Chimeric cell surface receptors and methods of use

Abstract

Provided are chimeric cell surface receptors comprising an extracellular domain of a cell surface receptor and an intracellular domain of a different cell surface receptor. In certain embodiments, a chimeric cell surface receptor of the present disclosure comprises an extracellular domain of a tumor necrosis factor receptor (TNFR) and an intracellular domain of a different TNFR or a receptor other than a TNFR. According to other embodiments, a chimeric cell surface receptor of the present disclosure comprises an extracellular domain of an interleukin receptor (ILR) and an intracellular domain of a different ILR or a receptor other than an ILR. Also provided are cells (e.g., immune effector cells) that express a chimeric cell surface receptor of the present disclosure. Also provided are methods of using such cells to treat a condition (e.g., cancer, infection, and / or the like) in a subject in need thereof.

Description

[0001] Atty. Docket: STAN-2194WO (S24-101)

[0002] CHIMERIC CELL SURFACE RECEPTORS AND METHODS OF USE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 701 ,237, filed September 30, 2024, which application is incorporated herein by reference in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (STAN-2194WO_SEQLIST; Size: 1 ,079,750 bytes; and Date of Creating: September 29, 2025) is herein incorporated by reference in its entirety.

[0007] INTRODUCTION

[0008] T-cell exhaustion is a broad term used to describe T cell dysfunction resulting from chronic stimulation. Exhausted T cells present with a distinct phenotype including overexpression of inhibitory markers such as PD-1 , LAG-3 and TIM-3 as well as impairment in their ability to release pro-inflammatory cytokines (IFNy and TNFa). Exhaustion commonly occurs in the tumor microenvironment where T cells suffer a loss of their cytotoxic function and become ineffective in their ability to kill cancerous cells.

[0009] Adoptive cell therapies, including chimeric antigen receptor (CAR) T cell therapy, engineered T cell receptor (TCR) T cell therapy, and the like, have been successful across multiple hematological cancers but robust responses against solid tumors remain elusive. A major barrier to clearance of solid tumors is intrinsic dysfunction or exhaustion of CAR T cells, where chronic antigen contact and subsequent signaling leads to reduced cytotoxicity. Myriad strategies, including genetic manipulation, receptor optimization and tuning of response to extracellular signals have aimed at preventing the development or decreasing the severity of this dysfunction. Yet, a broadly effective dysfunction-resistance strategy remains to be identified.

[0010] SUMMARY

[0011] Provided are chimeric cell surface receptors. The chimeric cell surface receptors comprise an extracellular domain of a cell surface receptor and an intracellular domain of a different cell surface receptor. In certain embodiments, a chimeric cell surface receptor of the present disclosure comprises an extracellular domain of a tumor necrosis factor receptor (TNFR) and an intracellular domain of a different TNFR or a receptor other than a TNFR. According to other embodiments, a chimeric cell surface receptor of the present disclosure comprises an extracellular domain of an interleukin receptor (ILR) and an intracellular domain of a different ILR or a receptor other than an ILR. Also provided are cells (e.g., immune effector cells) that express a chimeric cell surface receptor of the present disclosure. In some instances, such cells (e.g., T cells) exhibit enhanced function relative to control cells not expressing the chimeric cell surface Atty. Docket: STAN-2194WO (S24-101) receptor. Also provided are methods of using such cells to treat a condition (e.g., cancer, infection, and / or the like) in a subject in need thereof.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 : Summary table indicating that cell surface receptors are among a class of proteins that are highly enriched as previous functional hits in human and mouse T cells, identifying this class of proteins as comprising candidates for synthetic engineering to further enhance immune cell function.

[0014] FIG. 2A-2B: FIG. 2A: Schematic illustration of non-chimeric (“natural”) cell surface receptors and a chimeric cell surface receptor comprising heterologous extracellular and intracellular domains. FIG. 2B: Schematic illustration of a modular cloning strategy employed to generate a large library of combinatorially assembled chimeric cell surface receptors.

[0015] FIG. 3A-3B: FIG. 3A: Schematic illustration of the library generated according to the cloning strategy shown in FIG. 2B. FIG. 3B: A plot of the Log2 Fold change in barcode abundance between the output and input populations, showing a large number of chimeric cell surface receptors that allowed T cells to proliferate better than control cells in a repetitive stimulation assay.

[0016] FIG. 4: Results summary showing the top individual chimeric and natural cell surface receptors in the CD19 CAR driven Nalm6 repetitive stimulation assay. Of note, numerous synthetic surface receptors performed significantly better than controls as measured by proliferation in the repetitive stimulation assay (Log2 fold change). The top performing non- chimeric receptor was IL1 R1 .

[0017] DETAILED DESCRIPTION

[0018] Before the chimeric polypeptides, compositions and methods of the present disclosure are described in greater detail, it is to be understood that the chimeric polypeptides, compositions and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the chimeric polypeptides, compositions and methods will be limited only by the appended claims.

[0019] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the chimeric polypeptides, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the chimeric polypeptides, compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of Atty. Docket: STAN-2194WO (S24-101) the limits, ranges excluding either or both of those included limits are also included in the chimeric polypeptides, compositions and methods.

[0020] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the chimeric polypeptides, compositions and methods belong. Although any chimeric polypeptides, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the chimeric polypeptides, compositions and methods, representative illustrative chimeric polypeptides, compositions and methods are now described.

[0022] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present chimeric polypeptides, compositions and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0023] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0024] It is appreciated that certain features of the chimeric polypeptides, compositions and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the chimeric polypeptides, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present chimeric polypeptides, compositions and methods and are Atty. Docket: STAN-2194WO (S24-101) disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0025] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0026] CHIMERIC CELL SURFACE RECEPTORS

[0027] Numerous classes of proteins exist which manipulate cellular function to respond and adapt to changing environments and needs of an organism. These proteins can be organized into distinct families based on the function, location with the cell, and mechanisms of action. Currently, genetic manipulation of adoptive cell therapies, including gene deletion or knockdown and overexpression, is largely restricted to the endogenous genome. As such, resultant phenotypes predictably exist along the known continuum of canonical cell differentiation (e.g., T cell differentiation) and are commonly described as more “stem-like” or “effector-like”. These cells, however, tend to succumb to similar dysfunctional fates as their unmanipulated counterparts. An approach to this problem, then, is to deliberately engineer cells into a synthetic state where cells do not differentiate into a dysfunctional state in the face of chronic stimulation, but rather persist and remain cytotoxic.

[0028] In engineered immune cell therapies such as CAR T cell therapy, screening efforts have identified hundreds of protein targets that confer altered cell function when overexpressed or knocked out in human and mouse T cells. The inventors have identified two classes of proteins, transcription factors and surface receptors, that are highly enriched as functional “hits” in human and mouse T cells, identifying these classes of proteins as candidates for synthetic engineering of improved members to further enhance immune cell function.

[0029] Aspects of the present disclosure include chimeric cell surface receptors comprising an extracellular domain of a cell surface receptor and an intracellular domain of a different cell surface receptor. The chimeric cell surface receptors of the present disclosure are based in part on the rationale that installing synthetic surface receptors onto cells (e.g., T cells) would allow control over cell fate and uncover favorable, unevolved cell phenotypes.

[0030] As proof of concept, demonstrated herein is a subset of chimeric cell surface receptors that significantly enhance T cell function relative to T cells not expressing the chimeric cell surface receptors. These chimeric cell surface receptors represent a novel class of synthetic sequences conferring enhanced cellular function and, with the benefit of the present disclosure, are expected to have broad applications across cellular engineering and cellular therapeutic applications. Atty. Docket: STAN-2194WO (S24-101)

[0031] Details regarding chimeric cell surface receptors according to embodiments of the present disclosure will now be described.

[0032] The chimeric cell surface receptors are chimeric cell surface receptor polypeptides. The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alphaamino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, non-natural amino acids, or a combination thereof. As used herein, the term “chimeric” refers to a polypeptide composed of domains of different origins, e.g., different naturally occurring proteins. A chimeric molecule, as a whole, is non-naturally occurring, e.g., synthetic or recombinant, although the domains which comprise the chimeric molecule can be naturally occurring.

[0033] In some aspects, provided are chimeric cell surface receptors comprising an extracellular domain of a cell surface receptor and an intracellular domain of a different cell surface receptor. In certain embodiments, a chimeric cell surface receptor of the present disclosure comprises an extracellular domain of a tumor necrosis factor receptor (TNFR) and an intracellular domain of a different TNFR or a receptor other than a TNFR. In some instances, the extracellular domain is of TNFR superfamily member 5 (TNFRSF5), TNFR superfamily member 6 (TNFRSF6), TNFR superfamily member 12A (TNFRSF12A), or TNFR superfamily member 17 (TNFRSF17). According to some embodiments, the extracellular domain is of TNFRSF6.

[0034] When the extracellular domain of a chimeric cell surface receptor is of a TNFR, in certain embodiments, the intracellular domain is of a different TNFR. Non-limiting examples of such intracellular domains include an intracellular domain of TNFR superfamily member 4 (TNFRSF4), TNFRSF5, TNFR superfamily member 7 (TNFRSF7), TNFR superfamily member 11 A (TNFRSF11 A), TNFRSF12A, TNFR superfamily member 13B (TNFRSF13B), TNFR superfamily member 13C (TNFRSF13C), TNFR superfamily member 14 (TNFRSF14), TNFRSF17, TNFR superfamily member 25 (TNFRSF25), or TNFR superfamily member 27 (TNFRSF27).

[0035] When the extracellular domain of a chimeric cell surface receptor is of a TNFR, in some instances, the intracellular domain is of a receptor other than a TNFR. In certain embodiments, such an intracellular domain is of an interleukin (IL) receptor. Non-limiting examples of such intracellular domains include an intracellular domain of IL2RA, IL5RA, IL6R, IL12RB1 , IL15RA, IL18R1 , IL20RA, or IL27RA. In other embodiments, when the extracellular domain is of a TNFR and the intracellular domain is of a receptor other than a TNFR, the intracellular domain is of B- and T-lymphocyte attenuator (BTLA), CD4, CD28, colony stimulating factor 2 receptor subunit alpha (CSF2RA), colony stimulating factor 3 receptor (CSF3R), Inducible T-cell COStimulator (ICOS), programmed cell death protein 1 (PDCD1 ), or transforming growth factor beta receptor 3 (TGFRBR3). Atty. Docket: STAN-2194WO (S24-101)

[0036] According to some embodiments, a chimeric cell surface receptor of the present disclosure comprises an extracellular domain of an interleukin receptor (ILR) and an intracellular domain of a different ILR or a receptor other than an ILR. In some instances, the extracellular domain is of IL1 R1 , IL7R, or IL9R.

[0037] When the extracellular domain of a chimeric cell surface receptor is of an ILR, in certain embodiments, the intracellular domain is of a different ILR. Non-limiting examples of such intracellular domains include an intracellular domain of IL17RE.

[0038] When the extracellular domain of a chimeric cell surface receptor is of an ILR, in certain embodiments, the intracellular domain is of a receptor other than an ILR. In some instances, the intracellular domain of a receptor other than an ILR is the intracellular domain of a TNFR. When the extracellular domain is of an ILR and the intracellular domain of a TNFR, the intracellular domain is of TNFRSF3.

[0039] Non-limiting examples of particular chimeric cell surface receptors of interest include those comprising: an extracellular domain of TNFRSF6 and an intracellular domain of IL15RA; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF5; an extracellular domain of TNFRSF6 and an intracellular domain of CD28; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF14; an extracellular domain of TNFRSF6 and an intracellular domain of PDCD1 ; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF12A; an extracellular domain of TNFRSF6 and an intracellular domain of TGFBR3; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF25; an extracellular domain of TNFRSF6 and an intracellular domain of ICOS; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF27; an extracellular domain of TNFRSF6 and an intracellular domain of BTLA; an extracellular domain of TNFRSF6 and an intracellular domain of CSF2RA; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF13B; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF4; an extracellular domain of TNFRSF6 and an intracellular domain of IL12RB1 ; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF17; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF7; an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF13C; an extracellular domain of TNFRSF6 and an intracellular domain of IL6R; an extracellular domain of TNFRSF6 and an intracellular domain of IL27RA; an extracellular domain of TNFRSF6 and an intracellular domain of IL5RA; an extracellular domain of TNFRSF6 and an intracellular domain of IL18R1 ; an extracellular domain of TNFRSF6 and an intracellular domain of CD4; an extracellular domain of TNFRSF6 and an intracellular domain of CSF3R; an extracellular domain of TNFRSF12A and an intracellular domain of IL2RA; an extracellular domain of TGFBR1 and an intracellular domain of BTLA; an extracellular domain of IL7R and an intracellular domain of IL17RE; an extracellular domain of CD28 and an intracellular domain of TNFRSF10D; an extracellular domain of IL9R and an intracellular domain of TNFRSF3; an extracellular domain of TNFRSF12A and an intracellular domain of TNFRSF1 1 A; Atty. Docket: STAN-2194WO (S24-101 ) an extracellular domain of TNFRSF5 and an intracellular domain of IL20RA; or an extracellular domain of TNFRSF17 and an intracellular domain of IL12RB1 .

[0040] The amino acid sequences of the extracellular and intracellular domains referenced herein are known for various mammals. For example, the amino acid sequences of mouse and human cell surface receptors are known and readily available from resources such as UniProt, the National Center for Biotechnology Information (NCBI) (e.g., RefSeq), and the like. In addition, the amino acid sequences of exemplary cell surface receptors, extracellular and intracellular domains thereof, and exemplary signal peptides for cell surface expression are provided in Table 1 below. Table 1 - Amino Acid Sequences of Cell Surface Receptors, Domains Thereof and Signal Peptides Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101) Atty. Docket: STAN-2194WO (S24-101)

[0041] As will be appreciated, the extracellular domain and / or intracellular domains of a chimeric cell surface receptor of the present disclosure may comprise the wild-type (e.g., wild-type human) amino acid sequences of their respective cell surface receptor, or alternatively, one or both of the extracellular and / or intracellular domains may comprise a variant amino acid sequence relative to the wild-type domain sequence. When one or both of the extracellular and / or intracellular domains comprise a variant amino acid sequence, the amino acid sequence may comprise 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the corresponding wild-type domain. A chimeric polypeptide that comprises one or two variant domains will retain receptor and signaling activity. When a population of T cells expresses a chimeric cell surface receptor comprising one or two variant domains, in some instances, the T cell population exhibits at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of a desirable property exhibited by a T cell population expressing a chimeric polypeptide comprising the same extracellular and intracellular domains as the variant domain(s), but where the extracellular and intracellular domains comprise the wild-type domain sequences. Non-limiting examples of desirable properties include enrichment in an in vitro repetitive stimulation assay (see, e.g., Example 1 below), reduced exhaustion as determined in vitro or in vivo as compared to control T cells not expressing the chimeric cell surface receptor, enhanced efficacy (e.g., enhanced anti-tumor and / or anti-viral infection efficacy) as determined in vitro or in vivo as compared to control T cells not expressing the chimeric cell surface receptor, and / or the like.

[0042] In certain embodiments, a variant chimeric cell surface receptor comprises one or more amino acid substitutions, deletions, or truncations (e.g., a truncation at the N-terminus and / or C- terminus of the domain). In some instances, one or more amino acid substitutions (e.g., each amino acid substitution) are conservative substitutions. Conservative substitutions are shown in Atty. Docket: STAN-2194WO (S24-101 )

[0043] Table 2 under the heading of “preferred substitutions.” More substantial changes are provided in Table 2 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into a polypeptide and the products screened for a desired activity, e.g., retained / improved ligand binding, retained / improved intracellular signaling, retained or enhanced reduction of exhaustion, retained or enhanced anti-tumor and / or anti-viral infection efficacy, and / or the like.

[0044] Table 2 - Amino Acid Substitutions Atty. Docket: STAN-2194WO (S24-101)

[0045] Amino acids may be grouped according to common side-chain properties:

[0046] (1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0047] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0048] (3) acidic: Asp, Glu;

[0049] (4) basic: His, Lys, Arg;

[0050] (5) residues that influence chain orientation: Gly, Pro;

[0051] (6) aromatic: Trp, Tyr, Phe.

[0052] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0053] NUCLEIC ACIDS, EXPRESSION CONSTRUCTS, CELLS AND COMPOSITIONS

[0054] Nucleic Acids and Expression Constructs

[0055] Aspects of the present disclosure further include nucleic acids and expression constructs. For example, provided are nucleic acids encoding any of the chimeric cell surface receptors of the present disclosure. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides, and may be produced enzymatically or synthetically. The term “nucleotide” is intended to include those moieties that contain not only the naturally occurring purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.

[0056] Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a chimeric cell surface receptor of interest provides a description of all the polynucleotides capable of encoding the chimeric cell surface receptor of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the polypeptides and domains disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the chimeric cell surface receptor of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of Atty. Docket: STAN-2194WO (S24-101) polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences of the cell surface receptors and domains thereof set forth in Table 1 . Non-limiting examples of nucleotide sequences encoding the cell surface receptors and domains thereof set forth in Table 1 are set forth in SEQ ID NOs: 430-858.

[0057] The nucleotide sequences of the nucleic acids of the present disclosure may be codon- optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a polypeptide to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding a polypeptide may be codon- optimized for optimal production from the host organism selected for expression, e.g., human cells, such as human immune cells (e.g., human T cells).

[0058] Also provided are expression constructs comprising any of the nucleic acids of the present disclosure. As used herein, an “expression construct” is a circular or linear polynucleotide (a polymer composed of naturally occurring and / or non-naturally occurring nucleotides) comprising a region that encodes a natural or chimeric cell surface receptor of the present disclosure, operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the polypeptide is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoter, enhancer, etc., present in the expression construct. By “heterologous” in this context is meant the regulatory element (e.g., promoter) to which the nucleic acid is operably linked is a regulatory element which is not operably linked to the nucleic acid in nature.

[0059] In some embodiments, expression of the cell surface receptor may be controlled by one or more endogenous regulatory elements, e.g., promoter, enhancer, etc., at or near a genomic locus into which the expression construct is inserted or knocked in. In some instances, the one or more endogenous regulatory elements are of the T-cell receptor a constant (TRAC) locus. For example, the nucleic acid encoding the cell surface receptor may be knocked into TRAC locus, e.g., integrated in-frame at exon 1 of the TRAC locus as described, e.g., by Eyquem et al. (2017) Nature 543:1 13-1 17 and Roth et al. (2018) Nature 559(7714) :405-409.

[0060] The expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression constructs may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the first subunit, Atty. Docket: STAN-2194WO (S24-101) the second subunit, or both. The expression constructs optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0061] To obtain high levels of expression of a cloned nucleic acid it is common to construct expression constructs which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli 's also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the polypeptide are available using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used. Transducing cells with nucleic acids (e.g., expression constructs) can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector.

[0062] In certain embodiments, upon delivery of an expression construct to cells, one or more of the expression constructs are episomal (e.g., extra-chromosomal), where by “episome” or “episomal” is meant a polynucleotide that replicates independently of the cell’s chromosomal DNA. A non-limiting example of an episome that may be employed is a plasmid.

[0063] According to some embodiments, upon delivery of an expression construct to cells, the expression construct integrates (e.g., by insertion or knock-in) into the genome of the cell. In certain embodiments, the expression construct is adapted for site-specific integration into the genome. For example, an expression construct may be adapted for site-specific integration into the genome, where the site-specific integration inactivates a gene within the genome of the cell. Functional integration of an expression construct may be achieved through various means, including through the use of integrating vectors, including viral and non-viral vectors. In some instances, a retroviral vector, e.g., a lentiviral vector, may be employed. In some instances, a non-retroviral integrating vector may be employed. An integrating vector may be contacted with the cells in a suitable transduction medium, at a suitable concentration (or multiplicity of infection), and for a suitable time for the vector to infect the target cells, facilitating functional integration of the expression construct. Non-limiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated virus (AAV) vectors, hybrid Ad-AAV vector systems, and the like.

[0064] In certain embodiments, an expression construct of the present disclosure comprises a ribosome skipping site to allow for polycistronic expression of a natural or chimeric cell surface receptor and one or more additional polypeptides, e.g., a recombinant receptor (e.g., a CAR) as Atty. Docket: STAN-2194WO (S24-101) described elsewhere herein. A non-limiting example of a suitable ribosome skipping site which may be incorporated into an expression construct is the P2A ribosome skipping site from porcine teschovirus.

[0065] Also provided by the present disclosure are messenger RNAs (mRNAs) encoding any of the natural or chimeric cell surface receptors of the present disclosure. In some instances, the mRNAs are encapsulated within particles, e.g., lipid nanoparticles. Such mRNAs and particles find use, e.g., for expression of a polypeptide ex vivo or in vivo upon delivery of the mRNA to cells of a subject. Strategies for delivering mRNA-based therapeutics are described, e.g., in Kowalski et al. (2019) Mol Ther. 27(4) :710-728; and Hou et al. (2021 ) Nature Reviews Materials 6:1078-1094.

[0066] Cells and Compositions

[0067] Aspects of the present disclosure further include cells comprising a nucleic acid of the present disclosure, as well as cells comprising an expression construct of the present disclosure. In certain embodiments, the cells are prokaryotic cells (e.g., bacteria), yeast cells, insect (e.g, drosophila) cells, amphibian (e.g., frog, e.g., Xenopus) cells, plant cells, etc. According to some embodiments, the cells are mammalian cells. Mammalian cells of interest include human cells, rodent cells, and the like.

[0068] In some embodiments, the cells are immune cells, e.g., human immune cells. Non-limiting examples of immune cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the cells are T cells. When the immune cells comprise T cells, the T cells may comprise one or any combination of naive T cells (TN), cytotoxic T cells (TCTL), memory T cells (TMEM), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), tissue resident memory T cells (TRM), effector T cells (TEFF), regulatory T cells (TREGS), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha beta T cells (Tap), gamma delta T cells (TVB).

[0069] Approaches for introducing the nucleic acid or expression construct into cells of interest are known and may include contacting a population of cells with the nucleic acid or expression construct under conditions in which the nucleic acid or expression construct is delivered to cells of the population of cells. The contacting step may comprise contacting the population of cells with the nucleic acid or expression construct, e.g., by combining the cells and the nucleic acid or expression construct in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of the nucleic acid or expression construct into cells of the population of cells.

[0070] A variety of suitable approaches and conditions for the delivery of nucleic acids and expression constructs to cells are known. According to some embodiments, delivery is carried out by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, and / or the like. In certain embodiments, the nucleic acid Atty. Docket: STAN-2194WO (S24-101) or expression construct is introduced into cells of the population of cells by AAV transduction. The AAV vector may comprise ITRs from AAV2, and a serotype from any one of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10. According to some embodiments, the AAV vector comprises ITRs from AAV2 and a serotype from AAV6. In certain embodiments, the nucleic acid or expression construct is introduced into the cells (e.g., T cells) by lentiviral or retroviral transduction. The lentiviral vector backbone may be derived from HIV-1 , HIV-2, visna- maedi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (Bl V), or simian immunodeficiency virus (SIV). The lentiviral vector may be integration competent or an integrase deficient lentiviral vector (TDLV). In one embodiment, IDLV vectors including an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) are employed. In certain embodiments, a cell of the present disclosure expresses the natural or chimeric cell surface receptor.

[0071] According to some embodiments, a cell of the present disclosure expresses the natural or chimeric cell surface receptor and also expresses a recombinant receptor on the surface of the cell. Non-limiting examples of such recombinant receptors include a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), a synthetic notch receptor (synNotch), a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.

[0072] As used herein, with respect to a protein, the term “recombinant” means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein, and cells or organisms that express the protein. With respect to a nucleic acid, the term “recombinant” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant.

[0073] In certain embodiments, the recombinant receptor (e.g., a CAR) comprises an extracellular binding domain that binds a tumor antigen expressed on the surface of a cancer cell. Non-limiting examples of such tumor antigens include 5T4, AXL receptor tyrosine kinase (AXL), B7-H3, B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS- Atty. Docket: STAN-2194WO (S24-101) like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1 ), GD2 ganglioside (“GD2”), glycoprotein non-metastatic B (GPNMB), guanylate cyclase 2 C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal- associated membrane protein 1 (LAMP-1 ), Lewis Y, LIV-1 , leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1 ), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), programmed cell death receptor ligand 1 (PD-L1 ), programmed cell death receptor ligand 2 (PD- L2), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1 ), tissue factor (TF), T cell immunoglobulin and mucin protein-1 (TIM-1 ), Tn antigen, trophoblast cell-surface antigen (TROP-2), Wilms’ tumor 1 (WT1 ), and VEGF-A.

[0074] As described above, according to some embodiments, the recombinant receptor may be a CAR. The extracellular binding domain of the CAR may comprise a single chain antibody. The single-chain antibody may be a monoclonal single-chain antibody, a chimeric single-chain antibody, a humanized single-chain antibody, a fully human single-chain antibody, and / or the like. In one non-limiting example, the single chain antibody is a single chain variable fragment (scFv). In some embodiments, the extracellular binding domain of the CAR is a single-chain version (e.g., an scFv version) of an antibody approved by the United States Food and Drug Administration and / or the European Medicines Agency (EMA) for use as a therapeutic antibody. Non-limiting examples of single-chain antibodies which may be employed when the protein of interest is a CAR include single-chain versions (e.g., scFv versions) of Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab,

[0075] Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab,

[0076] Panitumumab, Patritumab, Pritumumab, Radretumab, Ramucirumab, Rilotumumab,

[0077] Robatumumab, Seribantumab, Tarextumab, Teprotumumab, Tovetumab, Vantictumab,

[0078] Vesencumab, Votumumab, Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab, Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Cantuzumab, Citatuzumab, Clivatuzumab, Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Atty. Docket: STAN-2194WO (S24-101)

[0079] Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or an antigenbinding variant thereof.

[0080] When the cells are engineered to express a recombinant receptor on the surface thereof, the receptor may include one or more linker sequences between the various domains. A “variable region linking sequence” is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that includes the same light and heavy chain variable regions. A non-limiting example of a variable region linking sequence is a glycine-serine linker, such as a (648)3 linker as described above. In certain embodiments, a linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. In particular embodiments, the receptor (e.g., CAR) includes one, two, three, four, or five or more linkers. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more amino acids in length.

[0081] In some embodiments, when the cells are engineered to express a recombinant receptor on the surface thereof, the antigen binding domain of the receptor (e.g., CAR) is followed by one or more spacer domains that moves the antigen binding domain away from the cell surface (e.g., the surface of a T cell (e.g., a CD8+ or CD4+ T cell) expressing the receptor) to enable proper cell / cell contact, antigen binding and / or activation. The spacer domain (and any other spacer domains, linkers, and / or the like described herein) may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In some embodiments, the spacer domain includes the CH2 and / or CH3 of lgG1 , lgG4, or IgD. Illustrative spacer domains suitable for use in the receptors (e.g., CARs) described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD4, which may be wild-type hinge regions from these molecules or variants thereof. In certain embodiments, the hinge domain includes a CD8a hinge region. According to some Atty. Docket: STAN-2194WO (S24-101) embodiments, the hinge is a PD-1 hinge or CD152 hinge. In certain embodiments, the hinge is an lgG4 hinge.

[0082] The “transmembrane domain” (Tm domain) is the portion of the receptor (e.g., CAR) that fuses the extracellular binding portion and intracellular signaling domain and anchors the receptor to the plasma membrane of the cell (e.g., T-cell, such as a Treg). The Tm domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the Tm domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell receptor, CD35, CD3^, CD3y, CD36, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.

[0083] In one embodiment, a receptor (e.g., CAR) includes a Tm domain derived from CD28. In certain embodiments, a receptor includes a Tm domain derived from CD28 and a short oligo- or polypeptide linker, e.g., between 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the Tm domain and the intracellular signaling domain of the receptor. A glycine-serine linker may be employed as such a linker, for example.

[0084] The “intracellular signaling” domain of a receptor (e.g., a CAR) refers to the part of the receptor that participates in transducing the signal from binding to a target molecule / antigen into the interior of the cell to elicit cell function. Accordingly, the term “intracellular signaling domain” refers to the portion of a protein which transduces the signal and that directs the cell to perform a specialized function. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of a full-length intracellular signaling domain as long as it transduces the signal. The term intracellular signaling domain is meant to include any truncated portion of an intracellular signaling domain sufficient for transducing signal.

[0085] Signals generated through the T cell receptor (TCR) alone are insufficient for full activation of the T cell, and a secondary or costimulatory signal is also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR / CD3 complex) and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal. As such, a receptor (e.g., CAR) expressed by a genetically modified cell may include an intracellular signaling domain that includes one or more (e.g., 1 , 2, or more) “costimulatory signaling domains” and a “primary signaling domain.”

[0086] Primary signaling domains regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in a receptor of the present disclosure include those derived from FcRy, FcRp, CD3y, CD35, CD3s, CD3^, CD22, CD79a, CD79P, and CD665. In certain embodiments, Atty. Docket: STAN-2194WO (S24-101) a receptor includes a CD3^ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.

[0087] In some embodiments, when the cells of the present disclosure are engineered to express a recombinant receptor on the surface thereof, the receptor (e.g., CAR) includes one or more costimulatory signaling domains to enhance the efficacy and expansion of immune effector cells (e.g., T cells) expressing the receptor. As used herein, the term “costimulatory signaling domain” or “costimulatory domain” refers to an intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Example costimulatory molecules suitable for use in receptors contemplated in particular embodiments include TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1 BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70. In some embodiments, the receptor (e.g., CAR) includes one or more costimulatory signaling domains selected from the group consisting of 4-1 BB (CD137), CD28, and CD134, and a CD3^ primary signaling domain.

[0088] A recombinant receptor (e.g., CAR) may include any variety of suitable domains including but not limited to a leader sequence; hinge, spacer and / or linker domain(s); transmembrane domain(s); costimulatory domain(s); signaling domain(s) (e.g., CD3 domain(s)); ribosomal skip element(s); restriction enzyme sequence(s); reporter protein domains; and / or the like.

[0089] Also provided by the present disclosure are compositions comprising a population of the therapeutic immune cells of the present disclosure, e.g., any of the therapeutic immune cells described elsewhere herein.

[0090] Compositions suitable for adoptive cell therapy may be manufactured by expanding the therapeutic immune cells of the present disclosure. By “expanding” is meant the cells are cultured under conditions in which the cells proliferate. Suitable conditions may vary depending upon, e.g., the type of cells being expanded. Such conditions may include culturing the cell in a suitable container (e.g., a cell culture plate or well thereof, a cassette, tube, bottle or bag suitable for use in an automated therapeutic cell manufacturing system, e.g., a closed automated therapeutic cell manufacturing system such as the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, or the like), in suitable medium (e.g., cell culture medium, such as RPMI, DMEM, IMDM, MEM, DMEM / F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO2, e.g., 3% to 10%, such as 5%.

[0091] Methods for activating and expanding cells for therapy (e.g., therapeutic T cells and the like) are known in the art and are described, e.g., in U.S. Patent Nos. 6,905,874; 6,867,041 ; and 6,797,514; and PCT Publication No. WO 2012 / 079000, the contents of which are hereby Atty. Docket: STAN-2194WO (S24-101) incorporated by reference in their entirety. In the example of T cells, such methods may include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, the T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514, the contents of which are hereby incorporated by reference in their entirety.

[0092] In certain embodiments, the cells are expanded using an automated system designed for the manufacture of therapeutic cells. Non-limiting examples of such systems include the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, etc. Detailed guidance and protocols for manufacturing therapeutic cells on such systems are available from the providers of such systems.

[0093] Harvested therapeutic immune cell populations may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and / or device. Such populations of cells and pharmaceutical compositions may be prepared and / or used fresh or may be cryopreserved. In some instances, populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g, where the therapeutic cells are present in a suitable diluent and / or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media). Populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like.

[0094] In some instances, the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container. Cell therapy doses may be generated through a variety of methods. Aliquoting expanded populations of therapeutic cells into cell therapy doses may be performed by a variety of means. In some instances, a cell therapy dose includes, e.g., at least 10 million, at least 25 million, at least 50 million, at least 75 million, at least 100 million, at least 250 million, at least 500 million, at least 750 million, at least 1 billion, at least 2 billion, at least 3 billion, at least 4 billion, at least 5 billion, at least 6 billion, at least 7 billion, at least 8 billion, at least 9 billion, at least 10 billion, at least 15 billion, at least 20 billion, at least 30 billion, at least 40 billion, at least 50 billion, at least 60 billion, at least 70 billion, at least 80 billion, at least 90 billion, or at least 100 billion therapeutic cells. Atty. Docket: STAN-2194WO (S24-101)

[0095] In certain embodiments, the compositions may include the therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCI2, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3- aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

[0096] The compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control. An effective amount can be administered in one or more administrations.

[0097] The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and / or the like.

[0098] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0099] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0100] An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0101] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents Atty. Docket: STAN-2194WO (S24-101) may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0102] In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, tricresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v).

[0103] METHODS OF USE

[0104] Aspects of the present disclosure further include methods of using the cells of the present disclosure for therapy, e.g., to treat a cell proliferative disorder such as cancer, and / or to treat an infection such as a viral infection. In some instances, the cells are immune effector cells that express a natural or chimeric cell surface receptor of the present disclosure. According to some embodiments, the cells further express a recombinant receptor (e.g., a CAR) on their surface as described elsewhere herein.

[0105] In certain embodiments, provided are methods of administering an adoptive cell therapy to a subject (e.g., a human subject) having cancer. Such methods comprise administering to the subject a composition of the present disclosure (e.g., a composition comprising any of the therapeutic immune cells of the present disclosure) in an amount effective to treat the cancer.

[0106] According to some embodiments, the adoptive cell therapy is an adoptive T cell therapy, e.g., a CAR T cell therapy, a TIL therapy, a natural killer T (NKT) cell therapy, a therapy comprising administration of T cells expressing an engineered TCR, or the like. In some instances, the adoptive cell therapy is a natural killer (NK) cell therapy (e.g., a CAR NK cell therapy) or a macrophage therapy.

[0107] The therapeutic cells may be autologous / autogeneic (“self”) or non-autologous (“nonself”, e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells (e.g., T cells) are cells obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human.

[0108] As summarized above, in some embodiments, the subject has cancer. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Atty. Docket: STAN-2194WO (S24-101)

[0109] In some instances, the cancer comprises a solid tumor. According to some embodiments, the solid tumor is a carcinoma, lymphoma, blastoma, or sarcoma. When the solid tumor is a carcinoma, in certain embodiments, the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma.

[0110] According to some embodiments, the cancer comprises a hematological malignancy. For example, the subject treated by the methods of the present disclosure may have a hematological malignancy such as a leukemia, a lymphoma, or multiple myeloma.

[0111] According to some embodiments, the cancer of the subject is myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), multiple myeloma (MM), Non-Hodgkin’s lymphoma (NHL), non-small cell lung cancer, head and neck squamous cell carcinoma, gastroesophageal junction adenocarcinoma, gastric adenocarcinoma, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, chronic lymphocytic lymphoma (CLL), B cell lymphoma, lung adenocarcinoma, osteosarcoma, ovarian cancer, or leiomyosarcoma.

[0112] Particular examples of cancers which the subject may have include renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular Atty. Docket: STAN-2194WO (S24-101) breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactinsecreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, KRAS mutated colorectal cancer; colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgendependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterer); renal carcinoma; Wilms' tumor; and bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, Atty. Docket: STAN-2194WO (S24-101) adenocarcinoma, carcinosarcoma. In some embodiments, the cancer is myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinomas.

[0113] In certain embodiments, provided are methods of administering an adoptive cell therapy to a subject having an infectious disease. Such methods comprise administering to the subject a composition of the present disclosure (e.g., a composition comprising any of the therapeutic immune cells of the present disclosure) in an amount effective to treat the infectious disease. In some embodiments, the infectious disease is a viral infection. Non-limiting examples of viral infections treatable according to the methods of the present disclosure include Human Immunodeficiency Virus (e.g., in which the cells express a CAR targeting a CD4 binding site on gp-120), Hepatitis C Virus (e.g., in which the cells express a CAR targeting HCV E2 glycoprotein), Hepatitis B Virus (e.g., in which the cells express a CAR targeting S HBV surface protein or HBV surface antigen), and Human Cytomegalovirus infections (e.g., in which the cells express a CAR targeting Glycoprotein B or virally-encoded FcRs). In some instances, the infectious disease is an opportunistic fungal infection. In some embodiments, the opportunistic fungal infection is an invasive aspergillosis (e.g., Aspergillus fumigatus) infection, e.g., in which the cells express a CAR targeting p-glucan.

[0114] As demonstrated in the Experimental section below, the natural (e.g., IL1 R1 ) and chimeric cell surface receptors of the present disclosure find use in significantly enhancing T cell function relative to control T cells not expressing the natural or chimeric cell surface receptor. Accordingly, provided are methods in which the adoptive cell therapy exhibits: reduced (or prevented) T cell exhaustion as compared to an adoptive cell therapy employing T cells not expressing the natural or chimeric cell surface receptor; enhanced efficacy (e.g., enhanced anti-tumor and / or anti-viral infection efficacy) as compared to an adoptive cell therapy employing T cells not expressing the natural or chimeric cell surface receptor; and / or the like.

[0115] The adoptive cell therapy may be administered via a route of administration independently selected from parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, or epidural injection), oral, topical, or nasal administration.

[0116] An “amount effective to treat” a particular condition or a “therapeutically effective amount” may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a human subject / patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration Atty. Docket: STAN-2194WO (S24-101) of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a composition of the present disclosure includes from 1 x106to 5x1010of the therapeutic immune cells of the present disclosure.

[0117] By treatment is meant at least an amelioration of one or more symptoms associated with the condition (e.g., cancer, infectious disease, or the like) of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the condition, or at least the symptoms that characterize the condition. With respect to cancer, in some embodiments, the treatment is effective to slow the growth of a tumor, reduce the size of a tumor, and / or the like. With respect to an infectious disease, in some embodiments, the treatment is effective to slow the growth of the underlying pathogen, reduce the number of cells infected by the pathogen, and / or the like.

[0118] The following examples are offered by way of illustration and not by way of limitation.

[0119] EXPERIMENTAL

[0120] Example 1 - Production and Testing of a Chimeric Type 1 Transmembrane Receptor-Encoding T Cell Library

[0121] Described in this example is the production of a chimeric Type 1 transmembrane (TM) receptor-encoding T cell library. The chimeric Type 1 TM receptors expressed by cells of the library comprise an extracellular domain of a Type 1 TM receptor and an intracellular domain of a different Type 1 TM receptor (schematically illustrated in FIG. 2A).

[0122] As schematically illustrated in FIG. 2B, large scale libraries of extracellular and intracellular Type 1 TM domains were combinatorially assembled using an iterative, modular cloning strategy to create large libraries of chimeric surface receptors (sometimes referred to herein as “synthetic” surface receptors). Each member of the large library of chimeric surface receptors possessed a unique barcode allowing for each member to be individually tracked in large scale pooled experiments.

[0123] In particular, the present library included combinatorial chimeras of extracellular and intracellular domains from 88 naturally occurring single transmembrane pass human surface receptors including from: TCR Signaling Components (CD3Z, etc.); CD28 Family (CTLA4, PD1 , ICOS, BTLA); TNF Receptor Superfamily (LTBR, FAS, 41 BB, etc.); Type I Cytokine Receptors (IL2RA, IL7R, etc.); Type II Cytokine Receptors (INFAR1 , IL10RA, etc.); Ig Superfamily Cytokine Receptors (IL1 R1 , IL18R1 , etc.); IL17 Family Receptors (IL17RA, etc.); and TGFp Family Receptors (TGFBR2, etc.). Combinatorially recombining the extracellular and intracellular Atty. Docket: STAN-2194WO (S24-101) domains of these receptors (FIG. 3A) created a library with 7,744 chimeric human surface receptors, each uniquely barcoded, for functional testing in primary human T cells.

[0124] The combinatorial library was subjected to functional testing in a large scale pooled screen in primary human T cells in a model of T cell exhaustion. After combinatorial molecular construction of the synthetic surface receptor library, the library was introduced into the genome of primary human T cells using non-viral genome targeting and was integrated into the TRAC locus along with a Chimeric Antigen Receptor (CD19-28Z architecture), resulting in a pool of human T cells with their T cell receptors replaced by a CD19-28Z CAR with each cell also coexpressing one of the 7,744 chimeric surface receptors and its corresponding barcode.

[0125] The pool of modified human T cells was expanded for 5 days after editing, and then repetitively stimulated with a Nalm6 Leukemia target cancer cell line expressing CD19 at a 1 :8 effector to target ratio. The T cell pool was stimulated every two days with new Nalm6 leukemia target cells for a total of 10 days. Samples of the T cell pool were saved before the first target cell stimulation (“input population”) and after 10 days of repetitive stimulation (“output population”). Genomic DNA was extracted from each of these populations, and amplicon sequencing of the unique barcodes was performed on both populations. The entire experiment was performed in four unique healthy human donors. The Log2 Fold change in barcode abundance between the output and input populations is displayed (FIG. 3B), showing a large number of synthetic surface receptors that allowed T cells to proliferate better than control cells in the repetitive stimulation assay.

[0126] Shown in FIG. 4 are the top individual chimeric surface receptors across four donors in the CD19 CAR driven Nalm6 repetitive stimulation assay. Of note, numerous synthetic surface receptors performed significantly better than controls, in particular synthetic surface receptors bearing a TNFRSF6 (FAS) extracellular domain. The top performing non-chimeric (“natural”) surface receptor was the interleukin 1 receptor component (IL1 R1 ).

[0127] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same Atty. Docket: STAN-2194WO (S24-101 ) function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

Atty. Docket: STAN-2194WO (S24-101)WHAT IS CLAIMED IS:1 . A chimeric cell surface receptor comprising: an extracellular domain of a tumor necrosis factor receptor (TNFR); and an intracellular domain of a different TNFR or a receptor other than a TNFR.

2. The chimeric cell surface receptor of claim 1 , wherein the extracellular domain is of TNFR superfamily member 5 (TNFRSF5), TNFR superfamily member 6 (TNFRSF6), TNFR superfamily member 12A (TNFRSF12A), or TNFR superfamily member 17 (TNFRSF17).

3. The chimeric cell surface receptor of claim 1 , wherein the extracellular domain is of TNFRSF6.

4. The chimeric cell surface receptor of any one of claims 1-3, wherein the intracellular domain is of a different TNFR.

5. The chimeric cell surface receptor of claim 4, wherein the different TNFR is TNFR superfamily member 4 (TNFRSF4), TNFRSF5, TNFR superfamily member 7 (TNFRSF7), TNFR superfamily member 11 A (TNFRSF11 A), TNFRSF12A, TNFR superfamily member 13B (TNFRSF13B), TNFR superfamily member 13C (TNFRSF13C), TNFR superfamily member 14 (TNFRSF14), TNFRSF17, TNFR superfamily member 25 (TNFRSF25), or TNFR superfamily member 27 (TNFRSF27).

6. The chimeric cell surface receptor of any one of claims 1 -3, wherein the intracellular domain is of a receptor other than a TNFR.

7. The chimeric cell surface receptor of claim 6, wherein the intracellular domain is of an interleukin (IL) receptor.

8. The chimeric cell surface receptor of claim 7, wherein the IL receptor is IL2RA, IL5RA, IL6R, IL12RB1 , IL15RA, IL18R1 , IL20RA, or IL27RA.

9. The chimeric cell surface receptor of claim 6, wherein the intracellular domain is of BTLA, CD4, CD28, CSF2RA, CSF3R, ICOS, PDCD1 , or TGFRBR3.

10. A chimeric cell surface receptor comprising: an extracellular domain of an interleukin receptor (ILR); and an intracellular domain of a different ILR or a receptor other than an ILR.Atty. Docket: STAN-2194WO (S24-101)11 . The chimeric cell surface receptor of claim 10, wherein the extracellular domain is of IL1 R1 , IL7R, or IL9R.

12. The chimeric cell surface receptor of claim 10 or 11 , wherein the intracellular domain is of a different ILR.

13. The chimeric cell surface receptor of claim 12, wherein the different ILR is IL17RE.

14. The chimeric cell surface receptor of claim 10 or 11 , wherein the intracellular domain is of a receptor other than an ILR.

15. The chimeric cell surface receptor of claim 14, wherein the intracellular domain is of a TNFR.

16. The chimeric cell surface receptor of claim 15, wherein the TNFR is TNFRSF3.

17. A chimeric cell surface receptor comprising an extracellular domain of TNFRSF6 and an intracellular domain of a receptor chosen from IL15RA, TNFRSF5, CD28, TNFRSF14, PDCD1 , TNFRSF12A, TGFBR3, TNFRSF25, ICOS, TNFRSF27, BTLA, CSF2RA, TNFRSF13B, TNFRSF4, IL12RB1 , TNFRSF17, TNFRSF7, TNFRSF13C, IL6R, IL27RA, IL5RA, IL18R1 , CD4, or CSF3R.

18. A chimeric cell surface receptor comprising:(a) an extracellular domain of TNFRSF6 and an intracellular domain of IL15RA;(b) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF5;(c) an extracellular domain of TNFRSF6 and an intracellular domain of CD28;(d) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF14;(e) an extracellular domain of TNFRSF6 and an intracellular domain of PDCD1 ;(f) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF12A;(g) an extracellular domain of TNFRSF6 and an intracellular domain of TGFBR3;(h) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF25;(i) an extracellular domain of TNFRSF6 and an intracellular domain of ICOS;(j) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF27;(k) an extracellular domain of TNFRSF6 and an intracellular domain of BTLA;(l) an extracellular domain of TNFRSF6 and an intracellular domain of CSF2RA;(m)an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF13B;(n) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF4;Atty. Docket: STAN-2194WO (S24-101)(o) an extracellular domain of TNFRSF6 and an intracellular domain of IL12RB1 ;(p) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF17;(q) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF7;(r) an extracellular domain of TNFRSF6 and an intracellular domain of TNFRSF13C;(s) an extracellular domain of TNFRSF6 and an intracellular domain of IL6R;(t) an extracellular domain of TNFRSF6 and an intracellular domain of IL27RA;(u) an extracellular domain of TNFRSF6 and an intracellular domain of IL5RA;(v) an extracellular domain of TNFRSF6 and an intracellular domain of IL18R1 ;(w) an extracellular domain of TNFRSF6 and an intracellular domain of CD4;(x) an extracellular domain of TNFRSF6 and an intracellular domain of CSF3R;(y) an extracellular domain of TNFRSF12A and an intracellular domain of IL2RA;(z) an extracellular domain of TGFBR1 and an intracellular domain of BTLA;(aa) an extracellular domain of IL7R and an intracellular domain of IL17RE;(bb) an extracellular domain of CD28 and an intracellular domain of TNFRSF10D;(cc) an extracellular domain of IL9R and an intracellular domain of TNFRSF3;(dd) an extracellular domain of TNFRSF12A and an intracellular domain ofTNFRSF11A;(ee) an extracellular domain of TNFRSF5 and an intracellular domain of IL20RA; or (ff) an extracellular domain of TNFRSF17 and an intracellular domain of IL12RB1 .

19. A nucleic acid encoding the chimeric cell surface receptor of any one of claims 1-18.

20. A cell comprising the nucleic acid of claim 19, wherein the cell expresses the chimeric cell surface receptor.21 . A cell comprising a nucleic acid encoding interleukin 1 receptor (IL1 R1 ), wherein the nucleic acid is operably linked to a heterologous promoter.

22. The cell of claim 20 or 21 , wherein the nucleic acid is operably linked to an endogenous promoter.

23. The cell of claim 22, wherein the endogenous promoter is of the T-cell receptor a constant ( TRAC) locus.

24. The cell of claim 20 or 21 , wherein the nucleic acid is operably linked to an exogenous promoter.

25. The cell of claim 24, wherein the exogenous promoter is a constitutive promoter.Atty. Docket: STAN-2194WO (S24-101)26. The cell of claim 24, wherein the exogenous promoter is an inducible promoter.

27. The cell of any one of claims 20-26, wherein the cell is a human cell.

28. The cell of any one of claims 20-27, wherein the cell is an immune effector cell.

29. The cell of claim 28, wherein the immune effector cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a macrophage.

30. The cell of any one of claims 20-29, wherein the cell comprises a nucleic acid encoding a recombinant receptor, wherein the cell expresses the recombinant receptor on its surface.31 . The cell of claim 30, wherein the recombinant receptor is a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), a synthetic notch (synNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.

32. The cell of claim 30, wherein the recombinant receptor is a CAR.

33. The cell of claim 28 or 29, wherein the cell does not comprise a nucleic acid encoding a recombinant receptor.

34. The cell of claim 33, wherein the cell is a tumor infiltrating lymphocyte (TIL).

35. A method of making the cell of one of claims 20-34, comprising stably introducing into the cell the nucleic acid of claim 19.

36. A composition comprising a population of cells as defined in any one of claims 20-34.

37. The composition of claim 36, wherein the composition is formulated for administration to a subject in need thereof.

38. A method comprising administering an effective amount of the composition of claim 37 to a subject in need thereof.

39. The method according to claim 38, wherein the subject has cancer and the method is for treating the cancer.Atty. Docket: STAN-2194WO (S24-101 )40. The method according to claim 38 or 39, wherein the composition comprises a population of immune effector cells as defined in any one of claims 28-34. 41 . The method according to any one of claims 38-40, wherein the cells express a recombinant receptor as defined in claim 31 .

42. The method according to claim 41 , wherein the recombinant receptor is a CAR.

43. The method according to claim 41 or 42, wherein the subject has cancer, and wherein the recombinant receptor binds to a tumor antigen present on cancer cells in the subject.

Images