Chimeric immune cell receptors

Abstract

The present disclosure provides, among other things, methods and compositions useful in the engineering of chimeric switch receptors (CSRs) and / or natural killer (NK) cells. The present disclosure provides, among other things, sequences for use in stimulatory domains, such as CSR stimulatory domains that are engineered to promote expansion, persistence, and / or function of NK cells, especially under immunosuppressive conditions. The present disclosure further provides combinations of sequences for use in stimulatory domains, such as CSR stimulatory domains that are engineered to promote expansion, persistence, and / or function of NK cells, especially under immunosuppressive conditions.

Description

CHIMERIC IMMUNE CELL RECEPTORSCROSS-REFERENCE TO RELATED APPLICATION[1] This application claims the benefit of U.S. Provisional Application No.: 63 / 539,234, filed September 19, 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-01560.xml. The XML file, created on September 6, 2024, is 1,944,111 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.BACKGROUND[3] Natural killer (NK) cells are immune cells that can participate in efficient clearance of target cells. Natural functions of NK cells include, among other things, participation in immune responses against tumors and infections. NK cells can be engineered to express chimeric switch receptors (CSRs). Engineered NK 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 switch receptors (CSRs) and / or natural killer (NK) cells. The present disclosure provides, among other things, sequences for use in stimulatory domains, such as CSR stimulatory domains that are engineered to promote expansion, persistence, and / or function of NK cells. The present disclosure further provides combinations of sequences for use in stimulatory domains, such as CSR stimulatory domains that are engineered to promote expansion, persistence, and / or function of NK cells.[5] Without wishing to be bound by any particular scientific theory, the present disclosure is based in part on the observation that CSR stimulatory domains known in the art can include sequences and combinations of sequences that were not developed for use in NK cells and / or are not satisfactory for use in NK cells. 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 NK cells, including without limitationunexpectedly advantageous expansion, persistence, and / or function of engineered NK cells. Furthermore, it is recognized that sequences demonstrating enhanced function in NK cells can enhance function in other cell types in which these specific sequences have not yet been tested.[6] In at least one aspect, the present disclosure provides a chimeric switch receptor (CSR) including a target binding domain (e.g., a ligand-binding domain or an antigen-binding domain such as an antibody or antibody fragment), 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: 1-15 (see, e.g., Table 1). In at least one aspect, the present disclosure provides a chimeric switch receptor (CSR) including a ligand-binding domain, a transmembrane domain, and 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 a sequence selected from SEQ ID NOs: 16-491 (see, e.g., Table 2). In at least one aspect, the present disclosure provides a chimeric switch receptor (CSR) including a ligand-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: 492-967 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: 968-1443, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 2. In at least one aspect, the present disclosure provides a chimeric switch receptor (CSR) including a ligand-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 first domain sequence of Table 2 and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a second domain sequence of Table 2, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 2. 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 CSR includes a linker positioned between the ligand binding domain and the transmembrane domain. In various embodiments, the CSR includes a ligand binding domain that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 1444-1489. In various embodiments, the CSR ligand binding domain contains an N-terminal signal peptide that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 1492). In various embodiments, the CSR includes a transmembrane domain that has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from VTGISLLPPLGVAISVIIIFY (SEQ ID NO 1490) or ILYILVPSIAIPLVIACLFFLG (SEQ ID NO 1491). In various embodiments, the transmembrane domain and ligand-binding domains are separated by a linker disclosed herein, e.g., a flexible linker.[7] In at least one aspect, the present disclosure provides a stimulatory region including 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: 1-15. 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: 16-491. 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: 492-967 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: 968- 1443, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 2. 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 first domain sequence of Table 2 and the second stimulatory sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a second domain sequence of Table 2, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 2. 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 / orwherein 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. In various embodiments, the stimulatory region is operably linked with a target binding domain (e.g., a ligand-binding domain or an antigen-binding domain such as an antibody or antibody fragment). In various embodiments, the ligand-binding domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 1444-1489. In various embodiments, the stimulatory region is operably linked with a transmembrane domain. In various embodiments, the transmembrane domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs 1490-1491. In various embodiments, the transmembrane domain and ligand-binding domains are separated by a flexible linker.[8] In at least one aspect, the present disclosure provides an engineered immune cell including a chimeric switch receptor (CSR) of the present disclosure and / or a stimulatory region of the present disclosure. In various embodiments, the cell is an NK cell. In various embodiments, the cell is a CD56+ cell. In various embodiments, the CD56+ 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 switch receptor (CSR) of the present disclosure and / or a stimulatory region of the present disclosure. In various embodiments, the cell is an NK cell. In various embodiments, the cell is a CD56+ cell. In various embodiments, the CD56+ 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, EGFR, 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 an autoimmune condition 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 autoimmune condition is selected from lupus, rheumatoid arthritis, ulcerative colitis, type I diabetes, systemic sclerosis, multiple sclerosis, Sjogren’s Syndrome, lupus nephritis, and myositis. In various embodiments, the administration is intravenous.DEFINITIONS

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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 abinding 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 natural immunoglobulins 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.

[0018] 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.

[0019] 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.

[0020] 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 are characteristic 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Chimeric switch receptor'. As used herein, “Chimeric switch receptor” or “CSR” refers to an engineered protein that includes (i) an extracellular domain that includes a moiety that binds a target ligand; (ii) a transmembrane domain; and (iii) an intracellular signaling domain that sends activatingsignals when the CSR is stimulated by binding of the extracellular binding moiety with a target ligand. A T cell, for example, that has been genetically engineered to express a chimeric switch receptors may be referred to as a CSR T cell. Thus, for example, when certain CSRs are expressed by a T cell, binding of the CSR extracellular binding moiety with a target ligand can activate the T cell.

[0025] 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.

[0026] 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.

[0027] 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 themto 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 are associated 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.

[0028] 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.

[0029] 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 more amino 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.

[0030] 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.

[0031] 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 atherapeutically 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 formulated and / 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.

[0032] 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

[0033] Fig. 1 is a schematic of an exemplary general architecture of CSRs, in which CSRs can be designed to bind soluble or bound ligands.

[0034] Fig. 2 describes an exemplary CSR architecture in which CSRs that include combinations of distinct stimulatory sequences derived from different full protein domains are expressed in NK cells. NK cells are subsequently subjected to a serial restimulation assay.

[0035] Fig. 3 is a graph displaying data from an example in which CSRs that include a TGFbRII-derived ligand binding domain and distinct combinations of stimulatory sequences derived from different full protein domains (SEQ ID NOs 16-100) are expressed in NK cells that are subsequently subjected to a serial restimulation assay in the presence of 40 ng / mL TGFb. The chart depicts the log2 fold-change of NK cell numbers for NK cells expressing different CSR designs at day 11 versus day 0 after daily stimulation with Raji target cells at a 1:1 NK:Raji ratio.

[0036] Fig. 4 is a graph displaying data from an example in which CSRs that include a TGFbRII-derived ligand binding domain and distinct combinations of stimulatory sequencesderived from different full protein domains (SEQ ID NOs 101-200) are expressed in NK cells that are subsequently subjected to a serial restimulation assay in the presence of 40 ng / mL TGFb. The chart depicts the log2 fold-change of NK cell numbers for NK cells expressing different CSR designs at day 11 versus day 0 after daily stimulation with Raji target cells at a 1:1 NK:Raji ratio.

[0037] Fig. 5 is a graph displaying data from an example in which CSRs that include a TGFbRII-derived ligand binding domain and distinct combinations of stimulatory sequences derived from different full protein domains (SEQ ID NOs 201-300) are expressed in NK cells that are subsequently subjected to a serial restimulation assay in the presence of 40 ng / mE TGFb. The chart depicts the log2 fold-change of NK cell numbers for NK cells expressing different CSR designs at day 11 versus day 0 after daily stimulation with Raji target cells at a 1:1 NK:Raji ratio.

[0038] Fig. 6 is a graph displaying data from an example in which CSRs that include a TGFbRII-derived ligand binding domain and distinct combinations of stimulatory sequences derived from different full protein domains (SEQ ID NOs 301-377) are expressed in NK cells that are subsequently subjected to a serial restimulation assay in the presence of 40 ng / mE TGFb. The chart depicts the log2 fold-change of NK cell numbers for NK cells expressing different CSR designs at day 11 versus day 0 after daily stimulation with Raji target cells at a 1:1 NK:Raji ratio.

[0039] Fig. 7 is a graph displaying data from an example in which CSRs that include a TIGIT ligand binding domain and distinct combinations of stimulatory sequences derived from different full protein domains (SEQ ID NOs 378-491) are expressed in NK cells that are subsequently subjected to a serial restimulation assay against K562 target cells expressing the TIGIT ligand CD 155. The chart depicts the log2 fold-change of NK cell numbers for NK cells expressing different CSR designs at day 11 versus day 0 after daily stimulation with K562 target cells at a 1:1 NK:K562 ratio.DETAILED DESCRIPTION

[0040] The present disclosure provides, among other things, sequences for use in stimulatory domains of CSRs (which can be referred to herein as “stimulatory sequences”). In various embodiments, the present disclosure provides sequences for full CSRs including specific ligand-binding domains, transmembrane domains, and stimulatory domains. In various embodiments, the present disclosure provides sequences for use in stimulatory domains of CSRs that are particularly useful in engineering of NK cells. Accordingly, the presentdisclosure includes NK cells engineered to express CSRs (CSR-NK cells) including stimulatory sequences of the present disclosure.Chimeric Switch Receptors

[0041] CSRs are engineered proteins designed to redirect and amplify the response of immune cells in the presence of specific ligands. CSRs generally include three modules: an extracellular binding domain, a transmembrane domain, and one or more intracellular stimulatory sequences (see, e.g., Figs. 1 and 2). 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 CSR 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 CSR function cooperatively, those of skill in the art appreciate that each is an independently engineered and independently useful component.

[0042] An extracellular ligand binding domain can be or include a binding domain such as an antibody or antibody fragment, that specifically binds an antigen, or can be or include a binding domain derived from the extracellular domain of a native surface receptor that binds to a ligand that is soluble, presented on extracellular matrix (e.g., on a diseased cell or a neighboring cell). For example, an extracellular domain can be an scFv or nanobody that specifically binds a given antigen target, or an extracellular binding domain from an immune checkpoint or cytokine receptor that binds a soluble or cell-displayed ligand. In some embodiments, different leader peptide sequences can be used to dictate trafficking of the receptor to the cell membrane.

[0043] Transmembrane domains within a CSR 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. CSR 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.

[0044] Broadly, intracellular stimulatory sequences determine the signaling consequences of ligand binding, but in a way that is dependent on the extracellular receptor and target ligand. The first CSR stimulatory sequences described utilized co-stimulatory domains borrowed from Chimeric Antigen Receptor (CAR) designs and featured single signaling domains.

[0045] By contrast, the exploration of multiple stimulatory sequences in combination, and the interaction between stimulatory sequence function and choice of extracellular ligand context for CSR design has been limited.CSR-expressing immune cells

[0046] Without wishing to be bound by any particular scientific theory, CSRs function by tying a ligand-binding event to specific signaling activity in the immune cell. Such function can enhance immune cell function by converting otherwise inhibitory extracellular signals into positive signals, or by conferring a degree of context-dependent signaling to the immune cells. CSRs in T cells can be designed to convert otherwise suppressive signals into positive intracellular signals along the canonical T cell activation pathways. For example, CSR 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. CSRs can also be expressed in NK cells (canonically CD56+, CD3-). In the mammalian immune system, NK cells activate by summing signals across a wide range of primary and supporting activating receptors such as CD 16, NKG2D, NKp46, and 2B4. NK cells also express a broad range of inhibitory receptors such as TGFbRII, TIGIT, Fas, and KIRs, among others. These native activating receptors represent functional and signaling information that is distinct from that of canonical TCR signaling in T cells. Thus, the present disclosure includes the recognition that there exists a breadth of potential NK-specific CSR designs that incorporate features of NK cell activation, including features that fall outside of canonical NK activation pathways.Stimulatory Sequences

[0047] Without wishing to be bound by any particular scientific theory, binding of a target ligand initiates downstream CSR signaling events through recruitment of adapter and second messenger proteins to stimulatory sequences (including, e.g., domains and motifs associated with stimulatory activity). Downstream CSR 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. Legacy CSR 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.

[0048] Several alternative stimulatory domains have been explored in NK cells in other engineered receptor contexts, including CD28, 2B4, and 0X40. However, the present disclosure includes the recognition that there is a need for further, alternative, and / or improved stimulatory sequences for use in NK cells (e.g., NK-CSR cells), and that certain such sequences can be advantageously selected and / or derived from NK-native stimulatory domains. The present disclosure includes the recognition that such stimulatory sequences and combinations thereof can provide an increased diversity in CSR signaling and functional outcomes (e.g., in CSRs and / or for NK-CSR cells), and / or drive enhanced stimulation.

[0049] The present disclosure discloses stimulatory sequences and combinations thereof that are, e.g., particularly useful in engineering of CSRs for use in NK cells, and production of CSR-NK cells.

[0050] The present disclosure includes the discovery that certain stimulatory sequences identified herein as useful in CSRs and / or NK-CSR 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.

[0051] The present disclosure describes two categories of stimulatory sequences: those incorporating one or more full protein domains, and those incorporating one or more individual signaling motifs. Among stimulatory sequences representing one or more protein domains provided herein, various such domains are characterized by a certain biological function when present in cells, and combinations of full stimulatory sequence domains having certain such biological functions give rise to unexpectedly advantageous properties, e.g., for NK cell activation. CD40 (e.g. included in SEQ ID NOs 101-116) is essential for mediating a broad variety of immune and inflammatory responses via NFkB signaling. 4- 1BB (e.g. included in SEQ ID NOs 22-34) signaling results in increased NFkB pathway activation. DAP10 (e.g. included in SEQ ID NO 110) is involved in JAK3 / STAT5a and PI3K signaling. CD27 transduces signals that lead to the activation of NFkB and MAPK8 / JNK. CD 16 (e.g. included in SEQ ID NOs 182-185) domains contain immunomodulatory tyrosine activating motifs (IT AMs) and are a canonical route of NK cell activation. FCERG (e.g. included in SEQ ID NOs 172-180) activation domains also contain activating motifs and have been used as alternatives to CD3z in CSR designs.Table 1: Stimulatory sequence domainsTable 2: Stimulatory sequences derived from one or more stimulatory sequences

[0052] For the avoidance of doubt, Table 2 provides the sequences of stimulatory regions (SEQ ID NOs: 16-491) that include a first stimulatory domain sequence (SEQ ID NOs: 492- 967) and a second stimulatory domain sequence (SEQ ID NOs: 968-1443). Each of the stimulatory regions according to SEQ ID NOs: 16-491 consists of, from N terminus to Cterminus, (1) the indicated first domain sequence, (2) the GS linker, and (3) the indicated second domain sequence.

[0053] The present disclosure includes the recognition that stimulatory regions that include a first stimulatory domain sequence (SEQ ID NOs: 492-967) and a second stimulatory domain sequence (SEQ ID NOs: 968-1443), e.g., in combinations as set forth in rows of Tables 2, 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 2) 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 TCR and / or NK 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 can potentially limit steric hindrance effects that might otherwise hamper the downstream function driven by each sequence.

[0054] 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).

[0055] 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

[0056] CSR designs disclosed herein can drive diverse and useful cell states in engineered cell therapies. Enhancing the signaling and receptor space captured by CSR designs is a strategy for enhancing therapeutically relevant cell characteristics in a ligand-binding dependent manner. For example, transmembrane domains that modulate CSR presentation levels on the surface of immune cells can be used to tune the sensitivity of the immune cell response to different levels of a target (such as a ligand or antigen). Alternatively, stimulatory domains can 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 can be largely derived from native receptor sequences, or manipulated at the level of individual protein binding motifs. Furthermore, alternative extracellular receptor domains targeting different modes of immunosuppressive or context- specific ligands can be employed to tune cell therapy function to specific therapeutic contexts.

[0057] 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.

[0058] Fnl4-related domains as core stimulatory domains represent a strategy to improve NK cell expansion and survival in addition to cytotoxicity, owing to previous descriptions of Fnl4 driving non-canonical NFkB signaling in NK cells. The observed function of Fnl4- derived domains in combination with CD27, CD3z, JAK2, IE-7RA, 0X40 and CD40 domains, among others, demonstrates the ability to layer novel signaling cascades that drive pro-fitness effects.

[0059] Similarly, cytokine signaling related domains such as JAK1, JAK3, and IE-7RA represent a strategy to enhance NK cell expansion and survival in addition to cytotoxicity given previous descriptions of various JAK / STAT pathway signaling driving NK cellproliferation and homeostasis. Further, these domains represent a strategy for mimicking cytokine-dependent signaling absent an external cytokine signal.

[0060] CSR designs can utilize a diverse array of ligand-binding domains, e.g., where ligand binding stimulates and / or activates a host cell, e.g., via the CSR’s stimulatory domains. In some embodiments, a corresponding ligand binding event in non-engineered mammalian (e.g., human) cells (e.g., between a receptor including a wild-type sequence that corresponds to the ligand-binding domain, and a natural ligand of that receptor) is recognized as having at least certain inhibitory functions (e.g., functions that suppress cell activation). Example surface receptor ligand-binding domains that could be used in CSRs include TGFbRII, TGFbRI, TIGIT, PD-1, TIM-3, LAG-3, CD96, CD-161, NKG2A, KIR, CD47, SIRPa, CD24, PODXL, CAV1, CTLA-4, CD200R1, BTLA, BTN3A1, VISTA, IL-lORa, IL-4Ra, CD94, Siglec-3, Siglec-9, CD161, LAIR1, KLRG1, LILRB1, CEACAM-1, FAS, TNFRSF21, TRAILR2, TRAILR1, CD120a, TRAMP, DR-6, DNAM-1, NKG2D, CD16, NKp30, NKp44, NKp46, CRTAM, NKp80, CD28, 4-1BB, Fnl4, CD40, and CD40L, or ligand-binding domains thereof. The native versions of these receptors encode both inhibitory and activating functions. Thus, various CSRs disclosed herein convert inhibitory signals into activating signals or alter the nuanced signaling properties of the activating ligands.

[0061] CSRs can be expressed in cells in multiple schemes, including with or without a chimeric antigen receptor (CAR) co-expressed. Because NK cells feature a native activating receptor repertoire, CSRs can be used to enhance the native signaling, or drive orthogonal signaling to that achieved through the native activating receptors, such as that promoting the persistence of the cell. Alternatively, CSRs can be co-expressed along with CARs. In this case, the CAR is driving activation of the cell. The CSR can be used to enhance that signaling or to complement it in order to achieve an independent outcome, such as enhancing the persistence or metabolism of the cell. The option to layer different effects across different receptors in the same cell is an advantageous cell engineering strategy in that specific functions such as activation or proliferation can become separably controllable across distinct ligand-binding events, and not all functions must be encoded in a single receptor.

[0062] CSRs can be useful in, for example, cell therapy applications in which the cell must operate in a suppressive environment, such as in a solid tumor. In these cases, a CSR can be used to convert suppressive signals to activating signals and promote therapeutic function. Alternatively, in autoimmune cell therapy contexts, a CSR can be used to convert a pro- inflammatory signal to an anti-inflammatory signal.

[0063] In various embodiments, a sequence and / or CSR of the present disclosure can be used for treatment of cancer. For example, a sequence and / or CSR of the present disclosure, or a composition or method including such a sequence and / or CSR, can be useful in a method of reducing size of solid tumors, inhibiting cancer growth or metastasis, treating various lymphatic cancers, and / or prolonging the survival time of mammals (including humans) suffering from cancer. Examples of cancers and cancer conditions for which a sequence and / or CSR of the present disclosure, or a composition or method including such a sequence and / or CSR, can be useful include, but are not limited to, cancers of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves and other parts of the CNS, such as glioblastomas or medulloblastomas); head and / or neck cancer, breast cancers, cancers of the circulatory system (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular cancers, and tumor-associated vascular tissue); cancers of the blood and lymphatic system (e.g., Hodgkin's disease, Non-Hodgkin's disease lymphoma, Burkitt's lymphoma, AIDS-related lymphomas, malignant immunoproliferative diseases, multiple myeloma, and malignant plasma cell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell type, leukemia of unspecified cell type, unspecified malignant neoplasms of lymphoid, haematopoietic and related tissues, such as diffuse large cell lymphoma, T-cell lymphoma or cutaneous T-cell lymphoma); cancers of the excretory system (e.g., kidney, renal pelvis, ureter, bladder, and other urinary organs); cancers of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal); cancers involving the liver and intrahepatic bile ducts, gall bladder, and other parts of the biliary tract, pancreas, and other digestive organs; cancers of the oral cavity (e.g., lip, tongue, gum, floor of mouth, palate, parotid gland, salivary glands, tonsil, oropharynx, nasopharynx, puriform sinus, hypopharynx, and other sites of the oral cavity); cancers of the reproductive system (e.g., vulva, vagina, Cervix uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); cancers of the respiratory tract (e.g., nasal cavity, middle ear, accessory sinuses, larynx, trachea, bronchus and lung, such as small cell lung cancer and non-small cell lung cancer); cancers of the skeletal system (e.g., bone and articular cartilage of limbs, bone articular cartilage and other sites); cancers of the skin (e.g., malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and cancers involving other tissues including peripheral nerves and autonomic nervous system, connective and softtissue, retroperitoneoum and peritoneum, eye and adnexa, thyroid, adrenal gland, and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasms of other sites.

[0064] In various embodiments, a sequence and / or CSR of the present disclosure can be used for treatment of an autoimmune disease. In various embodiments, a sequence and / or CSR of the present disclosure can be used for treatment of an autoimmune disease selected from, e.g., lupus nephritis, rheumatoid arthritis, type I diabetes, multiple sclerosis, Hashimoto thyroiditis, pernicious anemia, primary biliary cirrhosis, Sjogren syndrome, Addison disease, celiac disease, reactive arthritis, myasthenia gravis, dermatomyositis, Graves disease, systemic sclerosis, limbic encephalitis, pemphigus vulgaris, and Devic’s syndrome.EXAMPLES

[0065] The present Examples demonstrate that compositions and methods of the present disclosure can impart enhanced functionality in immune cells. These Examples demonstrate, among other things, the ability to drive enhanced proliferation of CSR-NK cells upon repeated antigen exposure, which addresses a commonly cited barrier to CSR-NK function therapeutically.Example 1: Functional assessment of CSRs by serial restimulation assay in the context of TGFb immunosuppression

[0066] The present Example demonstrates that specific CSR sequences (SEQ ID NOs: 1-13 and 16-377) drive enhanced cell proliferation of NK cells, in the context of an 11 day serial restimulation with Raji tumor cells under otherwise suppressive TGFb incubation. In the present Example, full CSR designs included an N-terminal signal (SEQ ID NO: 1492), a TGFbRII extracellular domain (SEQ ID NO 1450), the TGFbRII transmembrane domain (SEQ ID NO 1490), and stimulatory sequences.

[0067] In these experiments, NK cells were transduced with different CSRs including different stimulatory sequences or combinations thereof, and co-cultured with cells from the Raji CD 19+ tumor cell line in high TGFb conditions (40 ng / mL) at a 1:1 ratio to create a tumor- mimicking suppressive cytokine stress. In the first experiments, NK cells killed the Raji cells, after which additional target cells were added to the cultures to maintain a 1:1 ratio. Each day, the NK cells were counted in order to gauge the degree of NK expansion in conditions of daily antigen refresh and at 11 days, remaining NK cells were sequenced toidentify enriched CSR designs that persisted. In a second experiment, an identical co-culture was used, but acute cytotoxicity after 4 hours was measured via CD107a staining of NK cells followed by sequencing for enriched CSR designs. Fig. 2 depicts log2-fold-change enrichment values for top CSR designs, where that enrichment is seen in either the serial restimulation or acute cytotoxicity formats. SEQ ID NOs: 1-13 and 16-377 demonstrated positive enrichment over either the 11 day serial restimulation experiment or 4 hour acute cytotoxicity experiment and are depicted in Figure 3. Notably, screening experiments identifying these sequences additionally included approximately 10,000 CSR sequences that did not demonstrate positive expansion. Such effects demonstrate therapeutic utility at least in that serial killing of tumor cells in the presence of immunosuppressive TGFb enhances the therapeutic area-under-the-curve of NK cell therapies, which is broadly advantageous in therapeutic contexts and particularly advantageous for treatment of solid tumors that are high in TGFb.Example 2: Functional assessment of CSRs by serial restimulation assay in the context of TIGIT immunosuppression

[0068] In this Example, CSR designs featured specific stimulatory sequences representing combinations of individual signaling motifs incorporated into a CSR with an N-terminal signal (SEQ ID NO: 1492), a TIGIT extracellular domain (SEQ ID NO: 1449) and the ROR2 transmembrane domain (SEQ ID NO 1491). This overall structure is employed to demonstrate the utility of stimulatory sequences based on signaling motifs.

[0069] In these experiments, NK cells were transduced with different CSR designs using the TIGIT ectodomain (SEQ ID NO 1445) and co-cultured with cells from the K562 cell line that were engineered to express high levels of the TIGIT ligand CD155 at a 1:1 ratio to create a tumor-mimicking immune checkpoint stress. In the first experiments, NK cells killed the K562 cells, after which additional target cells were added to the cultures to maintain a 1:1 ratio. Each day, the NK cells were counted in order to gauge the degree of NK expansion in conditions of daily antigen refresh and at 11 days, remaining NK cells were sequenced to identify enriched CSR designs that persisted. In a second experiment, an identical co-culture was used, but acute cytotoxicity after 4 hours was measured via CD107a staining of NK cells followed by sequencing for enriched CSR designs. Fig. 3 depicts log2-fold-change enrichment values for top CSR designs, where that enrichment is seen in either the serial restimulation or acute cytotoxicity formats. CSRs containing stimulatory sequences with SEQ ID NOs: 14-15 and 378-491 demonstrated positive enrichment over either the 11 day serialrestimulation experiment or 4 hour acute cytotoxicity experiment and are depicted in Figure 4. Notably, screening experiments identifying these sequences additionally included approximately 10,000 CSR sequences that did not demonstrate positive expansion. Such effects represent potential therapeutic utility in that metabolically sustainable serial killing of tumor cells enhances the therapeutic area-under- the-curve of NK cell therapies, which is broadly advantageous in therapeutic contexts and particularly advantageous for treatment of in solid tumors in which tumor cells express the immune checkpoint ligand CD155 and induce the inhibition of anti-tumor cytotoxicity.Materials and Methods for ExamplesStimulatory Sequence Cloning Strategy

[0070] A nested Golden Gate cloning strategy was used to create strings of stimulatory sequences in a CSR 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.Lentivirus Production

[0071] The above transfer plasmid encoding the CSR 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 NK cells by staining for CSR and observing transduction efficiency by flow cytometry.NK Cell Transduction

[0072] NK cells were seeded in NK 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 disCSRding the transduction media and adding fresh NK media. Cells were subsequently expanded using K562 feeder cells.NK Serial Restimulation Experiment

[0073] On Day 0, transduced CSR-NK cells were co-cultured with Raji or CD155+ K562 cells at a 1:1 ratio in basal NK media containing human serum AB and 5u / mL IL-2. Each subsequent day, NK and Raji cells were counted, and new Raji cells were added to reset the 1 : 1 NK:Raji ratio. NK cell counts at each timepoint represent the degree of NK expansion in continuous antigen-exposure conditions. For TGF-b experiments, TGF-b was added to the media to achieve a final concentration of 40 ng / mL at DO.NK Acute Cytotoxicity Experiment

[0074] Transduced CSR-NK cells were co-cultured with Raji or CD155+ K562 cells at a 1:1 ratio in basal NK media containing human serum AB and 5u / mL IL-2. After 4 hours of coculture, cells were removed from culture and live CD56+, CD107a+ cells were sorted, followed by Illumina sequencing. For TGF-b experiments, TGF-b was added to the media to 5 days prior to co-culture, achieving a final concentration of 40 ng / mL.Table 3: Full-length CSR Designs Used in ExamplesTable 4: Exemplary Ligand-binding Domains for use in CSRsTable 5: Transmembrane Domain Sequences Used in Example CSRsOTHER EMBODIMENTS

[0075] 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 as support 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 switch receptor (CSR) comprising a ligand-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: 1-15.

2. A chimeric switch receptor (CSR) comprising a ligand-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: 16- 491.

3. A chimeric switch receptor (CSR) comprising a ligand-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: 492-967 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: 968-1443, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 2 .

4. The CSR of claim 3, 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.

5. The CSR of claims 1-4, wherein the ligand-binding domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 1444-1489.

6. The CSR of any one of claims 1-5, wherein the transmembrane domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs 1490-1491.

7. The CSR of any one of claims 1-6, wherein the transmembrane domain and ligandbinding domains are separated by a flexible linker.

8. A stimulatory region comprising 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: 1-15.

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: 16-491.

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: 492-967 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: 968-1443, optionally wherein the first stimulatory sequence and second stimulatory sequence are each present in a row of Table 2.

11. The stimulatory region of claim 10, 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.

12. The stimulatory region of any one of claims 8-11, wherein the stimulatory region is operably linked with a ligand-binding domain, optionally wherein the ligand-binding domain comprises a native surface receptor extracellular domain.

13. The stimulatory region of claim 12, wherein the ligand-binding domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected fromSEQ ID NOs: 1444-1489.

14. The stimulatory region of any one of claims 8-13, wherein the stimulatory region is operably linked with a transmembrane domain.

15. The stimulatory region of claim 14, wherein the transmembrane domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs 1490-1491.

16. The stimulatory region of claim 14 or 15, wherein the transmembrane domain and ligand-binding domains are separated by a flexible linker.

17. An engineered immune cell comprising a chimeric switch receptor (CSR) according to any one of claims 1-7 or a stimulatory region according to any one of claims 8-16.

18. The engineered immune cell of claim 17, wherein the cell is an NK cell.

19. The engineered immune cell of claim 17 or 18, wherein the cell is a CD56+ cell.

20. The engineered immune cell of claim 19, wherein the CD56+ cell is differentiated from an induced pluripotent stem cell (iPSC), embryonic stem cell (ESC), or CD34+ progenitor cell (HSPC).

21. A method of producing an engineered immune cell, the method comprising contacting the immune cell with a nucleic acid encoding a chimeric switch receptor (CSR) according to any one of claims 1-7 or a stimulatory region according to any one of claims 8-16.

22. The method of claim 21, wherein the cell is an NK cell.

23. The method of claim 21 or 22, wherein the cell is a CD56+ cell.

24. The method of claim 23, wherein the CD56+ cell is differentiated from an induced pluripotent stem cell (iPSC), embryonic stem cell (ESC), or CD34+ progenitor cell.

25. The method of any one of claims 21-24, wherein the contacting comprises viral delivery of the nucleic acid to the cell.

26. The method of any one of claims 21-24, wherein the contacting comprises non- viral delivery of the nucleic acid to the cell.

27. 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 17-20.

28. The method of claim 27, wherein the cancer is a solid tumor.

29. The method of claim 28, 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.

30. The method of claim 27, wherein the cancer is a liquid cancer.

31. The method of claim 30, 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).

32. The method of any one of claims 27-31, 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.

33. The method of any one of claims 27-32, wherein the administration is intravenous.

34. The method of any one of claims 27-32, wherein the administration is peri-tumoral.

35. The method of any one of claims 27-32, wherein the administration is intra-tumoral.

36. A method of treating an autoimmune condition in a subject in need thereof, the method comprising administering to the subject an engineered immune cell according to any one of claims 17-20.

37. The method of claim 36, wherein the autoimmune condition is selected from lupus, rheumatoid arthritis, ulcerative colitis, type I diabetes, systemic sclerosis, multiple sclerosis, Sjogren’s Syndrome, lupus nephritis, and myositis.

38. The method of claim 36 or 37, wherein the administration is intravenous.