and the like are understood to be non-limiting. The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited. Therefore, for example, the phrase “wherein the lever extends vertically” means “wherein the lever extends substantially vertically” so long as a precise vertical arrangement is not necessary for the lever to perform its function. [0037] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude 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, “one or more” is understood, unless such interpretation is nonsensical in context. The terms “comprise”, “have”, “include” and “contain” (and their variants) are open- ended linking verbs and allow the addition of other elements when used in a claim.

[0038] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise.

[0039] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±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%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0040] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed 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 is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0041] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant. [0042] As used herein, the term "subject" can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an 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., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, a “subject” as disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, rodent, or other. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term "patient" includes human and veterinary subjects. In some aspects of the disclosure method, the subject has been diagnosed with a disorder of uncontrolled cellular proliferation, e.g., a cancer, prior to the administering step.

[0043] As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease.

[0044] As used herein, the term "prevent" or "preventing" refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where “reduce,” “inhibit” or “prevent” are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

10

SUBSTITUTE SHEET (RULE 26) [0045] As used herein, the term "diagnosed" means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, the term "diagnosed with a need for treatment of a disorder of uncontrolled cellular proliferation" refers to having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition of uncontrolled cellular proliferation, e.g. , a cancer, that can be treated by various therapeutic agents or methods, including, but not limited to, the disclosed compounds and/or products of the disclosed methods of making described herein.

[0046] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intra-aural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0047] The term "contacting" as used herein refers to bringing a disclosed compound or construct and a target such as a cell(s), a target protein(s), or other biological entity/entities together in such a manner that the compound or construct can affect the activity of the target, either directly; e.g., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.

[0048] As used herein, the terms "effective amount" and "amount effective" refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose

11

SUBSTITUTE SHEET (RULE 26) level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.

[0049] As and if used herein, "kit" means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0050] As and if used herein, "instruction(s)" means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website,

12

SUBSTITUTE SHEET (RULE 26) or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.

[0051] As and if used herein, the terms "therapeutic agent" include any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14^th edition), the Physicians' Desk Reference (64^th edition), and The Pharmacological Basis of Therapeutics (12^th edition) , and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double- and single-stranded molecules, gene constructs, expression vectors, antisense molecules

13

SUBSTITUTE SHEET (RULE 26) and the like), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas. The term therapeutic agent also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or prodrugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0052] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0053] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

[0054] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0055] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein.

Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The presently-disclosed subject matter will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

14

SUBSTITUTE SHEET (RULE 26) [0057] Figure 1A compares the mechanisms of classical MHC and MR1 cell surface expression.

[0058] Figure IB shows MR1 expression in various tumorigenic and non-tumorigenic cell lines as % surface M 1.

[0059] Figure 2A illustrates the distinctions between a traditional CAR-T architecture and a TCR-based CAR-T architecture according to the present disclosure.

[0060] Figure 2B shows the architecture of a TCR-based MR1 -CAR-T architecture according to the present disclosure.

[0061] Figure 2C graphically shows high expression levels of the TCR-based MRl-CAR-T of Figure 2B in virally transduced T-cells.

[0062] Figure 2D shows high cellular expansion of the TCR-based MR1 -CAR-T of Figure 2B after transduction.

[0063] Figure 3A graphically illustrates effective in vitro targeting and killing of various tumor cell lines by a TCR-based MR1-CAR-T according to the present disclosure.

[0064] Figure 3B shows specificity of a TCR-based MR1 -CAR-T according to the present disclosure against a parent AML cell line compared to an AML cell line lacking expression of b2-microglobulin (KO).

[0065] Figure 3C shows TCR-based MRl-CAR-T-induced degranulation in several cell lines, measured as CD 107a expression.

[0066] Figure 3D shows TCR-based MR1 -CAR-T -induced interferon-gamma induction by several cell types compared to non-transduced cells.

[0067] Figure 4A depicts the architecture of a conventional MRl-CAR-T.

[0068] Figure 4B shows potency of the conventional MRl-CAR-T of Figure 4A against AML, PC, and GBM cell lines.

[0069] Figure 5A illustrates reduced tumor growth over 4 weeks in an in vivo AML tumor model (Molml3) in mice treated with a TCR-based MRl-CAR-T compared to mice treated with control T-cells and untreated mice.

[0070] Figure 5B shows improved survival of mice treated with TCR-based MRl-CAR-T according to the present disclosure in the in vivo AML tumor model compared to mice treated with control T-cells and untreated mice. [0071] Figure 5C shows improved clearance of CD33+ cells from blood of mice treated with TCR-based MR1-CAR-T according to the present disclosure compared to mice treated with control T-cells and untreated mice.

[0072] Figure 6 graphically shows a comparison of tumor volume in an in vivo gastric tumor model (SNU-16 implantation) in mice treated with TCR-based MR1-CAR-T according to the present disclosure compared to mice treated with vehicle or non-transduced T cells.

[0073] Figure 7 graphically shows killing efficiency of embodiments of TCR-based MR1- CAR-T according to the present disclosure against Molml3, HL-60, Panel, and U87jos cell lines.

[0074] Figure 8A graphically compares killing efficiency of an embodiment of TCR-based MRl-CAR-T and a conventional MR1-CAR-T against a brain tumor cell line (U87).

[0075] Figure 8A graphically compares killing efficiency of an embodiment of TCR-based MRl-CAR-T and a conventional MR1-CAR-T against a pancreatic tumor cell line (Panel).

[0076] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

DETAILED DESCRIPTION

[0077] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0078] To address the above-summarized and other shortcomings in prior art CAR-T therapies, we confirmed MR1 expression in several tumor cell lines and designed multiple novel MRl-CARs including CARs having a traditional CAR architecture (traditional MR1-CAR) and CARs that utilize the T-cell receptor (TCR) machinery (TCR-based MR1-CAR). PBMC used in this study were obtained from healthy donors. For experiments on functional evaluation 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 described in more detail below.

MATERIALS AND METHODS

DNA constructs

[0079] At a high level, in order to develop the disclosed TCR-based MR1-CAR the variable regions of the TCR were combined in frame with the light and heavy chain (VL, VH) of an MR1 antibody (Chua W-J et al. J Immunol. 2011 April 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 contained CD28 transmembrane domains and CD28 co-stimulatory domains (Chua W-J et al. J Immunol. 2011 April 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(l):81-96).

[0080] Example 1. In one embodiment termed MR4 (see Figure 2B), a TCR-based MR1 CAR construct according to the present disclosure comprises the mouse TCR-alpha (SEQ ID NO: 8) and mouse TCR-beta (SEQ ID NO:6) having the VL domains of TCR-alpha and TCR- beta are respectively substituted by the VL domain (SEQ ID NO: 1) and VH domain (SEQ ID NO:2) of mouse anti-MRl antibody. The construct further comprises CD8 peptide signal (SEQ ID NO:5) sequences, and a furin P2A self-cleaving peptide separating the TCR-VH and TCR- VL spliced sequences. As is known, a furin site ensures cleavage of the polypeptide by endogenous cell proteases, creating the two independent proteins (TCR-a and TCR-b). The P2A is a self-cleaving sequence that further ensures this cleavage between the two different proteins occurs. Putting both Furin and P2A together ensured that the cleavage would be successful, creating in this case two independent chains TCR-alpha and TCR-beta. It will be appreciated that this construct could be modified by using human TCR-alpha and TCR-beta sequences substituted by VL domain (SEQ ID NO: 1) and VH domain (SEQ ID NO:2) of mouse anti-MRl antibody or other non-human anti-MRl antibody VL and VH domain sequences to provide a humanized CAR sequence.

[0081] Example 2. In another possible embodiment termed MR3.11, a traditional MR1 CAR construct according to the present disclosure comprises a CD8 peptide signal sequence (SEQ ID NO:5), a VL domain sequence (SEQ ID NO: 1) and a VH domain sequence (SEQ ID NO:2). The construct further comprises two linker sequences (SEQ ID NO:3 between VH and VL and GGG between a CD28 co-stimulatory domain and 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 costimulatory sequence (SEQ ID NO: 12). The construct further comprises a CD3 TCR-zeta chain sequence (SEQ ID NO: 13), and may optionally include a V5 tag (SEQ ID NO:9) useful for confirmation of successful detection of the construct.

[0082] Example 3. In another possible embodiment termed MR3. 12, a traditional MR1 CAR construct according to the present disclosure comprises a CD8 peptide signal sequence (SEQ ID NO:5), a VL domain sequence (SEQ ID NO: 1) and a VH domain sequence (SEQ ID NOT). The construct further comprises two linker sequences (SEQ ID NO:3 between VH and VL and GGG between a 4- IBB costimulatory domain and a CD3 TCR-zeta domain) and an IgG4 hinge sequence (SEQ ID NOTO). The construct further comprises a CD4 transmembrane domain sequence (SEQ ID NO: 14) and a 4-1BB costimulatory sequence (SEQ ID NO: 15), and may optionally include a V5 tag (SEQ ID NO: 9) useful for confirmation of successful detection of the construct.

[0083] Example 4. In another possible embodiment termed MR3.13, a traditional MR1 CAR construct according to the present disclosure comprises a CD8 peptide signal sequence (SEQ ID NO:5), a VL domain sequence (SEQ ID NOT) and a VH domain sequence (SEQ ID NOT). The construct further comprises two linker sequences (SEQ ID NOT between VH and VL and and GGG between a CD28 co-stimulatory domain and a CD3 TCR-zeta domain) and a CD8 stem sequence (SEQ ID NO: 16) provided as a ‘stem’ or spacer, i.e., an extra-cellular linker that is in this case very long and has the ability to anneal with the other CAR-T chains expressed, producing what is called a dimer. Sometimes CAR-T need a long ‘stem’, whereas in other situations a shorter ‘stem’ is required. This dimer between both CAR-T (a dimer is basically both CAR-T chains close next to each other) ensures target binding and strengthens intracellular activation signals. The construct further comprises a CD8 stem sequence (SEQ ID NO: 16) provided as a short stem or spacer linker sequence, a CD28 transmembrane domain sequence (SEQ ID NO: 11), and a CD28 costimulatory sequence (SEQ ID NO: 12). The construct further comprises a CD3 TCR-zeta chain sequence (SEQ ID NO: 13), and may optionally include a V5 tag (SEQ ID NO:9) useful for confirmation of successful detection of the construct.

[0084] Example 5. In another possible embodiment termed MR3.14, a traditional MR1 CAR construct according to the present disclosure comprises a CD8 peptide signal sequence (SEQ ID N0:5), a VL domain sequence (SEQ ID NO: 1) and a VH domain sequence (SEQ ID NO:2). The construct further comprises two linker sequences (SEQ ID NO:3 between VH and VL and GGG between a 4- IBB co-stimulatory domain and a CD3 TCR-zeta domain) and a CD8 stem sequence (SEQ ID NO: 16). The construct further comprises a CD4 transmembrane domain sequence (SEQ ID NO: 14) and a 4- IBB costimulatory sequence (SEQ ID NO: 15). The construct further comprises a CD3 TCR-zeta chain sequence (SEQ ID NO: 13), and may optionally include a V5 tag (SEQ ID NO:9) useful for confirmation of successful detection of the construct.

Lentivirus production

[0085] For lentivirus production, 293T cells (ATCC) were transfected with Trans-TI (Mirus, Madison WI). Medias were changed after 24h, sodium butyrate at 4mM added and all medias were harvested after 48 hours. The viral particles were 0.45um-filtered and purified with either lentifuge following manufacturer’s instructions (Cellecta, Mountain View, CA) or by ultracentrifugation using a Beckman Coulter for 2 hours at 98kg. Viral pellets were resuspended in T- cell media and frozen at -80C immediately until use.

CAR-T cell production

[0086] PBMC were obtained from healthy donor blood samples, purified using a Ficoll gradient, and depleted of monocytes by adherence by plating for 2 hours at a 5M/mL density in 10-cm dishes. T-cells were then activated using Trans Act (Miltneyi, Gaithersburg, MD) for 48h following manufacturer’s instructions. Cells were exposed to lentivirus for 24h and expanded for 7 days, with media replacement (RPMI, 10% FBS and pen strep) every 2-3 days.

Cytotoxic assays

[0087] For the lactate dehydrogenase (LDH) assay (Takara, San Jose, CA), 25xl0³ target cancer cells were plated in a 96-well plate with 150uL media (RPMI, 10% FBS and pen strep). MR1-CAR-T were added at Effector: Target (ET) ratios of 1: 1, 5:1 and 10:1 and incubated overnight at 37 ° C. The next day, the LDH assay was performed with minor modifications: 50uL of cell sup media were mixed with 50uL of HBSS and lOOuL of LDH mix added. The assay was read using a Victor 300X spectrophotometer using 450nm absorbance after a 9-11 minute incubation time. The killing percentage was calculated following the manufacturer’s protocol.

RESULTS

MR1 expression is low across several tumor cell lines and difficult to detect [0088] MR1 differs from classical MHC in that it is monomorphic, highly conserved across species and presents non-proteinaceous metabolites compared to classical MHC which present peptides. Both classical MHC and MR1 require the expression of b2microglobulin to achieve optimal surface expression in cells (see Figure 1A). Expression of MR1 was assessed by flow cytometry (Attune NX, ThermoFisher Scientific) in several tumor cell lines and non- tumorigenic cell lines. 1 million cells were stained for 15 minutes in 50uL volume using l-2uL of MR1 antibody. Cells were washed 3 times with ImL wash buffer, resuspended in lOOuL staining solution and run through a flow cytometer. As shown in Figure IB, MR1 expression was relatively high when quantified as % surface MR1 but did not reach 100% due to the antigen’s low presence on the surface of tumor cells.

TCR-based MR1-CAR-T is expressed in T-cells at high levels

[0089] The architecture of conventional CAR-T compared to TCR-based CAR-T is shown in Figure 2A. To create the disclosed non-conventional TCR-based MR1-CAR-T, the variable regions of a TCR were swapped for the heavy and light chain of an anti-MRl antibody (Figure 2B). The murine constant regions of the TCR were used to avoid mispairing with endogenous human TCRs. The novel TCR-based MR1-CAR-T expressed very efficiently in virally- transduced T-cells compared to untransduced T cells (Figure 2C), and was shown to undergo robust expansion after transduction (see Figure 2D).

TCR-based MR1-CAR-T kills tumor cell lines regardless of MR1 expression levels

[0090] Using calcein-Am and LDH assays, we tested the killing efficacy of the novel TCR- based MR1 -CAR-T of Example 1 against a wide array of different tumor cell lines. As shown in Figure 3 A, the TCR-based MR1 -CAR-T is effective at killing many tumor cell lines including hard to kill Glioblastoma Multiforme (GBM) and Pancreatic cancer (PC) cell lines. TCR-based MR1 -CAR-T also shows specificity, since an AML cell line lacking b2-microglobulin (b2M - which as noted above is required for MR1 surface expression) is not killed by TCR-based MR1- CAR-T in comparison to its parental cell line (Figure 3E)

[0091] The novel TCR-based MR1 -CAR-T also induced degranulation as measured by CD 107a expression (3uL of CD107-PE antibody [R&D # IC4800P] was incubated overnight along the mixture of effector cells and target cells. After 24h, the cells were collected, washed with FACS buffer and flowed using Attune flow cytometer or Aria-SORP flow cytometer) (Figure 3F) and released INF-gamma (INF-y ELISA kit from R&D following manufacturers’ recommended protocol) when exposed to several tumor cell lines (Figure 3G). INF-gamma is a known correlate of cytotoxicity.

TCR-based MR1-CAR-T shows improved potency over conventional MR1-CAR-T in vitro

[0092] We also tested killing efficacy of the conventional MR1 -CAR-T cells against AML, PC and GBM cell lines. Though effective, the conventional MR1-CAR-T cells were less effective against PC and GBM cell lines (Figure 4) compared to the TCR-based MR1 -CAR-T cells tested in Figure 3. A direct comparison of the efficacy of each CAR-T type against PC and GBM cell lines is shown in Figure 8A-B. As is clearly shown, while each CAR-T cell type was effective, the cells expressing the TCR-based MR1 -CAR-T were significantly more effective in killing glioblastoma and pancreatic tumor cells than cells expresing the conventional MR1-CAR- T.

TCR-based MR1-CAR-T shows robust tumor control in an in vivo AML tumor model

[0093] We next assessed the efficacy of the TCR-based MR1 -CAR-T against an AML tumor model in vivo. The model used was Molm-13, which is a known tumor model for AML. Molm- 13, as is known, is a type of liquid cancer of monocytic origin which can lead to circulating disease in mice. Molm-13 express a luciferase gene, and so their presence can be detected by bioluminescent imaging. Untreated mice injected with Molml3 cells died within 23 days, while mice treated with control T-cells (T-cells lacking gene modification, but otherwise purified and expanded identically to MR1 -CAR-T cells from the same donor) showed slightly lesser survival rates. 100% of the Molm 13 -injected mice treated with TCR-based MR1-CAR-T survived for 40 days (see Figure 5B). Blood collected from the untreated mice showed the presence of malignant human CD33+ Molml3 cells while TCR-based MR1-CAR-T treated mice showed complete eradication (undetectable AML cells in blood) of human CD33+ cells (Figure 5C).

MR1-CAR-T shows in vivo activity against MR1 gastric tumor model

[0094] We also assessed the efficacy of the TCR-based MR1 -CAR-T cells in an in vivo gastric tumor murine model wherein mice were injected with SNU-16 cells. 8 million SNU16 were injected subcutaneously into each side of the mouse flank. 11 days later tumor growth was assessed as to size between 100-300mm³. At day 11, 3 million MR1-CAR-T cells were injected via tail injection in the mice. Tumor growth was monitored for 41 days. Tumor size was assessed using a caliper, to provide 3-dimensional measurements, i.e., height, width, and depth. As shown in Figure 6, in mice treated with non-transduced T-cells, tumors grew significantly compared to mice treated with vehicle only (No T-cells). However, mice treated with TCR-based MR1-CAR-T were able to control tumor growth up to 44 days.

DISCUSSION

[0095] This study demonstrates that the non-conventional TCR-based MR1-CAR-T disclosed herein is able to very effectively target the MR1 surface protein present in many tumor cell lines despite the relatively low amount of this protein present on the cell surface. As a result, this novel TCR-based MR1-CAR-T is able to effectively destroy many tumor cells in vitro. To support our in vitro results, we used in vivo mouse tumor models to investigate the potential of TCR-based MR1-CAR-T to stall the growth of two tumor types: AML and gastric tumors. TCR- based MR1-CAR-T effectively reduced tumor growth/volume and improved survival rates of mice in the in vivo models. MR 1 -CAR based on a traditional CAR architecture was also shown to be effective in targeting MR1 cell-surface protein on tumor cell lines.

[0096] Accordingly in the present disclosure is provided a novel MR1-CAR which, when expressed by a suitable immune cell, allows targeting and killing numerous types of cancerous tumor cells in vivo and in vitro. Indeed, the skilled artisan will appreciate that the disclosed novel MR1-CAR will be expected to be efficacious, and therefore finds utility, in treatment of any type of cancer cell which expresses some level of cell-surface MR1, including without intending any limitation lung cancers, renal cancers, kidney cancers, uterine cancers, bowel cancers, cancers of the cervix, colon cancers, gastric cancers, head and neck cancers, and all liquid malignancies including lymphomas.

[0097] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation. Obvious modifications and variations are possible in light of the above teachings. All such modifications and variations are within the scope of the appended claims when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled.

Claims

WHAT IS CLAIMED

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

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

3. The MR1-CAR of claim 2, wherein the VH domain comprises a sequence having SEQ ID NO:2, or a sequence having at least 85% identity to 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 a TCR-alpha and a TCR-beta sequence each having a variable domain portion substituted respectively by the VL domain of the anti-MRl antibody and the VH domain of the anti-MRl antibody to provide the antigen recognition domain.

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

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

8. The MR1-CAR of claim 1, further comprising additional domains selected from the group consisting of: at least one linker sequence joining the VL and VH domains, 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 traditional MR1-CAR architecture.

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

10. The MR1-CAR of claim 8, wherein the transmembrane domain is selected from the group consisting of: CD28, CD3 epsilon, 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 a sequence having at least 85% identity thereto.

12. The MR1-CAR of claim 8, wherein the one or more costimulatory sequences are selected from the group consisting of one or more of: CD3-zeta, 0X40, CD27, CD28, CD5, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1; CDl la/CD18), ICOS (CD278), 4- 1BB (CD137) or a functional variant thereof, BTLA, a Toll ligand receptor, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, a ligand that specifically binds with CD83 and CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1 (LFA-1), CD2, CD5, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, Cytokine receptor 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 costimulatory sequences comprises one or more of SEQ ID NO: 12 and SEQ ID NO: 15, or a sequence having at least 85% identity thereto.

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

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

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

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

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

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

20. The modified cytolytic/cytotoxic immune cell of claim 19, wherein the T-cell or the NK cell is derived from a population of peripheral blood mononuclear cells (PBMC).

21. A pharmaceutical composition comprising a plurality of modified cytolytic immune cells according to claim 18, and one or more of a pharmaceutically or veterinary acceptable carrier, a diluent, an adjuvant, and an excipient.

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

23. The pharmaceutical composition of claim 22, wherein the disease or disorder is a cancer characterized by cancer cell surface expression of MR1.

24. The pharmaceutical composition of claim 23, wherein the cancer is selected from the group consisting of a brain cancer, a pancreatic cancer, a leukemia, a gastric cancer, a breast cancer, a melanoma, a prostate cancer, and a myeloma.

25. The pharmaceutical composition of claim 24, wherein the gastric cancer is selected from the group consisting of a carcinoma and a gastric carcinoma, and the leukemia is an acute myeloid leukemia.

26. A method for treating a disease or disorder characterized by uncontrolled cellular proliferation, comprising a step of administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 21.

27. The method of claim 26, wherein the disease or disorder is a cancer characterized by cancer cell surface expression of MR1.

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

29. The method of claim 28, wherein the gastric cancer is selected from the group consisting of a carcinoma and a gastric carcinoma, and the leukemia is an acute myeloid leukemia.