Immune cell stimulatory sequences

Abstract

The present disclosure provides, among other things, methods and compositions useful in the engineering of chimeric antigen receptors (CARs) and / or T cells. The present disclosure provides, among other things, sequences for use in stimulatory domains, such as CAR stimulatory domains that are engineered to promote expansion, persistence, and / or function of T cells. The present disclosure further provides combinations of sequences for use in stimulatory domains, such as CAR stimulatory domains that are engineered to promote expansion, persistence, and / or function of T cells.

Description

IMMUNE CELL STIMULATORY SEQUENCESCROSS-REFERENCE TO RELATED APPLICATION[1] This application claims the benefit of U.S. Provisional Application No.: 63 / 532,575, filed August 14, 2023, the content of which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING[2] This application contains a Sequence Listing that has been submitted electronically as an XML file named GBB-01660.xml. The XML file, created on August 12, 2024, is 212,635 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.BACKGROUND[3] T cells are immune cells that can participate in efficient clearance of target cells. Natural functions of T cells include, among other things, participation in immune responses against tumors and infections. T cells can be engineered to express chimeric antigen receptors (CARs). Engineered T cells have been used, e.g., in tumor immunotherapy.SUMMARY[4] The present disclosure provides, among other things, methods and compositions useful in the engineering of chimeric antigen receptors (CARs) and / or T cells. The present disclosure provides, among other things, sequences for use in stimulatory domains, such as CAR stimulatory domains that are engineered to promote expansion, persistence, and / or function of T cells. The present disclosure further provides combinations of sequences for use in stimulatory domains, such as CAR stimulatory domains that are engineered to promote expansion, persistence, and / or function of T cells.[5] Without wishing to be bound by any particular scientific theory, the present disclosure is based in part on the observation that CAR stimulatory domains known in the art can include sequences and combinations of sequences that are well known to those of skill in the art, and in some cases have become standard, but are not necessarily optimal and / or satisfactory for use all CAR, CAR T, and / or T cell applications. The present disclosure includes the recognition that sequences of the present disclosure for use in stimulatory domains, and combinations thereof, provide unexpected advantages in engineered T cells, including without limitation unexpectedly advantageous expansion, persistence, and / or function of engineered T cells. Furthermore, it is recognized that sequences demonstratingenhanced function in T cells could enhance function in other cell types in which presently disclosed sequences have not yet been tested.[6] In at least one aspect, the present disclosure provides a chimeric antigen receptor (CAR) including an antigen-binding domain, a transmembrane domain, and at least a first stimulatory sequence, wherein the stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NOs: 1-48 (see, e.g., Table 1). In at least one aspect, the present disclosure provides a chimeric antigen receptor (CAR) including an antigen-binding domain, a transmembrane domain, and a stimulatory region including a first stimulatory sequence and a second stimulatory sequence, wherein the first stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 49-96 and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 97-144, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 1. In various embodiments, the stimulatory region includes a linker positioned between the first stimulatory sequence and the second stimulatory sequence, optionally wherein the linker is a flexible linker and / or wherein the amino acids between the first stimulatory sequence and the second stimulatory sequence consist or consist essentially of the linker. In various embodiments, an exemplary linker can have or include the amino acid sequence GS.[7] In at least one aspect, the present disclosure provides a stimulatory region including a first stimulatory sequence and a second stimulatory sequence, wherein the stimulatory region has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with both the first and second stimulatory a sequences selected from SEQ ID NOs: 1-48. In at least one aspect, the present disclosure provides a stimulatory region including a first stimulatory sequence and a second stimulatory sequence, wherein the first stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 49-96 and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 97-144, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 1. In various embodiments, the stimulatory region includes a linker positioned between the first stimulatory sequence and the second stimulatory sequence, optionally wherein the linker is a flexible linker and / or wherein the amino acids between the first stimulatory sequence and the second stimulatory sequence consist or consist essentially of the linker. In various embodiments, an exemplary linker can have or include the aminoacid sequence GS. In various embodiments, the stimulatory region is operably linked with an antigen-binding domain. In various embodiments, the stimulatory region is operably linked with a transmembrane domain.[8] In at least one aspect, the present disclosure provides an engineered immune cell including a chimeric antigen receptor (CAR) of the present disclosure and / or a stimulatory region of the present disclosure. In various embodiments, the cell is a T cell. In various embodiments, the cell is a CD3+ cell. In various embodiments, the CD3+ cell is differentiated from an induced pluripotent stem cell (iPSC), embryonic stem cell (ESC), or CD34+ progenitor cell (HSPC).[9] In at least one aspect, the present disclosure provides a method of producing an engineered immune cell, the method including contacting the immune cell with a nucleic acid encoding a chimeric antigen receptor (CAR) of the present disclosure and / or a stimulatory region of the present disclosure. In various embodiments, the cell is a T cell. In various embodiments, the cell is a CD3+ cell. In various embodiments, the CD3+ cell is differentiated from an induced pluripotent stem cell (iPSC), embryonic stem cell (ESC), or CD34+ progenitor cell. In various embodiments, the contacting includes viral delivery of the nucleic acid to the cell. In various embodiments, the contacting includes non-viral delivery of the nucleic acid to the cell.

[0010] In at least one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method including administering to the subject an engineered immune cell of the present disclosure. In various embodiments, the cancer is a solid tumor. In various embodiments, the solid tumor is of a cancer selected from colorectal cancer, ovarian cancer, non small cell lung cancer, glioblastoma, triple negative breast cancer, hepatocellular carcinoma, prostate cancer, melanoma, small cell lung cancer, head and neck cancer, and pancreatic cancer. In various embodiments, the cancer is a liquid cancer. In various embodiments, the liquid cancer is selected from acute myeloid leukemia (AML), multiple myeloma, acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). In various embodiments, the cancer expresses a biomarker selected from Her2, EGER, CD19, BCMA, Mucl, CD20, Mesothelin, GPC3, Rorl, MAGE-A4, PRAME, NY-ESO-1, and PSA. In various embodiments, the administration is intravenous. In various embodiments, the administration is peri-tumoral. In various embodiments, the administration is intra-tumoral.

[0011] In at least one aspect, the present disclosure provides a method of treating autoimmune diseases in a subject in need thereof, the method including administering to thesubject an engineered immune cell of the present disclosure. In various embodiments, the autoimmune condition is selected from lupus, rheumatoid arthritis, ulcerative colitis, type I diabetes, systemic sclerosis, and multiple sclerosis.

[0012] For the avoidance of doubt, references to regions comprising a “first” sequence and a “second” sequence encompass embodiments in which the first and second sequences are present, from N-terminus to C-terminus, in the provided order (first before second) or in an alternative order (second before first), except as expressly provided herein and optionally with intervening amino acids (e.g., a linker as provided herein).DEFINITIONS

[0013] A, An, The, Or: As used herein, “a”, “an”, and “the” refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” discloses embodiments of exactly one element and embodiments including more than one element. As used herein, the terms “or” and “and / or”, as conjunctions in a list of at least two elements, encompass and disclose embodiments in which the listed elements are included in the alternative, together, or in any combination.

[0014] About: As used herein, term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0015] Administration: As used herein, the term “administration” typically refers to administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition.

[0016] Amino acid: in its broadest sense, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D- amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.“Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with a typical or canonical amino acid structure. For example, in some embodiments, an amino acid can be modified by methylation, amidation, acetylation,pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification can, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” can be used to refer to a free amino acid; in some embodiments it can be used to refer to an amino acid residue of a polypeptide.

[0017] Antibody’. As used herein, the term “antibody” refers to a polypeptide that includes one or more immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). Thus, the term antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents including the same. Antibodies can be naturally occurring immunoglobulins (e.g., generated by an organism reacting to an antigen). Synthetic, non-naturally occurring, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0018] As is well known in the art, typical human immunoglobulins are approximately 150 kD tetrameric agents that include two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a “Y-shaped” structure. Typically, each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain includes three CH domains: CHI, CH2 and CH3. A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.” Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs arearranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of a heavy and / or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells e.g., effector cells and / or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. When naturalimmunoglobulins fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0019] In some embodiments, an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody). In some embodiments, an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers. Moreover, the term “antibody” can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and / or functional features including without limitation intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (“SMIPsTM”), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies,, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®s, CARs, engineered TCRs, and antigenbinding fragments of any of the above.

[0020] In various embodiments, an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain. In some embodiments, an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0021] An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g., alpha (a), delta (5), epsilon (e), gamma (y) and mu (p)). IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. As used herein, a “light chain” can be of a distinct type, e.g., kappa (K) or lambda (I), based on the amino acid sequence of the light chain constant domain. In some embodiments, an antibody has constant region sequences that arecharacteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally-produced immunoglobulins are glycosylated, typically on the CH2 domain. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[0022] Antibody fragment'. As used herein, an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigenbinding portion or variable region thereof. An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.

[0023] Between or From'. As used herein, the term “between” refers to content that falls between indicated upper and lower, or first and second, boundaries (or “bounds”), inclusive of the boundaries. Similarly, the term “from”, when used in the context of a range of values, indicates that the range includes content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries.

[0024] Cancer: As used herein, the term “cancer” refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they display an abnormally elevated proliferation rate and / or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a cancer can include one or more tumors. In some embodiments, a cancer can be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a cancer can be or include a solid tumor. In some embodiments, a cancer can be or include a hematologic tumor.

[0025] Chimeric antigen receptor'. As used herein, “Chimeric antigen receptor” or “CAR” refers to an engineered protein that includes (i) an extracellular domain that includes a moiety that binds a target antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain that sends activating signals when the CAR is stimulated by binding of the extracellular binding moiety with a target antigen. A T cell that has been genetically engineered to express a chimeric antigen receptors may be referred to as a CAR T cell. Thus, for example, when certain CARs are expressed by a T cell, binding of the CAR extracellular binding moiety with a target antigen can activate the T cell. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors.

[0026] Domain: The term “domain” as used herein refers to a section or portion of an entity. In some embodiments, a “domain” is associated with a particular structural and / or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and / or functional feature. Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and / or imparts on the recipient entity one or more structural and / or functional features that characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, -helix character, P-sheet character, coiled-coil character, random coil character, etc.), and / or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.). In some embodiments, a domain is or includes a characteristic portion or characteristic sequence element.

[0027] Engineered’. As used herein, the term “engineered” refers to the aspect of having been manipulated by human intervention. For example, a polynucleotide is considered to be “engineered” when two or more sequences, that are not linked together in that order in nature, are manipulated by human intervention to be linked to one another in the engineered polynucleotide. Those of skill in the art will appreciate that an “engineered” nucleic acid or amino acid sequence can be a recombinant nucleic acid or amino acid sequence. In some embodiments, an engineered polynucleotide includes a coding sequence and / or a regulatory sequence that is found in nature operably linked with a first sequence but is not found in nature operably linked with a second sequence, which is in the engineered polynucleotide and operably linked in with the second sequence by human intervention. In some embodiments, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution, deletion, or mating). As is common practice and is understood by those of skill in the art, progeny or copies, perfect or imperfect, of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the direct manipulation was of a prior entity.

[0028] Operably linked’. As used herein, “operably linked” refers to the association of at least a first element and a second element such that the component elements are in a relationship permitting them to function in their intended manner. For example, a nucleic acid sequence or amino acid sequence is operably linked with another sequence if it modifies the expression, structure, or activity of the linked sequence, e.g., in an intended manner. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if the regulatory sequence and coding sequence areassociated in a manner that permits control of expression of the coding sequence by the regulatory sequence. In some embodiments, an "operably linked" regulatory sequence is directly or indirectly covalently associated with a coding sequence (e.g., in a single nucleic acid). In some embodiments, a regulatory sequence controls expression of a coding sequence in trans and inclusion of the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement of operable linkage. In many cases, two amino acid sequences are operably linked if they are expressed as a single polypeptide.

[0029] Polypeptide: As used herein, “polypeptide” refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through human intervention. In some embodiments, a polypeptide may be or include of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may be or include only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide can include D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may include only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., one or more amino acid side chains, e.g., at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, at non-terminal amino acids, or at any combination thereof. In some embodiments, such pendant groups or modifications may be selected from acetylation, amidation, lipidation, methylation, phosphorylation, glycosylation, glycation, sulfation, mannosylation, nitrosylation, acylation, palmitoylation, prenylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may include a cyclic portion.

[0030] In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure to indicate a class of polypeptides that share a relevant activity or structure. For such classes, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class. For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that can in some embodiments be or include a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and in some instances up to 20 or moreamino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide can be or include a fragment of a parent polypeptide. In some embodiments, a useful polypeptide may be or include a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0031] Subject: As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human, rat, or mouse). In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, a human subject can be interchangeably referred to as a “patient” or “individual.” A subject administered an agent associated with treatment of a disease, disorder, or condition with which the subject is associated can be referred to as a subject in need of the agent, i.e., as a subject in need thereof.

[0032] Therapeutically effective amount: As used herein, “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that a therapeutically effective amount does not necessarily achieve successful treatment in every particular treated individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulatedand / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0033] Treatment'. As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or of a subject who exhibits only early signs of the disease, disorder, or condition. Alternatively or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Fig. 1 is a schematic of an exemplary general architecture of CARs.

[0035] Fig. 2 describes an exemplary CAR architecture of a CAR that includes a combination of distinct stimulatory sequences derived from different full protein domains.

[0036] Fig. 3 is a graph displaying data from an example in which T cells engineered to express CARs that include combinations of distinct stimulatory sequences derived from different full protein domains are subjected to serial stimulation. The chart depicts the log2 fold-change of T cell numbers for T cells expressing different CAR designs at day 11 versus day 0 after daily stimulation with Raji target cells at a 1:1 T:Raji ratio.DETAILED DESCRIPTION

[0037] The present disclosure provides, among other things, sequences for use in stimulatory domains of CARs (which can be referred to herein as “stimulatory sequences”). In various embodiments, the present disclosure provides sequences for use in stimulatory domains of CARs that are particularly useful in engineering of T cells. Accordingly, the present disclosure includes T cells engineered to express CARs (CAR-T cells) including stimulatory sequences of the present disclosure.Chimeric Antigen Receptors

[0038] CARs are engineered proteins designed to redirect and / or amplify the response of immune cells against cells expressing specific antigen targets. CARs generally include three modules: an extracellular binding domain, a transmembrane domain, and one or more intracellular stimulatory sequences (see, e.g., Fig. 1). As those of skill in the art will appreciate, extracellular binding domains, transmembrane domains, and intracellular stimulatory sequence(s) are modular at least in that sequences of each can be independently engineered and / or that a functional CAR can be produced by independent selection of sequences for each. Accordingly, although an extracellular binding domain, a transmembrane domain, and intracellular stimulatory sequence(s) of a CAR function cooperatively, those of skill in the art appreciate that each is an independently engineered and independently useful component.

[0039] An extracellular binding domain can be or include a binding domain such as an antibody or antibody fragment, that specifically binds an antigen. For example, an extracellular domain can be an scFv or nanobody that specifically binds a given antigen target.

[0040] Transmembrane domains within a C AR molecule can serve to connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the expressed molecule in a cell’s membrane. CAR transmembrane domains can be derived from transmembrane domains of proteins such as CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22: CD33, CD37, CD64, CD80, CD86, CD 134, CD 137 and CD 154.

[0041] Broadly, intracellular stimulatory sequences determine the signaling consequences of antigen binding. The first intracellular stimulatory sequences were designed to model TCR signaling in T cells, and subsequent generations (e.g., including multiple stimulatory sequences) have likewise been developed for use in T cells. First generation CARs utilized the cytoplasmic region of CD3 as a stimulatory sequence. Second generation CARs utilized CD3 in combination with cluster of differentiation 28 (CD28) or 4-1BB (CD137), while third generation CARs have utilized CD3 in combination with CD28 and 4- IBB within intracellular effector domains. However, the diversity of stimulatory domains, and combinations thereof, explored in CARs is limited.CAR-T cells

[0042] Without wishing to be bound by any particular scientific theory, CARs function by tying an antigen-binding event to specific signaling activity in the immune cell. CARs in T cells can be viewed as synthetic TCRs, where the predominant signaling driven by the CAR falls along the canonical T cell activation pathways. For example, CAR designs featuring the CD3z domain activate T cells via PLCg-dependent signaling that drives Jun and Stat3- mediated transcriptional activation. Inclusion of co-stimulatory domains such as 4- IBB and CD28 drive additional pro-survival signaling through the PI3K and Jnk pathways.

[0043] However, increasing the functional persistence of CAR-T cells is an important goal of cell therapy engineering efforts, especially in oncology settings where CAR-T cells must serially kill large numbers of tumor cells in order to drive therapeutic efficacy. Incorporation of diverse signaling domains into the CARs of CAR-T cells can drive alternative signaling outcomes and potentially enhance the persistence of CAR-T cells, yet the exploration of such diverse domains has been shallow. The present disclosure provides, among other things, non canonical CAR stimulatory sequence domains that enhance the functional persistence of CAR-T cells.Stimulatory Sequences

[0044] Without wishing to be bound by any particular scientific theory, binding of a target antigen initiates downstream CAR signaling events through recruitment of adapter and second messenger proteins to stimulatory sequences (including, e.g., domains and motifs associated with stimulatory activity). Downstream CAR signaling events can cause activation of cells in which they occur, where activation can include one or more of differentiation, proliferation and / or activation or other effector functions. Canonical CAR designs (e.g., for use in CAR-T cells) feature stimulatory sequences that activate Src family protein tyrosine kinases. For example, such stimulatory sequences are found in the cytoplasmic tails of various CD3 chains in T cells, as well as in those of NK receptors such as CD16.

[0045] Several alternative stimulatory domains have been explored in T cells, including 2B4, ICOS, and 0X40, each in combination with CD3zeta. However, the present disclosure includes the recognition that there is a need for further, alternative, and / or improved stimulatory sequences for use in CARs and / or T cells (e.g., CAR-T cells), and that certain such sequences can be advantageously selected and / or derived from other immune cell-native stimulatory domains. The present disclosure includes the recognition that such stimulatory sequences and combinations thereof could provide an increased diversity in CAR signalingand functional outcomes (e.g., in CARs and / or for CAR-T cells), and / or drive enhanced stimulation, differentiation, proliferation and / or activation.

[0046] The present disclosure provides stimulatory sequences and combinations thereof that are, e.g., particularly useful in engineering of CARs for use in T cells, and production of CAR-T cells.

[0047] The present disclosure includes the discovery that certain stimulatory sequences identified herein as useful in CARs and / or T cells are unexpectedly characterized by (e.g., having, or derived from domains having) certain shared features and / or biological functions. The present disclosure further includes the discovery that combinations of stimulatory sequences that include a first stimulatory sequence characterized by a first feature and / or biological function and a second stimulatory sequence characterized by a second feature and / or biological function can be particularly advantageous.

[0048] The present disclosure describes stimulatory regions and / or stimulatory sequences that include one or more full protein domains and / or motifs. Among stimulatory regions and / or sequences that include one or more protein domains and / or motifs provided herein, various such domains and motifs can be characterized by certain biological functions when present in cells. Moreover, in various embodiments, a stimulatory region can include a combination of full protein domains and / or motifs having certain such biological functions that together give rise to unexpectedly advantageous properties, e.g., for T cell differentiation, proliferation and / or activation. IL7RA (e.g. included in SEQ ID NO 1) drives supportive cytokine signaling in T and NK cells. TIGIT and PDCD1 (e.g. included in SEQ ID NOs 8, 11) are inhibitory domains that in combination with CD3zeta activating domains potentially regulate the strength of the activation signal to increase cell persistence. NKp65 and NKp80 (e.g. included in SEQ ID NOs 4, 7, and 9) are activating receptors associated with Natural Killer (NK) cells. DAP12 (e.g. included in SEQ ID NOs 19) is involved in JAK3 / STAT5a and PI3K signaling. FCER1G (e.g. included in SEQ ID NO 44) activation domains contain activating motifs and have been used as alternatives to CD3z in CAR designs. Fnl4 and TANK (e.g. included in SEQ ID NOs 32, 34) are TNF-associated domains implicated in promoting proliferation in NK cells. TLR4 (e.g. included in SEQ ID NOs 23, 47) is a pattern recognition receptor associated with recognizing endotoxin. Accordingly, the present disclosure includes combinations of such domains (e.g., combinations set forth in Table 1) that can provide unexpectedly advantageous properties, e.g., for T cell differentiation, proliferation and / or activation.Table 1: Stimulatory sequence domains / motifs

[0049] The present disclosure provides stimulatory regions that include a first stimulatory domain sequence (SEQ ID NOs: 49-96) and a second stimulatory domain sequence (SEQ ID NOs: 97-144), e.g., in a combination of a first stimulatory domain and a second stimulatory domain set forth in rows of Table 1 (e.g., in any orientation or spacing), and that such stimulatory regions can give rise to unexpectedly advantageous properties, e.g., for T cell differentiation, proliferation and / or activation. For example, in various embodiments, a first stimulatory domain can be N-terminal of a second stimulatory domain, and in others a first stimulatory domain can be C-terminal of a second stimulatory domain, and in any of the various embodiments domains can be adjacent or optionally separated by a linker. For the avoidance of doubt, Table 1 further provides the sequences of stimulatory regions (SEQ ID NOs: 1-48) that include a first stimulatory domain sequence (SEQ ID NOs: 49-96) and a second stimulatory domain sequence (SEQ ID NOs: 97-144). Each of the stimulatory regions according to SEQ ID NOs: 1-48 consists of, from N terminus to C terminus, (1) the indicated first domain sequence, (2) the GS linker, and (3) the indicated second domain sequence.

[0050] The present disclosure includes the recognition that stimulatory regions that include a first stimulatory domain sequence (SEQ ID NOs: 49-96) and a second stimulatory domain sequence (SEQ ID NOs: 97-144), e.g., in combinations as set forth in rows of Table 1, do not require a linker to function in the manner provided herein. The present inventors have discovered that stimulatory regions without a linker (i.e., where the sequence of a first stimulatory domain sequence of the present disclosure is directly joined to a second stimulatory domain sequence of the present disclosure, e.g., in a combination set forth in a row of Table 1) are useful and advantageous for use as disclosed herein. The present inventors have further discovered that stimulatory regions that include a linker between a first stimulatory domain sequence of the present disclosure and a second stimulatory domain sequence of the present disclosure can demonstrate further increased stimulatory activity(e.g., when included in a T cell) as compared to a reference sequence without such a linker. Without wishing to be bound by any particular scientific theory, separating multiple stimulatory sequences on the same receptor using short, flexible peptide linkers could potentially limit steric hindrance effects that might otherwise hamper the downstream function driven by each sequence.

[0051] Linkers of the present disclosure include sequences that are useful to connect different elements to one another. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains (e.g., first and second stimulatory domain sequences) can include a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide including a linker element can have an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1 121-1123).

[0052] In some embodiments, a polypeptide linker can be at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In certain embodiments, a polypeptide linker can be at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids in length. In some embodiments, a polypeptide linker can have a length that is within a range having a lower bound selected from 1, 2, 3, 4, or 5 amino acids and an upper bound selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids. In certain embodiments, amino acids of a linker include or consistent of one or both of glycine and serine. The present disclosure exemplifies, without limitation, use of flexible linkers, such as the minimal flexible linker GS.Applications

[0053] CAR designs disclosed herein could drive diverse and useful cell states in engineered cell therapies. Enhancing the signaling space captured by CAR designs is a strategy for enhancing therapeutically relevant cell characteristics in an antigen-binding dependent manner. For example, transmembrane domains that modulate CAR presentation levels on the surface of immune cells could be used to tune the sensitivity of the immune cell response todifferent levels of antigen. Alternatively, stimulatory domains could be used not only to activate the cell, but also to stimulate signaling pathways that increase cell metabolic fitness in suppressive tumor microenvironments. Such domains could be largely derived from native receptor sequences, or manipulated at the level of individual protein binding motifs.

[0054] While certain signaling domains have been characterized previously, such as CD40, CD28, 4- IBB, and DAP10, specific stimulatory sequences featuring combinations of stimulatory sequences drive potentially therapeutically useful cell states in ways that are challenging to predict a priori.

[0055] Strong T cell activation via CD3zeta domains has been associated with limited T cell functional persistence, also known as exhaustion. Strategies to moderate CD3zeta signaling have been suggested in order to decrease exhaustion. The present disclosure recognize that combination of a CD3zeta domain with a domain derived from canonically inhibitory receptors TIGIT and PDCD1 in the present disclosure can provide a tuning mechanism that continues to support T cell differentiation, proliferation and / or activation but reduces, moderates, and / or eliminates exhaustion associated with CD3zeta activation. Additional exemplary inhibitory endodomains are listed in Table 2.Table 2: Inhibitory endodomain sequences

[0056] The present disclosure further recognizes that mimicking cytokine signaling through, for example, the JAK / STAT signaling pathway is another mechanism for increasing functional persistence as evidenced, e.g., by data relating to IL7RA and DAP12 stimulatory sequences in the present disclosure. Driving cytokine signaling through an antigen-binding event is advantageous over overexpression of cytokines owing to safety concerns over unregulated or misregulated cytokine levels.

[0057] While T cells canonically signal through the TCR pathway, other immune cell types, such as NK cells activate via broad sets of activating receptors that signal through pathways that partially overlap with the downstream TCR pathway. The present disclosure provides, among other things, CAR designs, stimulatory regions, and / or sequences useful for T cell (e.g., CAR T cell) differentiation, proliferation and / or activation that include NK cell activating receptors such as NKp80 and NKp65. Without wishing to be bound by any particular scientific theory, the present disclosure therefore provides CAR designs, stimulatory regions, and / or sequences that can drive T cell activation via non canonical signaling axes, and in various embodiments decoupel exhaustion from activation. The present disclosure provides the recognition that activating receptor endodomains, including NKp80, NKp65, and further NK cell activating receptor endodomains including those provided in Table 3, can contribute to enhanced CAR-T function.Table 3: Exemplary NK cell activating receptor end-domainsEXAMPLES

[0058] The present Example demonstrates that compositions and methods of the present disclosure can support differentiation, proliferation and / or activation of T cells, and / or impart enhanced functionality in immune cells. The present Examples demonstrate the ability of stimulatory sequences and regions provided herein to drive enhanced proliferation of CAR-T cells, including upon repeated antigen exposure. Among the demonstrated characteristics of compositions and methods provided herein, continued proliferation and / or survival over repeated antigen exposure demonstrates unexpected advantages that address a recognized barrier to the utility of CAR-T cells, e.g, in therapeutic contexts.Example 1: Functional assessment of CAR-T stimulatory sequences by serial restimulation assay

[0059] The present Example demonstrates that specific stimulatory sequences representing individual or combinations of full protein domains such as SEQ ID NOs: 1-48 drive enhanced cell expansion, persistence (e.g., non-exhaustion or reduced exhaustion), and / or function of T cells, as compared to a reference, in the context of an 11 day serial restimulation with Raji tumor cells. In the present Example, full CAR designs included a CD 19 binder, CD28 transmembrane domain, and stimulatory sequences representing pairwise combinations of full protein domains (Fig. 2).

[0060] In these experiments, T cells were transduced with different CD19-targeted CARs including different stimulatory sequences or combinations thereof, and co-cultured with cells from the Raji CD19+ tumor cell line. T cells killed the Raji cells, after which additional target cells were added to the cultures. Each day, the T cells were counted in order to gauge the degree of CAR-T expansion in conditions of daily antigen refresh (Fig. 3). SEQ ID NOs: 1-48 demonstrated positive expansion, persistence, and / or function after 11 days in at least one of the culture conditions. Notably, screening experiments identifying these sequences additionally included 2500 stimulatory sequences that did not demonstrate positive expansion, persistence, or function (data not shown). Accordingly, the advantages evidenced by the present Example are an unexpected characteristic of sequences disclosed herein. The present data demonstrate therapeutic utility of sequences disclosed in the present application, at least in that metabolically sustainable serial killing of tumor cells enhances the therapeutic area-under- the-curve of CAR-T cell therapies, which is broadly advantageous in therapeutic contexts and particularly advantageous for treatment of solid tumors.Materials and Methods for Examples

[0061] Stimulatory Sequence Cloning Strategy

[0062] A nested Golden Gate cloning strategy was used to create strings of stimulatory sequences in a CAR backbone. Golden Gate cloning sites for PaqCl and Esp31 Type IIS restriction enzymes, as well as stop codons, were included in the backbone. Cloning of stimulatory sequences was performed serially, first utilizing synthesized gene fragments in the insertion at the PaqCl site, followed by insertion of a second synthesized gene fragment in the Esp31 site.

[0063] Lentivirus Production

[0064] The above transfer plasmid encoding the CAR was co-transfected along with Rev, envelope and gag / pol encoding plasmids into Takara Lenti-X 293 cells. After three days of incubation, at 37C, the supernatant was harvested and concentrated 100X using Lenti-X. Virus was subsequently titrated in T cells by staining for CAR and observing transduction efficiency by flow cytometry.

[0065] T Cell Transduction

[0066] T cells were seeded in T culture media containing IL-2 and lOug / mL polybrene before adding virus at the desired concentration. Plates were spun at 1200xg for 30 min at 32C, then resuspended by pipetting up and down. After incubation at 37C for 1 hr, plates were again spun at 1200xg for 10 min at 32C before discarding the transduction media and adding fresh T media. Cells were subsequently expanded using K562 feeder cells.

[0067] T Serial Restimulation Experiment

[0068] On Day 0, transduced CAR-T cells were co-cultured with Raji tumor cells at a 1:1 ratio in basal T media containing human serum AB and 5u / mL IL-2. Each subsequent day, T and Raji cells were counted, and new Raji cells were added to reset the 1:1 T:Raji ratio. T cell counts at each timepoint represent the degree of T expansion in continuous antigen-exposure conditions.OTHER EMBODIMENTS

[0069] It will be appreciated that the scope of the present disclosure is to be defined by that which may be understood from the disclosure and claims rather than by the specific embodiments that have been presented by way of example. Elements described with respect to one aspect or embodiment of the present disclosure are also contemplated with respect to other aspects or embodiments of the present disclosure. For example, elements of claims that depend directly or indirectly from a certain independent claim presented herein serve assupport for those elements being presented in additional dependent claims of one or more other independent claims. Throughout the description, where compositions or methods are described as having, including, or comprising specific elements, compositions that consist essentially of, consist of, or do not comprise the recited elements are likewise hereby disclosed. All references cited herein are hereby incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and a stimulatory region comprising a first stimulatory sequence and a second stimulatory sequence, wherein the stimulatory region has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 1- 48.

2. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and a stimulatory region comprising a first stimulatory sequence and a second stimulatory sequence, wherein the first stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 49-96 and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 97-144, optionally wherein the first stimulatory sequence and second stimulatory sequence are present in a row of Table 1.

3. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and a stimulatory region comprising a first stimulatory sequence and a second stimulatory sequence, wherein the first stimulatory sequence is or comprises an intracellular domain of a T cell inhibitory receptor, and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a CD3zeta domain according to SEQ ID NO 49.

4. The CAR of claim 3, wherein the first stimulatory sequence is selected from a PDCD1, TIGIT, TIM3, TGFBRI, TGFBRII, CTLA4, LAG3, BTLA, or CD160 domain, optionally wherein the first stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 56, 59, and 145-150.

5. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and a stimulatory region comprising a first stimulatory sequence anda second stimulatory sequence, wherein at least one stimulatory sequence is or comprises an intracellular domain of an NK cell activating receptor.

6. The CAR of claim 5, wherein the at least one stimulatory sequence is selected from a NKp30, NKp40, NKp44, NKp46, NKp65, NKp80, Fnl4, SLAMF6, SLAMF7, CRTAM, FcR gammallla, DAP 10, DAP 12, or 2B4 domain optionally wherein the at least one stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 52, 70, 80, 81, 99, 101, 120, 124,115, and 151-156.

7. The CAR of claim 5 or claim 6, wherein the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 49- 156 and / or wherein the second stimulatory sequence comprises a stimulatory sequence selected from a 4- IBB stimulatory sequence and a CD28 stimulatory sequence.

8. The CAR of any one of claims 2-7, wherein the stimulatory region comprises a linker positioned between the first stimulatory sequence and the second stimulatory sequence, optionally wherein the linker is a flexible linker and / or wherein the amino acids between the first stimulatory sequence and the second stimulatory sequence consist or consist essentially of the linker.

9. A stimulatory region comprising a first stimulatory sequence and a second stimulatory sequence, wherein the stimulatory region has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with both the first and second stimulatory a sequences selected from SEQ ID NOs: 1-48.

10. A stimulatory region comprising a first stimulatory sequence and a second stimulatory sequence, wherein the first stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 49-96 and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 97-144, optionally wherein the first stimulatory sequence and second stimulatory sequence are present in a row of Table11. A stimulatory region comprising a first stimulatory sequence and a second stimulatory sequence, wherein the first stimulatory sequence is or comprises an intracellular domain of a T cell inhibitory receptor, and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a CD3zeta domain according to SEQ ID NO 49.

12. The stimulatory region of claim 11, wherein the first stimulatory sequence is selected from a PDCD1, TIGIT, TIM3, TGFBRI, TGFBRII, CTEA4, EAG3, BTEA, or CD160 domain, optionally wherein the first stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 56, 59, and 145-150.

13. A stimulatory region comprising a first stimulatory sequence and a second stimulatory sequence, wherein at least one stimulatory sequence is or comprises an intracellular domain of an NK cell activating receptor.

14. The stimulatory region of claim 13, wherein the at least one stimulatory sequence is selected from a NKp30, NKp40, NKp44, NKp46, NKp65, NKp80, Fnl4, SEAMF6, SEAMF7, CRTAM, FcR gammallla, DAP10, DAP12, or 2B4 domain optionally wherein the at least one stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 52, 70, 80, 81, 99, 101, 120, 124,115, 151-156.

15. The stimulatory region of claim 13 or 14, wherein the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 49-156 and / or wherein the second stimulatory sequence comprises a stimulatory sequence selected from a 4- IBB stimulatory sequence and a CD28 stimulatory sequence.

16. The stimulatory region of any one of claims 10-15, wherein the stimulatory region comprises a linker positioned between the first stimulatory sequence and the second stimulatory sequence, optionally wherein the linker is a flexible linker and / or wherein the amino acids between the first stimulatory sequence and the second stimulatory sequence consist or consist essentially of the linker.

17. The stimulatory region of any one of claims 9-16, wherein the stimulatory region is operably linked with an antigen-binding domain.

18. The stimulatory region of any one of claims 10-16, wherein the stimulatory region is operably linked with a transmembrane domain.

19. An engineered immune cell comprising a chimeric antigen receptor (CAR) according to any one of claims 1-8 or a stimulatory region according to any one of claims 9-18.

20. The engineered immune cell of claim 19, wherein the cell is a T cell.

21. The engineered immune cell of claim 19 or 20, wherein the cell is a CD3+ cell.

22. The engineered immune cell of claim 21, wherein the CD3+ cell is differentiated from an induced pluripotent stem cell (iPSC), embryonic stem cell (ESC), or CD34+ progenitor cell (HSPC).

23. A method of producing an engineered immune cell, the method comprising contacting the immune cell with a nucleic acid encoding a chimeric antigen receptor (CAR) according to any one of claims 1-8 or a stimulatory region according to any one of claims 9-18.

24. The method of claim 23, wherein the cell is a T cell.

25. The method of claim 23 or 24, wherein the cell is a CD3+ cell.

26. The method of claim 25, wherein the CD3+ cell is differentiated from an induced pluripotent stem cell (iPSC), embryonic stem cell (ESC), or CD34+ progenitor cell.

27. The method of any one of claims 23-26, wherein the contacting comprises viral delivery of the nucleic acid to the cell.

28. The method of any one of claims 23-26, wherein the contacting comprises non- viral delivery of the nucleic acid to the cell.

29. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an engineered immune cell according to any one of claims 19-22.

30. The method of claim 29, wherein the cancer is a solid tumor.

31. The method of claim 30, wherein the solid tumor is of a cancer selected from colorectal cancer, ovarian cancer, non small cell lung cancer, glioblastoma, triple negative breast cancer, hepatocellular carcinoma, prostate cancer, melanoma, small cell lung cancer, head and neck cancer, and pancreatic cancer.

32. The method of claim 31, wherein the cancer is a liquid cancer.

33. The method of claim 32, wherein the liquid cancer is selected from acute myeloid leukemia (AML), multiple myeloma, acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL).

34. The method of any one of claims 29-33, wherein the cancer expresses a biomarker selected from Her2, EGFR, CD19, BCMA, Mucl, CD20, Mesothelin, GPC3, Rorl, MAGE- A4, PRAME, NY-ESO-1, and PSA.

35. The method of any one of claims 29-34, wherein the administration is intravenous.

36. The method of any one of claims 29-34, wherein the administration is peri-tumoral.

37. The method of any one of claims 29-34, wherein the administration is intra-tumoral.

38. A method of treating autoimmune disease in a subject in need thereof, the method comprising administering to the subject an engineered immune cell according to any one of claims 19-22.

39. The method of claim 38, wherein the autoimmune condition is selected from systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, systemic sclerosis, multiple sclerosis, Sjogren’s syndrome, immune thrombocytopenia, dermatomyositis, myasthenia gravis, pemphigus vulgaris, autoimmune thyroiditis, antiphospholipid syndrome, Guillain-Barre syndrome, Addison’s disease, lupus nephritis, membranous glomerulopathy.

40. The method of claim 38, wherein the antigen-binding domain binds a target antigen selected from CD 19, CD20, BCMA, and PD1.

41. The method of any one of claims 38-40, wherein the administration is intravenous.

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