Fusion proteins for enhancing t-cell therapies

Abstract

The present disclosure relates generally to, inter alia, novel recombinant polypeptides for enhancing T cell functions. The disclosure also provides compositions and methods useful for of the development of immuno-therapeutics, including recombinant nucleic acids and recombinant cells, e.g., recombinant immune cells. Also provided are compositions and methods useful for preparing such recombinant cells, as well as methods and kits for the prevention and / or treatment of health conditions.

Description

Attorney Docket No.: 048536-807001 WOFUSION PROTEINS FOR ENHANCING T-CELL THERAPIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 63 / 733,927 filed on December 13, 2024, the disclosure of which is incorporated by reference herein in its entirety, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which is hereby incorporated herein by reference in its entirety. The accompanying Sequence Listing XML file, named “048536- 80700 lWO_Sequence Listing_ST26.XML,” was created on December 12, 2025 and is 704,773 bytes in size.FIELD

[0003] The present disclosure relates generally to the fields of oncology and immunotherapeutics, and particularly relates to compositions and methods for enhancing T cell therapeutics. The disclosure provides novel recombinant polypeptides for enhancing T cell functions, including proliferative capacity, antigen sensitivity, and anti-tumor effect. Also disclosed are nucleic acid constructs, recombinant cells, and pharmaceutical compositions comprising such recombinant polypeptides, methods for preparing T cells for use in cell therapies, as well as methods and kits for the prevention and treatment of health conditions.BACKGROUND

[0004] Recent advances in genetic engineering are enabling the development of novel gene therapies and engineered-cell therapies for the treatment of many diseases and health disorders. In particular, uses of recombinant cells expressing engineered immune receptors, such as chimeric antigen receptors (CARs) and high-affinity T cell receptors (TCRs), have emerged as a promising approach for immunotherapy and made headlines in clinical trials conducted by a number of pharmaceutical and biotechnology companies. For example, CAR- T cell therapy involves the introduction of a recombinant CAR that directs T cells to kill tumor cells expressing a particular antigen. CARs are antigen- specific recombinant receptors, which, in a single molecule, redirect the specificity and function of a number of immune cells, including T lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, and macrophages. In CAR-T cell therapy, the general premise for the use of CAR-T cells in cancer immunotherapy is to rapidly generate tumor-targeted T cells, bypassing the barriersAttorney Docket No.: 048536-807001 WO and incremental kinetics of active immunization, and eliminating MHC restriction in antigenrecognition. Once expressed in T cells, the CAR-modified T cells acquire supra-phy siological properties and act as “living drugs” that may exert both immediate and long-term effects.

[0005] However, the deployment of CAR-T cell therapies in humans continues to present significant challenges for clinical development. In particular, despite certain successes in treating patients, this modality still sometimes fails to achieve long-term responses. One reason is that after sustained killing, T cells can become dysfunctional. Additionally, if the antigen undergoes downregulation, the receptor can become ineffective at providing signals for T cell persistence or even responding to the tumor.

[0006] Accordingly, there remains a need in the art for alternative solutions to overcome these obstacles and achieve more effective adoptive T cell therapies and extend the reach of these therapeutics to more diseases and to treat more patients.SUMMARY

[0007] The present disclosure relates generally to, inter alia, novel recombinant polypeptides for enhancing T cell functions, including proliferative capacity, antigen sensitivity, and anti-tumor effect. As described in greater detail below, some aspects and embodiments of the present disclosure relate to the development of immuno-therapeutics, including recombinant polypeptides and pharmaceutical compositions including the same. Nucleic acids encoding these recombinant polypeptides are also provided. Recombinant cells, e.g., immune cells (e.g., T cells) including these recombinant polypeptides and nucleic acids are also provided. The disclosure further provides compositions and methods useful for preparing / making such recombinant cells, as well as methods and kits for the prevention and / or treatment of health conditions.

[0008] In one aspect, provided herein are various recombinant polypeptides including, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of ITK, SHP1, c-Cbl, LCK, FYN, ZAP70, and SYK; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SYK, LCK, FYN, ZAP70, LCK, and HPK1, wherein the first and the second polypeptide sequences are heterologous to one another.

[0009] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from ITK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK,Attorney Docket No.: 048536-807001 WOZAP70, PKC theta, LCK, and FYN. In some embodiments, (i) the first polypeptide sequence is derived from SHP1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, LCK, FYN, and ZAP70. In some embodiments, (i) the first polypeptide sequence is derived from c-Cbl and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of ZAP70, LCK, FYN, and SYK. In some embodiments, (i) the first polypeptide sequence is derived from a protein selected from the group consisting of LCK, FYN, ZAP70, and SYK; and (ii) the second polypeptide sequence is derived from HPKL In some embodiments, the recombinant polypeptides of the disclosure include a kinase activity. In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 31-46, 305, and 307.

[0010] In one aspect, provided herein are various recombinant polypeptides including, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of LAT and ITK; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, PKC theta, ZAP70, LCK, FYN, SYK, GRB2, GADS, S0S1, ADAP, PLCgl, ITK, VAV1, NCK1, AKT1, BTK, MAP4K3, PDK1, and PIK3R1 alpha, wherein the first and the second polypeptide sequences are heterologous to one another.

[0011] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from LAT and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, PKC theta, ZAP70, LCK, FYN, SYK, GRB2, GADS, S0S1, ADAP, PLCgl, ITK, VAV1, NCK1, AKT1, BTK, MAP4K3, PDK1, and PIK3R1 alpha. In some embodiments, (i) the first polypeptide sequence is derived from ITK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, PKC theta, ZAP70, LCK, FYN, GRB2, GADS, SLP76, ADAP, NCK1, MAP4K3, and PIK3R1 alpha. In some embodiments, the recombinant polypeptides include a transmembrane domain or a domain capable of tethering the recombinant polypeptide to a plasma membrane. In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 47-77.

[0012] In one aspect, provided herein are various recombinant polypeptides including, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP; and (b) aAttorney Docket No.: 048536-807001 WO second portion including a second polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP, wherein the first and the second polypeptide sequences are heterologous to one another. Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the recombinant polypeptide includes one or more additional domains capable of mediating protein-protein interaction (e.g., thereby increasing valency). Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 78-89.

[0013] In another aspect, provided herein are various recombinant polypeptides including, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of GADS, MAP4K1, GRB2, S0S1, PLCgl, VAV1, and NCK1; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, UK, MAP4K1, NCK1, S0S1, and VAV1, wherein the first and the second polypeptide sequences are heterologous to one another.

[0014] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from GADS and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, ITK, and MAP4K1. In some embodiments, (i) the first polypeptide sequence is derived from MAP4K1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of MAP4K1, S0S1, and VAV1. In some embodiments, (i) the first polypeptide sequence is derived from S0S1 and (ii) the second polypeptide sequence is derived from NCK1. In some embodiments, (i) the first polypeptide sequence is derived from GRB2 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76 and NCK1. In some embodiments, (i) the first polypeptide sequence is derived from PLCgl and (ii) the second polypeptide sequence is derived from SLP76. In some embodiments, (i) the first polypeptide sequence is derived from VAV1 and (ii) the second polypeptide sequence is derived from SLP76. In some embodiments, (i) the first polypeptide sequence is derived from NCK1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, MAP4K1, and S0S1. InAttorney Docket No.: 048536-807001 WO some embodiments, the recombinant polypeptides include a first additional domain capable of binding to protein(s) in the LAT / SLP76 signalosome (e.g., thereby conferring an “intra- signalosome adaptor activity”). In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 90-107. In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 41, 46, 54, 65, 68, 69, 77, 103, and 109.

[0015] In another aspect, provided herein are various recombinant polypeptides including, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of LCK and ZAP70; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, SOS1, VAV1, PLCgl, and NCK1, wherein the first and the second polypeptide sequences are heterologous to one another.

[0016] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from LCK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, S0S1, VAV1, PLCgl, and NCK1. In some embodiments, (i) the first polypeptide sequence is derived from ZAP70 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, S0S1, VAV1, PLCgl, and NCK1. In some embodiments, the recombinant polypeptides include (i) a first additional domain capable of binding to protein(s) in the LAT / SLP76 signalosome, and (ii) a second additional domain capable of binding to protein(s) outside of the LAT / SLP76 signalosome (e.g., thereby conferring an “extra-signalosome adaptor” activity). In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 108— 131.

[0017] In another aspect, provided herein are various recombinant polypeptides including, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from LAT; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, S0S1, MAP4K1, and ADAP, wherein the first and the second polypeptide sequences are heterologous to one another. Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, theAttorney Docket No.: 048536-807001 WO recombinant polypeptides further include a third polypeptide sequence derived from a protein selected from the group consisting of MAP4K1, ADAP, SLP76, and SOS1. In some embodiments, the third polypeptide sequence is operably linked to the C-terminus of the second polypeptide sequence. In some embodiments, the recombinant polypeptides include (i) a transmembrane domain, and (ii) one or more intrinsically disordered regions (IDRs). In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 132-143.

[0018] In certain embodiments of the disclosure, the recombinant polypeptides disclosed herein further include an amino acid sequence for the 6th Ankyrin Repeat (AR6) of the protein IKBOI, or a functional variant thereof, wherein the AR6 sequence or functional variant thereof mediates a targeted ubiquitin-independent degradation. In some embodiments, the AR6 sequence comprises or is a sequence selected from the group consisting of SEQ ID NOS: 297-299. In some embodiment, the AR6 sequence comprises or is the sequence of SEQ ID NO: 297.

[0019] In another aspect, provided herein are recombinant nucleic acids including a nucleic acid sequence encoding a recombinant polypeptide as disclosed herein. Non-limiting exemplary embodiments of the recombinant nucleic acids of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the nucleic acid sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 174-286, 306, and 308. In some embodiments, the nucleic acid sequence is operably linked to one or more heterologous nucleic acid sequences. In some embodiments, the one or more heterologous nucleic acid sequences comprises or is a nucleic acid control element. In some embodiments, the nucleic acid control element is selected from the group consisting of ribosomal binding sites, promoters, enhancer elements, activator elements, translational start sequences, translational termination sequences, transcription start sequences, transcription termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, replication elements, RNA processing and export elements, transposon sequences, transposase sequences, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences, boundary elements, locus control regions (LCR), matrix attachment regions (MAR), recombination or cassette exchange sequences, or any combination thereof.

[0020] In some embodiments, the heterologous nucleic acid sequence includes or is aAttorney Docket No.: 048536-807001 WO promoter. In some embodiments, the promoter includes or is an EFla promoter, CMV promoter, CAG promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD I la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD 161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL 17 A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NFAT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, or NR4A3 promoter. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the endogenous promoter comprises or is a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, a CD3z promoter, a CARD9 promoter, a CARDIO promoter, a CARD 11 promoter, a CARD 14 promoter, or a PIK3R3 promoter, a B2 microglobulin (B2M) promoter, or a class II transactivator (CIITA) promoter. In some embodiments, the heterologous nucleic acid sequence includes or is a synthetic promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a regulatable promoter, e.g., an inducible promoter or a repressible promoter.

[0021] In some embodiments, the one or more heterologous nucleic acid sequences comprises or is a coding sequence for a polypeptide of interest. In some embodiments, theAttorney Docket No.: 048536-807001 WO polypeptide of interest comprises or is a peptide linker sequence, a cleavable linker sequence, a secretion signal, a resistance marker, an anchoring peptide, a localization signal, a fusion tag, an affinity tag, a chaperonin, a protease, or any combination thereof.

[0022] In some embodiments, the recombinant nucleic acids of the disclosure further include a barcode molecule that identifies that recombinant polypeptide. In some embodiments, the recombinant nucleic acids of the disclosure further include nucleic acid sequences encoding for a ribosomal skipping peptide sequence. In some embodiments, the ribosomal skipping peptide sequence includes one or more ribosomal skipping peptide sequences from a porcine teschovirus- 1 2 A (P2A), a calcium-dependent serine endoprotease (furin), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof. In some embodiments, the ribosomal skipping peptide sequence includes a ribosomal skipping peptide sequence from a porcine teschovirus- 1 2A (P2A).

[0023] In some embodiments of the disclosure, the recombinant nucleic acids further include a second nucleic acid sequence encoding: (a) an engineered antigen receptor having specificity for a target ligand; and / or (b) a T cell receptor (TCR) having specificity for a target ligand. In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). In some embodiments, the recombinant nucleic acids further include a response element operably incorporated upstream of the coding sequence of the recombinant polypeptide, wherein the response element includes: (a) a cognate target sequence to which a transcriptional regulator binds; and / or an engineered responsive promoter operably linked to the cognate target sequence. In some embodiments, the transcriptional regulator is incorporated in the ICD of a Hybrid-R receptor as described herein. In some embodiments, the recombinant nucleic acids are incorporated into an expression cassette or an expression vector.

[0024] Accordingly, in another aspect, provided herein are vectors including a recombinant nucleic acid as disclosed herein. In some embodiments, the vector is an expression vector.

[0025] In some embodiments, the vector is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a RNA vector, a mRNA vector, a phagemid vector, a viral vector, a self-replicating RNA virus, a mRNA-packaging virus-like particle, or a Repackaging virus-like particle. In some embodiments, the viral vector is a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector. In some embodiments, the vector is formulated in a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a proteinAttorney Docket No.: 048536-807001 WO nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, a fusosome, an enveloped delivery vehicle, or an immune stimulating complex (ISCOM). In some embodiments, the LNP is a selective organ targeting LNP or an antibody-targeted LNP.

[0026] In yet another aspect, provided herein are recombinant cells including one or more of the following: (a) a recombinant polypeptide as described herein; (b) a recombinant nucleic acid as described herein; and (c) a vector as described herein. Non-limiting exemplary embodiments of the recombinant cells of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0027] In some embodiments, the recombinant cell is a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a gamma delta T cell (y5T), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, an engineered T cell including a transcriptional receptor, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, a naive T (TN) cell, a hematopoietic stem cell, or a progenitor cell of the lymphoid lineage. In some embodiments, the recombinant cell is a stem cell derived cell. In some embodiments, the recombinant cell has reduced / eliminated cell surface TCR expression, MHC-I expression, and / or MHC-II expression.

[0028] In some embodiments, the recombinant cell is a T cell. In some embodiments, the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell. In some embodiments, the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, bulk CD8+ T cells. In some embodiments, the CD4+ T helper lymphocyte cell is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

[0029] In some embodiments of the disclosure, the recombinant cell of the disclosure (e.g., recombinant T cell) further includes an additional recombinant nucleic acid moleculeAttorney Docket No.: 048536-807001 WO encoding: (a) an engineered antigen receptor having specificity for a target ligand; and / or (b) a T cell receptor (TCR) having specificity for a target ligand. In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR).

[0030] In some embodiments, the engineered antigen receptor is a hybrid-CAR receptor (Hybrid-R) comprising an intracellular domain (ICD) which comprises a transcriptional regulator, wherein binding of Hybrid-R to the target ligand results in cleavage of a ligandinducible proteolytic cleavage site and releases of the ICD. In some embodiments, the release of the ICD results in binding of the transcriptional regulator of the released ICD to a cognate target sequence that is operably incorporated upstream of the first recombinant nucleic acid molecule, wherein the binding of the transcriptional regulator to the cognate target sequence modulates transcription of the recombinant polypeptide.

[0031] In some embodiments, the target ligand is expressed on a tumor cell. In some embodiments, the target ligand is CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3e, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CDH17, CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, ROR1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPCAM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFRp, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, ROPN1, GPRC5D, CXORF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6- AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53,Attorney Docket No.: 048536-807001 WO p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML- IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

[0032] In some embodiments, the recombinant nucleic acid and / or the additional recombinant nucleic acid molecule are / is inserted in a specific site(s) in the genome of the recombinant cell. In some embodiments, the specific site(s) in the cell genome is / are independently selected from the list consisting of a T-cell receptor (TCR) locus, a CD3 locus, a B2 microglobulin (B2M) locus, a class II transactivator (CIITA) locus, a TRAC locus, and a safe harbor locus. In some embodiments, the safe harbor locus is AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, or SHS231 locus. In some embodiments, the expression of the recombinant polypeptide results in (i) increasing proliferative capacity, (ii) enhancing antigen- sensitivity, (iii) enhancing antitumor effect, (iv) altering T cell signaling through NF AT, NF-KB and / or AP-1 pathways, (v) altering cytokine or chemokine production, (vi) altering JAK / STAT signaling in T cells, (vii) altering co- stimulatory molecule signaling in T cells, (viii) altering RAS / MEK / ERK signaling in T cells, (ix) altering phospholipase C gamma signaling, (x) altering a transcription factor activity in T cells, and / or (xi) altering or enhancing in vivo persistence in tumors of the T cells including the recombinant polypeptide In some embodiments, the expression of the recombinant polypeptide in a T cell promotes the T cell’s in vivo proliferation. In some embodiments, the recombinant T cell has increased proliferative capacity, enhanced antigen sensitivity, enhanced anti-tumor effect, enhanced replicative lifespan, reduced exhaustion, decreased replicative senescence, reduced dysfunction, enhanced persistence, and / or increase intratumoral presence in vivo.

[0033] In a related aspect, some embodiments of the disclosure relate to cell cultures including at least one recombinant cell of the disclosure and a culture medium.

[0034] In one aspect, provided herein are pharmaceutical compositions including a pharmaceutically acceptable carrier and one or more of the following: (a) a recombinant polypeptide as described herein; (b) a recombinant nucleic acid as described herein; (c) a vector according as described herein; and (d) a recombinant cell as described herein. NonAttorney Docket No.: 048536-807001 WO limiting exemplary embodiments of the pharmaceutical compositions of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the composition includes a recombinant cell as described herein, a pharmaceutically acceptable carrier.

[0035] In another aspect, provided herein are methods of preparing / making a T cell for use in a cell therapy, the method including expressing in the T cell one or more of the following: (a) a recombinant polypeptide as described herein; (b) a recombinant nucleic acid as described herein; and (c) a vector as described herein. In some embodiments, the methods of preparing T cells as disclosed herein further include genetically modifying the T cell for expression of the recombinant polypeptide. In some embodiments, the methods further include introducing to the T cell a recombinant nucleic acid as described herein, or a vector as described herein. In some embodiments, the method includes one or more of the following: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, an RNA-guided nuclease including a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex including a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR- associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In some embodiments, the T cell is made using homology-directed repair (HDR)-mediated insertion. In some embodiments, the T cell is made in vivo, ex vivo, or in vitro. In some embodiments, the methods further include expressing in the T cell an engineered immune receptor that binds to a target ligand.

[0036] In yet another aspect, provided herein are methods of preventing and / or treating a health condition in a subject in need thereof, including administering to the subject a pharmaceutically effective amount of recombinant cells as disclosed herein. In some embodiments, the recombinant cells are allogeneic relative to the subject. In some embodiments, the recombinant cells are autologous relative to the subject.

[0037] In some embodiments, the health condition is a proliferative disorder (e.g., cancers), an infectious disease, an autoimmune disease, and / or inflammatory disease. In some embodiments, the proliferative disorder is a cancer. In some embodiments, the cancer expresses a ligand selected from the group consisting of CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, Claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24,Attorney Docket No.: 048536-807001 WOCD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD72, CD97, CEA, Claudin 6(CLDN6), CLECL1, CS-1, DLL-3, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GCC, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-P, PRAME, PRSS21, PSCA, PSMA, R0R1, SIRPa, SSEA-4, STEAP2, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Ax. In some embodiments, the cancer is small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, nonsmall cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head and neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

[0038] In another aspect, provided herein are kits for preventing or treating a health condition in a subject in need thereof, the kits include one or more of the following: (a) a recombinant polypeptide as disclosure herein; (b) a recombinant nucleic acid as disclosed herein; (c) a vector as described herein; and (d) a recombinant cell as described herein.

[0039] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects, and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIGS. 1A-1H depict a graphical representation of modular protein domain building blocks used to generate the library (see also Table 1). Text under horizontal bars indicates other molecules to which a given domain can bind.

[0041] FIGS. 2A-2E depict an overview of the new strategy for identifying genes that enhance therapeutic T cell activity. FIG. 2A: A pooled adeno-associated virus (AAV) homology-directed repair template (HDRT) library encoding synthetic fusion proteins was designed and targeted as a synthetic exon to the intron upstream of the TRAC locus in primary human T cells. The P2A sequence upstream of the library contains a nucleotide barcode that is made of synonymous codons. FIG. 2B: Various receptors one at a time were targeted to the safe harbor locus GSH6, which is within the pseudogene ZNF767P. Thus, the T cells underwent dual editing. FIG. 2C: Depiction of the types of exemplary antigen receptors evaluated in accordance to various embodiments of the disclosure (e.g., nonAttorney Docket No.: 048536-807001 WO exhaustive for the possible receptors that could be used). FIG. 2D: Timeline of cell manufacturing and in vivo screening. NSG mice were injected with NY-ESO-1+CD19+luciferase-expressing NALM6 cells, and four days later injected with CAR-T cells. FIG. 2E: Tumor burden was tracked by bioluminescence imaging. Mice underwent rechallenges with tumor cells to provide more selective pressure on the library. In this experiment, for each receptor, cells were produced from two peripheral blood mononuclear cell (PBMC) donors and assayed in two mice; in total, there were 5x2x2 = 20 mice. Right plot: Numbers of T cells obtained from bone marrow after take-down. Genomic DNA was extracted and prepared for next- generation sequencing of barcodes to quantify enrichment of library members as compared to pre-injection baseline.

[0042] FIGS. 3A-3B: Strategy to quantify library member abundances from amplification of genomically integrated cargo and next-generation sequencing. Each library member has a barcode that is a unique set of synonymous codons in a P2A sequence. FIG. 3B: Volcano plots depict enrichment of library members from the in vivo screen as compared to preinjection. One library member, ITK-ZAP70 (circle with arrow), consistently appeared as the top hit when paired with each of the antigen receptors tested. This strong proliferative advantage suggested a functional advantage that this gene could confer to engineered cells, e.g., CAR / TCR / HIT-T cells.

[0043] FIG. 4: In vitro screening with each receptor in T cells from two PBMC donors: 5x2=10 dots per position in the plots. Library members are ranked by average enrichment in each of the three screening conditions. The dynamic range is lower as compared to the in vivo screening, but certain library members do stand out as enriched. In condition A, ITK-ZAP70 does not confer a strong advantage, but several other genes do. Natural kinases and fusions containing their catalytic domain are generally present among the top hits. In this figure, the synthetic fusion ITKPH TH-SYK in the library (SEQ ID NO: 69) differs from the natural- derived fusion ITKPH-TH-SYKlntB’KDin subsequent figures. Abbreviations: intB is a portion of interdomain B, and KD is kinase domain.

[0044] FIG. 5: Diagram of transgenes in the 77MC-exon- targeted AAV HDRTs evaluated in arrayed validation, e.g., transgenes that are coexpressed with a 19BBz CAR in several subsequent figures. In some experiments, each transgene is coexpressed with a 19BBz CAR. NGFRt is a neutral control. ITK and ZAP70 are the genes from which the synthetic fusion ITK-ZAP70 (ITKPH TH SH3 SH2-ZAP70KD) is derived. ITK-SYK and CARD11-PIK3R3 are naturally occurring fusion proteins arising from chromosomal translocations. Some constructs were designed to determine which domains of the ITK-ZAP70 fusion are necessary for itsAttorney Docket No.: 048536-807001 WO potency-enhancing function.

[0045] FIG. 6: One-day cytotoxicity assay with a luminescent readout using CAR-T cells in co-culture with NALM6 cell lines expressing different levels of CD19 (three traces per plot), at different effector cell to target cell ratios. ITK-ZAP70 confers sensitivity that differs from the parent kinases. Plots display the mean ± S.E.M. from triplicate plating. TR AC-KO is the negative control. The top hit from the screen ITK-ZAP70 (ITKPH-TH’SH3’SH2-ZAP70KD) conferred enhanced sensitivity as compared to the base case (NGFRt) and the greatest sensitivity among the constructs evaluated.

[0046] FIG. 7: Intracellular staining and flow cytometry following a six-hour co-culture indicate that the enhanced antigen sensitivity conferred by ITK-ZAP70 (ITKPH-TH’SH3’SH2- ZAP70KD) is apparent in cytokine produced by the CAR-T cells and the CD8+subset in particular. Legend for each group of four bars, from left to right: T cells only; T cells and NALM6 CD I 9very low; T cells and NALM6 CD19low; T cells and NALM6 CD19hlgh. Plots display the mean ± S.E.M. for T cells from n = 3 PBMC donors.

[0047] FIG. 8: Avidity measurement using acoustic force spectroscopy (z-Movi). 19BBz is expressed with NGFRt, ITK-SYK, or ITK-ZAP70. n = 3 or 4 measurements per condition. 5- minute coincubation prior to applying a force ramp on the z-Movi instrument. *p<Q.Q5, **p<Q.Ql using a / -test. ITK-ZAP70 retains modest albeit significant avidity at low antigen density.

[0048] FIG. 9: Arrayed in vivo validation. Various fusion proteins described in FIG. 5 was tested with a 19BBz CAR (10A5 CAR-T cell dose) against NALM6 cells with regular (wild type) CD19WTexpression in NSG mice. Bioluminescence imaging and survival monitoring indicated that at least three fusions (ITKPH TH SH3 SH2-ZAP70KD, iTKPH TH SH3 SH2-ZAP70lntB-KD, and CARD11-PIK3R3) conferred tumor control. ITK-SYK, which is derived from a chromosomal translocation, conferred control but the mice soon reached end points. The two variations of ITK-ZAP70 conferred control and improved survival in this experiment.

[0049] FIG. 10: Take-down and flow cytometry analysis on day 12 after T cell injection, n = 3 mice per condition. Bars indicate mean ± S.E.M. The fusions tested confer a proliferative advantage, however, ITK-SYK leads to excessive proliferation.

[0050] FIG. 11A: In vivo experiment using 10A5 CAR T cells against CD19lowNALM6. ITK-ZAP70 confers an advantage at low antigen density. FIG. 11B: Takedown and flow cytometry analysis on day 11 after T cell injection, with n = 3-4 mice per takedown condition (mean ± S.E.M.)Attorney Docket No.: 048536-807001 WO

[0051] FIG. 12: Genes were implemented as inducible cargo downstream of the signaling of a CD19-targeting Hybrid-R. In these experiments, the Hybrid-R contained, on its intracellular tail, elements including a BB costimulatory domain, CD3z stimulatory domain, and HNF la-based transcription factor. In this circuit implementation, an HDRT for EF la- promoter-driven Hybrid-R was targeted to the B2M intron 1, and an HDRT for TCRa- promoter-driven expression of NGFRt (reporter) and inducible expression of a gene of interest and spQ8 (reporter) was targeted to TRAC exon 1. In these experiments, 2xlOA5 antigen-receptor-positive T cells were injected into mice bearing CD19WTNALM6.

[0052] FIG. 13: ITK-ZAP70degcontains the AR6 degron derived from IKB. The one-day in vitro cytotoxicity assay uses NALM6 lines with varied CD19 expression. Mean ± S.E.M. for triplicate plating.

[0053] FIGS. 14A-14B: In vivo experiments to assess the addition of the AR6 degron to ITK-ZAP70 (ITK-ZAP70deg) . In FIG. 14A, animals were administered CD19WTNALM6 tumor cells, and T cell injection doses were 2xlOA5 for TRAC-KO, 2xlOA5 for HIT, 5xlOA4 for 1928z, and 5xlOA4 for 1928zlXX. In FIG. 14B, animals were administered CD19lowNALM6 tumor cells, and T cell injection doses were 2xlOA5 for TRAC-KO and 2xlOA5 for HIT.

[0054] FIGS. 15A-15B: In vivo experiments to assess the ability of T cells comprising the ITK-ZAP70degto control additional tumor types. FIG. 15A In vivo experiments to assess T cells comprising the ITK-ZAP70degand a bb2121-scFv CAR targeting BCMA to control OPM2 multiple myeloma cells. FIG. 15B In vivo experiments to assess T cells comprising the ITK-ZAP70degand a 376.96-scFv CAR to control B7-H3 on A549 lung carcinoma cells.

[0055] FIG. 16: A one-day in vitro cytotoxicity assay using NALM6 lines with varied CD19 expression was performed using variations on the ITK-ZAP70 gene, in which the PH- TH portion of ITK origin was replaced with the PH-TH portion of other genes. Some variants (BTK-ITK-ZAP70, TEC-ITK-ZAP70) led to increased antigen sensitivity as compared to the NGFRt base case and approached that of ITK-ZAP70. Mean ± S.E.M. for triplicate plating.DETAILED DESCRIPTION OF THE DISCLOSURE

[0056] The present disclosure relates generally to, inter alia, novel recombinant polypeptides for enhancing T cell functions, including proliferative capacity, antigen sensitivity, and anti-tumor effect. As described in greater detail below, some aspects and embodiments of the present disclosure relate to the development of immuno-therapeutics, including recombinant polypeptides and pharmaceutical compositions including the same.Attorney Docket No.: 048536-807001 WONucleic acids encoding these recombinant polypeptides are also provided. Recombinant cells, e.g., immune cells (e.g., T cells) including these recombinant polypeptides and nucleic acids are also provided. The disclosure further provides compositions and methods useful for preparing / making such recombinant cells, as well as methods and kits for the prevention and / or treatment of health conditions.

[0057] As will be discussed more thoroughly herein, CAR-T cell therapy involves the introduction of a receptor that directs T cells to kill tumor cells expressing a particular antigen. Despite certain successes in treating patients, this modality still sometimes fails to achieve long-term responses. One reason is that after sustained killing, T cells can become dysfunctional1,2. Additionally, if the antigen undergoes downregulation3’5, the receptor can become ineffective at providing signals for T cell persistence or even responding to the tumor6,7. Much of the current understanding of the failure modes of CAR-T cells comes from how normal T cells become dysfunctional8and how CAR signaling compares to that of the TCR.9Through this work, it has become apparent that CARs are suboptimal at engaging the proximal proteins that mount the T cell anti-tumor response10’13.

[0058] T-cell lymphomas and other cancers have been found to express fusion proteins that provide a fitness advantage14’20. A recent study21found that introducing into CAR-T cells the gene CARD11-PIK3R3, which is a fusion that originally arose from chromosomal damage22, enhances signaling and benefits the CAR-T cells in pre-clinical cancer models without incurring runaway proliferation. In this way, by implementing a fusion protein in therapeutic cells, the tumor’s strategy is turned against tumors.

[0059] As described in greater detail in the Examples below, it is believed that the outcome with CARD11-PIK3R3 might represent a type of enhancement that is not limited to translocation-derived genes. The inventors investigated rationally designed fusion proteins assembled from domains of receptor-proximal proteins that are intended to assist receptors by augmenting signaling through the LAT / SLP76 signalosome: a macromolecular complex that nucleates at the cell membrane in response to TCR stimulus, acts as an upstream signaling hub, and controls downstream pathways23’26. The inability of CARs to properly engage this signalosome13supports the idea that proteins that rescue this gap could overcome CAR deficiencies. Such molecules might also benefit other types of receptors.DEFINITIONS

[0060] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonlyAttorney Docket No.: 048536-807001 WO understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0061] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B.”

[0062] The terms “cell,” “cell culture,” “cell line” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the originally cell, cell culture, or cell line.

[0063] As used herein, the term “chimeric antigen receptor” (CAR) refers to a polypeptide construct comprising at least an extracellular antigen-binding domain, a TMD and a cytoplasmic signaling domain (also referred to as “an intracellular signaling domain” or ICD). In some cases, the cytoplasmic signaling domain includes a functional signaling domain derived from a stimulatory molecule. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. Optionally, the ICD can further include one or more functional signaling domains derived from at least one costimulatory molecule.

[0064] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single- stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence" as used herein interchangeably refer to the sequence of a polynucleotide molecule.

[0065] The term “domain” refers to a distinct functional and / or structural unit within aAttorney Docket No.: 048536-807001 WO protein. Typically, a “domain” is a portion of a protein molecule that is sufficient or responsible for, or contributes measurably to, a particular function or interaction, and / or structural role within a protein. Similar domains (i.e., domains sharing structural, functional, and / or sequence homology) can exist within a single protein or can exist within distinct proteins having similar or different functions. A protein domain is often a conserved part of a given protein tertiary structure or sequence and, in many cases, can fold and function independently of the remainder of the protein or of a subunit thereof. As used herein, “domain” encompasses naturally occurring, modified, and synthetic (engineered or chimeric) domains, and can include domains that are continuous in the primary amino acid sequence or that are formed from residues that are non-contiguous in the primary sequence but come together in the folded structure.

[0066] The term “membrane-localization module” or “MLM” refers to a polypeptide segment of a recombinant polypeptide that is sufficient to mediate localization, enrichment, and / or retention of the recombinant polypeptide at a membrane of a cell, typically at a plasma membrane and / or at a receptor-proximal region that forms at the plasma membrane upon engagement of a receptor with its cognate ligand. An MLM can comprise one or more domains, as described herein, and / or sequence motifs that confer membrane association directly or indirectly, including, for example, pleckstrin homology (PH) domains, Tec homology (TH) regions, Src homology 2 (SH2) or Src homology 3 (SH3) domains, transmembrane segments (e.g., single-pass transmembrane anchors), lipidation motifs (e.g., palmitoylation or myristoylation motifs), and / or sequences that bind membrane- associated proteins or lipids. MLMs can be naturally occurring, modified, truncated, chimeric, and / or synthetic sequences, and may be contiguous or non-contiguous within the primary sequence of the recombinant polypeptide. In certain embodiments described herein, the MLM lacks a functional enzymatic domain.

[0067] The term “functional enzymatic domain” refers to a protein domain that confers enzymatic catalytic activity on a polypeptide in which it is present. A functional enzymatic domain typically comprises one or more amino acid residues that are necessary and sufficient for catalysis of a chemical transformation of a substrate (e.g., phosphoryl transfer, hydrolysis, oxidation-reduction, bond formation or cleavage) under suitable conditions. Non-limiting examples of functional enzymatic domains include kinase domains (e.g., serine / threonine or tyrosine kinase domains), phosphatase domains, protease domains, nuclease domains, deaminase domains, ligase domains, and transferase domains.

[0068] The term “signaling-effector module” or “SEM” refers to a polypeptide segment ofAttorney Docket No.: 048536-807001 WO a recombinant polypeptide that, when localized to a membrane (e.g., a plasma membrane or receptor-proximal region at the plasma membrane), is sufficient to potentiate, modulate, and / or reconstitute a kinase signaling cascade in a cell. In general, an SEM comprises (i) a kinase domain that directly catalyzes phosphorylation of one or more substrates in a signaling pathway and / or (ii) one or more non-enzymatic regulatory and / or adaptor domains that bind and recruit components of a kinase signaling cascade in a manner that promotes or amplifies signaling. Non-limiting examples of SEMs include segments comprising a Syk / ZAP70- family non-receptor tyrosine kinase, an AKT or PDK1 serine / threonine kinase, a class IA PI3K regulatory subunit (e.g., a segment comprising nSH2-iSH2-cSH2 domains), or a GRB2 adaptor (e.g., a segment comprising an N-terminal SH3 domain and optionally an SH2 domain and / or a C-terminal SH3 domain).

[0069] The term “operably linked,” as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. For example, an operably linkage between a polynucleotide of interest and a nucleic acid control element (for example, a regulatory sequence, e.g., a promoter) is functional link that allows for expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to the positioning of a regulatory sequence and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term "operably linked” denotes a configuration in which a regulatory sequence is placed at an appropriate position relative to a sequence that encodes a polypeptide such that the control sequence directs or regulates the expression or cellular localization of the mRNA encoding the polypeptide. Thus, a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence. Operably linked elements may be contiguous or non-contiguous. In the context of a polypeptide, e.g. CAR, “operably linked” refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g., different domains) to provide for a described activity of the polypeptide. In the present disclosure, various domains of the polypeptides of the disclosure may be operably linked to retain proper folding, processing, targeting, expression, binding, and other functional properties of the polypeptides in the cell. Operably linked domains of the polypeptides of the disclosure may be contiguous or noncontiguous (e.g., linked to one another through a linker).

[0070] The term “percent identity” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have aAttorney Docket No.: 048536-807001 WO specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Such modifications can occur naturally or synthetically. In some embodiments, sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J Mol Biol 215:403, 1990). In some embodiments, sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine structural similarity or identity amino acid sequences include those relying on positionspecific structure- scoring matrix (P3SM) that incorporates structure-prediction scores from Rosetta, as well as those based on a length-normalized edit distance as described previously in, e.g., Setliff et al., Cell Host & Microbe 23(6), May 2018.

[0071] The term “recombinant” nucleic acid molecule, polypeptide, and cell as used herein, refers to a nucleic acid molecule, polypeptide, and cell that has been altered through human intervention.

[0072] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human subjects) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term "non-human animals" includes all vertebrates, e.g., mammals, e.g., rodents,Attorney Docket No.: 048536-807001 WO e.g., mice, and non- mammals, such as non-human primates, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0073] The term "vector" is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. For example, a vector can be used as a gene delivery vehicle to transfer a gene into a cell. The transferred nucleic acid molecule is generally linked to, e.g., inserted into, the vector nucleic acid molecule. Generally, a vector is capable of replication when associated with the proper control elements. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An "expression vector" is a vector that includes a regulatory region, thereby capable of expressing DNA sequences and fragments in vitro and / or in vivo. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses. In some embodiments, a vector is a gene delivery vector.

[0074] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0075] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0076] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. AnyAttorney Docket No.: 048536-807001 WO listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0077] 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. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0078] It is appreciated that certain features of the disclosure, 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 disclosure, 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 pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.COMPOSITIONS OF THE DISCLOSURE

[0079] As described in greater detail below, one aspect of the present disclosure relates to novel recombinant polypeptides capable of enhancing T cell functions, including proliferative capacity, antigen sensitivity, and anti-tumor effect. In some embodiments, also provided areAttorney Docket No.: 048536-807001 WO(i) recombinant nucleic acids and vectors encoding such recombinant polypeptides, (ii) recombinant cells that have been engineered to express a recombinant polypeptide as disclosed herein, and (iii) pharmaceutical compositions including one or more of the recombinant polypeptides, nucleic acids, vectors, recombinant cells, and / or cell cultures of the disclosure, and a pharmaceutically acceptable carrier.RECOMBINANT POLYPEPTIDES

[0080] As discussed above, one aspect of the disclosure relates to novel recombinant polypeptides for enhancement of T cell functionality. In particular, in various embodiments, the recombinant polypeptides of the disclosure are made up of two or more heterologous polypeptide sequences that are operably linked to each other, whereby that combination is not normally found in nature. As such, the term "heterologous" refers to the relationship between two or more polypeptide sequences (or two or more nucleic acid sequences) that are derived from different sources. In some embodiments, the heterologous polypeptide sequences are covalently linked (e.g., fused in-frame) to one another.

[0081] Some embodiments of the disclosure provide recombinant polypeptides that include, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of ITK, SHP1, c-Cbl, LCK, FYN, ZAP70, and SYK; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SYK, LCK, FYN, ZAP70, LCK, and HPK1, wherein the first and the second polypeptide sequences are heterologous to one another.

[0082] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from ITK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, ZAP70, PKC theta, LCK, and FYN. In some embodiments, (i) the first polypeptide sequence is derived from SHP1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, LCK, FYN, and ZAP70. In some embodiments, (i) the first polypeptide sequence is derived from c-Cbl and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of ZAP70, LCK, FYN, and SYK. In some embodiments, (i) the first polypeptide sequence is derived from a protein selected from the group consisting of LCK, FYN, ZAP70, and SYK; and (ii) the second polypeptide sequence is derived from HPK1. In some embodiments, the recombinantAttorney Docket No.: 048536-807001 WO polypeptides of the disclosure include a kinase activity. In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 31-46, 305, and 307.

[0083] Some embodiments of the disclosure provide recombinant polypeptides that include, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of LAT and ITK; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, PKC theta, ZAP70, LCK, FYN, SYK, GRB2, GADS, SOS1, ADAP, PLCgl, ITK, VAV1, NCK1, AKT1, BTK, MAP4K3, PDK1, and PIK3R1 alpha, wherein the first and the second polypeptide sequences are heterologous to one another.

[0084] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from LAT and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, PKC theta, ZAP70, LCK, FYN, SYK, GRB2, GADS, S0S1, ADAP, PLCgl, ITK, VAV1, NCK1, AKT1, BTK, MAP4K3, PDK1, and PIK3R1 alpha. In some embodiments, (i) the first polypeptide sequence is derived from ITK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, PKC theta, ZAP70, LCK, FYN, SYK, GRB2, GADS, ADAP, NCK1, MAP4K3, and PIK3R1 alpha. In some embodiments, the recombinant polypeptides include a transmembrane domain or a domain capable of tethering the recombinant polypeptide to a plasma membrane. In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 47-77.

[0085] Some embodiments of the disclosure provide recombinant polypeptides that include, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP, wherein the first and the second polypeptide sequences are heterologous to one another. Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the recombinant polypeptide further includes a third polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP. In some embodiments, theAttorney Docket No.: 048536-807001 WO third polypeptide sequence is operably linked to the C-terminus of the second polypeptide sequence. In some embodiments, the recombinant polypeptide includes one or more additional domains capable of mediating protein-protein interaction (e.g., thereby increasing valency). Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 78-89.

[0086] Some embodiments of the disclosure provide recombinant polypeptides that include, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of GADS, MAP4K1, GRB2, S0S1, PLCgl, VAV1, and NCK1; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, ITK, MAP4K1, NCK1, S0S1, and VAV1, wherein the first and the second polypeptide sequences are heterologous to one another.

[0087] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from GADS and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, ITK, and MAP4K1. In some embodiments, (i) the first polypeptide sequence is derived from MAP4K1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of MAP4K1, S0S1, and VAV1. In some embodiments, (i) the first polypeptide sequence is derived from S0S1 and (ii) the second polypeptide sequence is derived from NCK1. In some embodiments, (i) the first polypeptide sequence is derived from GRB2 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76 and NCK1. In some embodiments, (i) the first polypeptide sequence is derived from PLCgl and (ii) the second polypeptide sequence is derived from SLP76. In some embodiments, (i) the first polypeptide sequence is derived from VAV1 and (ii) the second polypeptide sequence is derived from SLP76. In some embodiments, (i) the first polypeptide sequence is derived from NCK1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, MAP4K1, and S0S1. In some embodiments, the recombinant polypeptides include a first additional domain capable of binding to protein(s) in the LAT / SLP76 signalosome (e.g., thereby conferring an “intra- signalosome adaptor activity”). In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 90-107. InAttorney Docket No.: 048536-807001 WO some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 41, 46, 54, 65, 68, 69, 77, 103, and 109.

[0088] Some embodiments of the disclosure provide recombinant polypeptides that include, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from a protein selected from the group consisting of LCK and ZAP70; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, SOS1, VAV1, PLCgl, and NCK1, wherein the first and the second polypeptide sequences are heterologous to one another.

[0089] Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, (i) the first polypeptide sequence is derived from LCK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, S0S1, VAV1, PLCgl, and NCK1. In some embodiments, (i) the first polypeptide sequence is derived from ZAP70 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, S0S1, VAV1, PLCgl, and NCK1. In some embodiments, the recombinant polypeptides include (i) a first additional domain capable of binding to protein(s) in the LAT / SLP76 signalosome, and (ii) a second additional domain capable of binding to protein(s) outside of the LAT / SLP76 signalosome (e.g., thereby conferring an “extra-signalosome adaptor” activity). In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 108— 131.

[0090] Some embodiments of the disclosure provide recombinant polypeptides that include, in N- to C-terminal direction: (a) a first portion including a first polypeptide sequence derived from LAT; and (b) a second portion including a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, S0S1, MAP4K1, and ADAP, wherein the first and the second polypeptide sequences are heterologous to one another. Non-limiting exemplary embodiments of the recombinant polypeptides of this aspect and other aspects of the disclosure include one or more of the following features. In some embodiments, the recombinant polypeptides include (i) a transmembrane domain, and (ii) one or more intrinsically disordered regions (IDRs). In some embodiments, the recombinant polypeptides include the amino acid sequence selected from the group consisting of SEQ ID NOs: 132-143.Attorney Docket No.: 048536-807001 WO

[0091] Among provided embodiments are recombinant polypeptides that comprise, in N- to C-terminal direction, a first polypeptide segment that is a membrane-localization module (MLM) and a second polypeptide segment that is a signaling-effector module (SEM). In some embodiments, the MLM mediates localization of the recombinant polypeptide to a membrane of a cell, such as a plasma membrane and / or a receptor-proximal region at the plasma membrane that forms in response to engagement of a receptor with its cognate ligand. In some embodiments, the SEM, when so localized by the MLM, potentiates, modulates, and / or reconstitutes a kinase signaling cascade in the cell. In some embodiments, the MLM and SEM are directly contiguous in the primary amino acid sequence; in other embodiments, they are separated by a linker sequence, such as a flexible glycine / serine-rich linker of about 2 to about 50 amino acids.

[0092] In some embodiments, the MLM is an independent polypeptide segment that is sufficient to confer membrane association, enrichment, and / or retention on a polypeptide to which it is fused. In some embodiments, the MLM comprises one or more domains that mediate direct binding to membrane lipids (for example, phosphoinositides) and / or binding to membrane-proximal proteins within receptor signaling complexes. In some embodiments, the MLM comprises one or more domains selected from pleckstrin homology (PH) domains, Tec homology (TH) regions, Src homology 2 (SH2) domains, Src homology 3 (SH3) domains, and single-pass transmembrane segments, optionally in combination with cytosolic lipidation motifs (e.g., palmitoylation motifs) that promote stable association with the inner leaflet of the plasma membrane. In some embodiments, the MLM lacks a functional enzymatic domain and functions primarily as a localization and / or scaffolding module.

[0093] In some embodiments, the MLM comprises a PH domain. In some embodiments, the PH domain is a lipid-binding domain that recognizes phosphoinositide species enriched at the plasma membrane, such as phosphatidylinositol-3,4,5-trisphosphate (PIP3) and / or phosphatidylinositol-4,5-bisphosphate (PIP2). In some embodiments, the PH domain is derived from a Tec family kinase, such as ITK, BTK, TEC, or BMX, or is a variant thereof that retains the ability to bind one or more membrane phosphoinositides and thereby localize the MLM, and any fusion polypeptide comprising it, to the plasma membrane and / or to receptor-proximal microdomains. In some embodiments, the MLM is composed solely of a PH domain; in other embodiments, the PH domain is part of a multi-domain MLM as described herein.

[0094] In some embodiments, the MLM comprises a PH-TH region. In some embodiments, a PH-TH region comprises a PH domain as described above together with an adjacent TecAttorney Docket No.: 048536-807001 WO homology (TH) region from a Tec family kinase, such as ITK, BTK, TEC, or BMX. In some embodiments, the TH region comprises a proline-rich region and / or zinc-binding motifs that participate in interactions with small GTPases, adaptor proteins, and / or cytoskeletal elements. In some embodiments, cooperation between the PH domain and TH region stabilizes recruitment of the MLM to receptor-proximal signaling complexes, such as LAT-SLP-76 signalosomes, by combining lipid binding (via the PH domain) with protein-protein interactions (via the TH region). In some embodiments, the MLM further comprises one or both of an SH3 domain and an SH2 domain appended to the PH-TH core, for example in PH- TH-SH2 and / or PH-TH-SH2-SH3 arrangements.

[0095] In some embodiments, MLMs that comprise Src homology 2 (SH2) and / or Src homology 3 (SH3) domains mediate membrane localization and / or retention at least in part by binding to receptor- proximal adaptor proteins and signaling complexes. For example, in some embodiments, an SH2 domain within the MLM binds to phosphotyrosine-containing motifs on adaptor proteins such as LAT, SLP-76, or other components of a T cell receptor (TCR) signaling complex, while an SH3 domain binds to proline-rich motifs in SLP-76, ADAP, or other scaffolding proteins. In some embodiments, such interactions promote inducible recruitment of the MLM, and any SEM fused thereto, into a LAT-SLP-76 signalosome upon receptor engagement. In some embodiments, SH2- and / or SH3-containing MLMs may be composed of one or more SH2 and / or SH3 domains, or may be present in combination with other domains such as PH and / or TH domains (for example, PH-SH2, PH- SH3, or PH-TH-SH2-SH3 arrangements), thereby localizing chimeric polypeptides both by direct lipid binding and by multivalent engagement of receptor-proximal scaffolds. In some embodiments, particular PH-TH are provided by polypeptides comprising SEQ ID NO: 288, or sequences having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 288. In some embodiments, particular PH-TH-SH2-SH3 MLMs are provided by polypeptides comprising SEQ ID NO: 287, or sequences having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 287.

[0096] In some embodiments, the MLM comprises a single-pass transmembrane anchor derived from a transmembrane adaptor protein (TRAP). In some embodiments, such TRAP- derived MLMs include a type I transmembrane segment that spans the plasma membrane together with a cytosolic juxtamembrane region containing a palmitoylation motif (e.g., 2 or more cysteine residues) that promotes stable association with cholesterol-rich and / or receptor-proximal microdomains at the inner leaflet. In some embodiments, the TRAP isAttorney Docket No.: 048536-807001 WO selected from LAT, PAG / Cbp, LIME, SIT, TRIM, LAX, SCIMP, PRR7, LST1 / A, or a variant of any of the foregoing that retains palmitoylation-dependent membrane anchoring. In some embodiments, the MLM is characterized by a transmembrane domain and a cytosolic juxtamembrane palmitoylation motif comprising 2 or more cysteines. In some embodiments, the MLM includes a TRAP transmembrane segment and its juxtamembrane palmitoylation motif, and optionally a luminal N-terminal segment of about 20 amino acids or less (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 contiguous amino acid from the luminal N-terminal segment). In some embodiments, the TRAP is LAT and the MLM comprises the transmembrane domain of LAT (LAT™D), which provides a minimal singlepass membrane anchor that localizes the MLM-SEM fusion to LAT-enriched receptor- proximal membrane domains in T cells. In some embodiments, the MLM is or comprises the sequence of amino acids set forth in SEQ ID NO: 289, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 289.

[0097] In some embodiments, membrane localization or receptor-proximal enrichment mediated by a given MLM can be assessed experimentally. In some embodiments, an MLM (optionally fused to an SEM) is expressed as a fluorescently tagged fusion (e.g., fused to GFP or mCherry), and plasma-membrane localization is evaluated by confocal microscopy, total internal reflection fluorescence (TIRF) microscopy, or lattice light-sheet microscopy before and / or after receptor stimulation (for example, TCR stimulation in T cells or via an antigenspecific CAR stimulation). In some embodiments, the degree of membrane localization is quantified as a ratio of fluorescence at the plasma membrane relative to cytosolic fluorescence, or as co-localization with a membrane marker (e.g., a fluorescent membrane dye or a LAT reporter) using Pearson’s correlation coefficients. In some embodiments, membrane association is further confirmed by biochemical fractionation of cells into cytosolic and membrane fractions followed by immunoblotting for the MLM-containing fusion. In some embodiments, MLMs comprising PH or PH-TH domains exhibit increased plasma membrane localization relative to cytosolic control constructs lacking these domains, and MLMs comprising PH-TH-SH2-SH3 domains exhibit inducible enrichment at receptor- proximal microclusters (e.g., LAT-SLP-76 microclusters) upon receptor engagement.

[0098] In some embodiments, the SEM is an independent polypeptide segment that provides the signaling “effector” function of the chimeric recombinant polypeptide. In some embodiments, the SEM comprises a kinase domain that directly catalyzes phosphorylation of one or more substrates in a signaling pathway when the SEM is brought into proximity toAttorney Docket No.: 048536-807001 WO those substrates at the membrane by the MLM. In some embodiments, the SEM may also additionally comprise non-enzymatic regulatory and / or adaptor domains (for example, SH2 and / or SH3 domains, nSH2-iSH2-cSH2 domain clusters, or other interaction modules) that recruit and organize components of a kinase signaling cascade to promote or amplify signal transduction. In some embodiments, the SEM, by virtue of its domain composition, is configured to potentiate one or more of a Syk / ZAP70 non-receptor tyrosine kinase cascade, an AKT serine / threonine kinase cascade, a class IA PI3K cascade, or a Ras-Raf-MEK-ERK cascade.

[0099] In some embodiments, the SEM comprises a Syk / ZAP70-family non-receptor tyrosine kinase or a portion thereof comprising a kinase domain, with or without one or more interdomain regions such as interdomain B. In some embodiments, when localized by an MLM, such Syk / ZAP70 SEMs phosphorylate immunoreceptor tyrosine-based activation motifs (IT AMs), LAT, SLP-76, and / or other adaptor proteins, thereby initiating or amplifying downstream signaling. In some embodiments, the SEM comprises an AKT1 or PDK1 kinase domain, such that membrane localization facilitates engagement with PIP3, PIP2, and / or upstream kinases and substrates involved in AKT signaling. In some embodiments, the SEM comprises a class IA PI3K regulatory subunit fragment (e.g., a PIK3Rla nSH2-iSH2-cSH2 domain cluster) that recruits a PI3K catalytic subunit to the membrane and / or to receptor- proximal complexes, thereby driving localized PIP3 production. In some embodiments, the SEM comprises a GRB2 adaptor segment (e.g., an N-terminal SH3 domain with or without an SH2 and / or C-terminal SH3 domain) that, when localized by the MLM, recruits S0S1 or other effectors to promote Ras-Raf-MEK-ERK signaling. In some embodiments, the SEM domain comprises the amino acid sequence of any one of SEQ ID NO: 3, 4, 6, 19, 290, 291, 292, 293, 294, 295, or 296, or sequences having at least about 85, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoing.

[0100] In some embodiments, the SEM comprises a Syk / ZAP70-family non-receptor tyrosine kinase or a portion thereof comprising a kinase domain, with or without one or more interdomain regions such as interdomain B. In some embodiments, the SEM comprises a ZAP70 kinase domain, a ZAP70 interdomain B-kinase domain (ZAP70intB-KD), a SYK kinase domain, or a SYK interdomain B-kinase domain (SYKintB-KD). In some embodiments, the SEM comprises a ZAP70 kinase domain or a ZAP70 interdomain B-kinase domain (ZAP70intB-KD), for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 290, 291, or 292, or a sequence having at least about 85%, 86%, 87%, 88%,Attorney Docket No.: 048536-807001 WO89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the SEM comprises a SYK kinase domain or a SYK interdomain B- kinase domain (SYKintB-KD), for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 293, or 294, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, when localized by an MLM, such ZAP70- or SYK-based SEMs phosphorylate immunoreceptor tyrosine-based activation motifs (IT AMs), LAT, SLP- 76, and / or other adaptor proteins, thereby initiating or amplifying downstream signaling.

[0101] In some embodiments, the SEM comprises an AKT1 or PDK1 kinase domain, such that membrane localization facilitates engagement with PIP3, PIP2, and / or upstream kinases and substrates involved in AKT signaling. In some embodiments, such AKT1 and PDK1 SEMs are provided by truncated polypeptides comprising the amino acid sequences of SEQ ID NO: 295 (AKTltrunc, comprising a kinase domain and lacking a PH domain) or SEQ ID NO: 296 (PDKltrunc, comprising a kinase domain and lacking a PH domain), or sequences having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0102] In some embodiments, the SEM comprises a class IA PI3K regulatory subunit fragment, for example a PIK3Rla nSH2-iSH2-cSH2 domain cluster. In some embodiments, such SEM is provided by a polypeptide comprising the amino acid sequence of SEQ ID NO: 19 or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In any of such embodiments, the SEM is configured to recruit a PI3K catalytic subunit to the membrane and / or to receptor- proximal complexes, thereby driving localized PIP3 production.

[0103] In some embodiments, the SEM comprises a GRB2 adaptor segment, for example an N-terminal SH3 domain with or without an SH2 and / or C-terminal SH3 domain. In some embodiments, such SEM is provided by a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In any of such embodiments, such SEM is one in which when localized by the MLM, recruits S0S1 or other effectors to promote Ras-Raf-MEK-ERK signaling.

[0104] In some embodiments, the capacity of a polypeptide segment to act as an SEM is assessed functionally. In some embodiments, cells expressing an MLM-SEM fusion are stimulated through a receptor (for example, a TCR, CAR, or other antigen receptor), and activation of one or more kinase signaling cascades is measured using phospho-specificAttorney Docket No.: 048536-807001 WO readouts. Non-limiting examples include detection of phosphorylated ZAP70, SYK, LAT, SLP-76, ERK, AKT, S6, or other pathway components by immunoblotting or flow cytometry. In some embodiments, transcriptional reporters (for example, NFAT-, NF-KB-, or AP-l-driven reporter constructs) and / or cytokine secretion (for example, IE-2, IFN-y) are used as downstream functional readouts. In some embodiments, SEM function is inferred from an increase in pathway activation in cells expressing the MEM-SEM fusion as compared to cells expressing an MLM alone, an SEM alone, or control constructs lacking key catalytic or interaction domains. In some embodiments, SEMs comprising kinase domains show increased phosphorylation of their cognate substrates when localized by an MLM, whereas SEMs comprising adaptor modules such as PIK3Rla nSH2-iSH2-cSH2 or GRB2 show increased recruitment or activation of associated catalytic partners and downstream signaling.

[0105] In some embodiments, the MLM and SEM are heterologous to one another, meaning that they are derived from different parent proteins. In some embodiments, the MLM is derived from a Tec family kinase, such as ITK, BTK, TEC, or BMX, or from a transmembrane adaptor protein (TRAP), such as LAT, PAG / Cbp, LIME, SIT, TRIM, LAX, SCIMP, PRR7, or LST1 / A. In some embodiments, the SEM is derived from a kinase or adaptor of a Syk / ZAP70 non-receptor tyrosine kinase cascade, an AKT serine / threonine kinase cascade, a class IA phosphoinositide 3-kinase (PI3K) lipid-kinase cascade, or a Ras- Raf-MEK-ERK cascade. In some embodiments, the MLM lacks a functional enzymatic domain, whereas the SEM comprises one or more functional enzymatic domains, such as a kinase domain. In other embodiments, the SEM comprises primarily non-enzymatic adaptor and / or regulatory domains that nevertheless potentiate a kinase signaling cascade when localized by the MLM.

[0106] In some embodiments, the MLM and SEM are combined to form specific chimeric signaling polypeptides having defined N- to C-terminal architectures. In some embodiments, the recombinant polypeptide comprises an ITK-derived MLM fused to a ZAP70 SEM. In some embodiments, the recombinant polypeptide is an ITK PH-TH-SH2-SH3-ZAP70KD fusion (also referred to herein as “ITK PH-TH-SH2-SH3-ZAP70KD”), in which an ITK PH- TH-SH2-SH3 MLM is operably linked in N- to C-terminal direction to a ZAP70 kinase domain SEM. In some embodiments, the MLM portion comprises the sequence of SEQ ID NO: 287 or SEQ ID NO: 288 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto) and the SEM portion comprises the sequence of SEQ ID NO: 3 (or a sequence having at leastAttorney Docket No.: 048536-807001 WO about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto). In some embodiments, the full-length fusion polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 33, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the full-length fusion polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 305 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 305.

[0107] In some embodiments, the recombinant polypeptide comprises an ITK-derived MLM fused to a SYK SEM. In some embodiments, the recombinant polypeptide is ITK PH- TH-SYK, in which an ITK PH-TH MLM is linked in N- to C-terminal direction to a SYK SEM comprising a kinase domain. In some embodiments, the MLM portion comprises the sequence of SEQ ID NO: 287 or SEQ ID NO: 288 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto) and the SEM portion comprises the sequence of SEQ ID NO: 4, 293, or 294 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto). In some embodiments, the full- length fusion polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 69, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 69.

[0108] In some embodiments, an ITK PH-TH MLM is fused to a SYK interdomain B- kinase domain (SYKintB-KD) to provide an ITK PH-TH-SYKintB-KD fusion architecture. In some embodiments, the MLM portion of such a fusion comprises an ITK PH-TH segment, for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 287 or SEQ ID NO: 288 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto), and the SEM portion comprises a SYKintB-KD segment, for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 293 or SEQ ID NO: 294 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto). In some embodiments, the full-length fusion polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 307 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 307. This ITK-SYKintB-KD arrangement corresponds in part to a naturally occurring ITK-SYK fusion protein produced by a somaticAttorney Docket No.: 048536-807001 WO chromosomal translocation in certain T-cell lymphomas; however, the present disclosure encompasses engineered or recombinant ITK PH-TH-SYKintB-KD polypeptides that are expressed from a recombinant nucleic acid in a host cell. For the avoidance of doubt, the recombinant polypeptides of the invention do not include naturally occurring or endogenously generated ITK-SYK fusion proteins arising from somatic chromosomal translocations in mammalian cells.

[0109] In some embodiments, the recombinant polypeptide comprises a LAT™DMLM fused to a kinase or adaptor SEM. In some embodiments, the recombinant polypeptide is LAT™D-AKTltrunc, in which the MLM comprises a LAT transmembrane domain (e.g., SEQ ID NO: 289 or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto) and the SEM comprises an AKT1 kinase domain lacking the PH domain (e.g., SEQ ID NO: 295 or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto). In some embodiments, the full- length fusion polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 60, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 60. In some embodiments, the recombinant polypeptide is LAT™D-SYK, in which the MLM comprises LAT™Dand the SEM comprises SYK, for example as set forth in SEQ ID NO: 50, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the recombinant polypeptide is LAT™D-PIK3Rla, in which LATTMDis fused to a PIK3Rla nSH2-iSH2-cSH2 SEM, for example yielding a fusion polypeptide having the amino acid sequence of SEQ ID NO: 64, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the recombinant polypeptide is LAT™D-GRB2, for example comprising the amino acid sequence of SEQ ID NO: 51, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the recombinant polypeptide is LATTMD-PDK1, for example comprising the amino acid sequence of SEQ ID NO: 63, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0110] In some embodiments, the MLM comprises an ITK PH-TH domain and the SEM comprises a PIK3Rla regulatory fragment. In some embodiments, the recombinantAttorney Docket No.: 048536-807001 WO polypeptide is ITK PH-TH-PIK3Rla, in which an ITK PH-TH MLM is fused to a PIK3Rla nSH2-iSH2-cSH2 SEM. In some embodiments, the full-length polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 76, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 76.

[0111] In some embodiments, the recombinant polypeptide is any as disclosed herein. In some embodiments, the recombinant polypeptide comprises the sequence of amino acids set forth in any one of SEQ ID NOs: 31-143, 305, and 307; or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 31-143, 305, and 307. In some embodiments, nucleic acids encoding any of the recombinant polypeptides described herein are provided. In some embodiments, such nucleic acids comprise or consist of the nucleotide sequences set forth in any one of SEQ ID NOs: 174-286, or sequences having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 174- 286, 306, and 308. In some embodiments, such nucleic acids are codon-optimized for expression in a mammalian cell, such as a human T cell, and are operably linked to one or more regulatory elements (e.g., promoters, enhancers, polyadenylation signals, and / or ribosomal skipping sequences) as described elsewhere herein. Table 1 depicts exemplary features of any of the provided recombinant polypeptides and encoding nucleic acids.

[0112] In some embodiments, the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 33, 69, 50, 60, 64, 76, 51, or 63, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoing.

[0113] In some embodiments, the recombinant polypeptide is ITK PH-TH-SH2-SH3- ZAP70KD comprising the sequence of amino acids set forth in SEQ ID NO: 33, or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33.

[0114] In some embodiments, the recombinant polypeptide includes a polypeptide designated ADAP-12234valency-increased, GADSSH2-SLP76disordered, HPKldisordered- SLP76SH2, NCKlSH3-SH3-SH3-SLP76disordered, LCKCD4-CD8-interactors-SH3-SH2- ITKSH3-SH2, and LAT™D-HPKldisordered-SLP76disorderedA,I. In some embodiments, the recombinant polypeptide comprise the sequences of SEQ ID NO: 87, 91, 94, 105, 111, or 137, respectively, or sequences having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NO: 87,Attorney Docket No.: 048536-807001 WO91, 94, 105, 111, or 137. In some embodiments, the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 87, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 87. In some embodiments, the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 91, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 91. In some embodiments, the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 94, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 94. In some embodiments, the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 105, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 105. In some embodiments, the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 111, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 111. In some embodiments, the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 137, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 137.

[0115] In certain embodiments of the disclosure, the recombinant polypeptides disclosed herein further include an amino acid sequence for the 6th Ankyrin Repeat (AR6) of the protein IKBO., or a functional variant thereof, wherein the AR6 sequence or functional variant thereof mediates a targeted ubiquitin-independent degradation by the proteasome, controlling NF-KB activation. This degron (specifically residues like 251-262) is crucial for regulating IKBO.'S short half-life, allowing for timely NF-KB release, and can trigger degradation of other proteins when attached. More information in this regard can be found in, e.g., Fortman KT et al. J Mol Biol. 2015 August 28; 427(17): 2748-2756.

[0116] In some embodiments, the AR6 sequence comprises or is the sequence selected from the group consisting of SEQ ID NOS: 297-299. In some embodiment, the AR6 sequence comprises or is the sequence of SEQ ID NO: 297.

[0117] One skilled in the art will appreciate that the complete amino acid sequence of a recombinant polypeptide of the disclosure can be used to construct a back-translated gene. For example, a DNA oligomer containing a nucleotide sequence coding for a givenAttorney Docket No.: 048536-807001 WO recombinant polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired recombinant polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0118] In addition to generating desired recombinant polypeptides expression of nucleic acid molecules that have been engineered by recombinant molecular biological techniques, a subject recombinant polypeptide in accordance with the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those of skill in the art.Attorney Docket No.: 048536-807001WOTABLE 1 : Brief description for the modular protein domain building blocks used to generate a library of fusion proteins in accordance with various non-limiting embodiments of the disclosure (see also, e.g., Example 1 and FIGS. 1A-1H). Provided are exemplary non-fusion proteins or fusion proteins, in which two or more heterologous polypeptide sequences are covalently linked (e.g., fused in-frame) to one another. The amino acid sequences and polynucleotide sequences of the fusions proteins are provided in the Sequence Listing. Amino acid positions in each of the polypeptide sequences are numbered relative to the native full-length sequences.Attorney Docket No.: 048536-807001WOAttorney Docket No.: 048536-807001WOAttorney Docket No.: 048536-807001WOAttorney Docket No.: 048536-807001WOAttorney Docket No.: 048536-807001WOAttorney Docket No.: 048536-807001WOAttorney Docket No.: 048536-807001 WO

[0119] Once assembled (e.g., by synthesis, recombinant methodologies, site-directed mutagenesis or other suitable techniques), the DNA sequences encoding a recombinant polypeptide as disclosed herein can be inserted into an expression vector and operably linked to an expression control sequence appropriate for expression of the recombinant polypeptide in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is known in the art, in order to obtain high expression levels of a transfected gene in a host, take should be taken to ensure that the gene is operably linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.NUCLEIC ACID MOLECULES AND VECTORS

[0120] In discussed above, the recombinant polypeptides as described herein can be obtained by expression of a recombinant nucleic acid or vector (e.g., expression vector). Accordingly, one aspect of the disclosure relates to a recombinant nucleic acids and vectors including a nucleic acid sequence that encodes a recombinant polypeptide of the disclosure.

[0121] In some embodiments, the recombinant nucleic acids of the disclosure can be configured as expression cassettes or vectors containing these nucleic acid molecules operably linked to one or more heterologous nucleic acid sequences such as, for example, regulatory sequences which allow in vivo expression of the receptor in a host cell.

[0122] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules that are generally between about 5 Kb and about 50 Kb, for example between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0123] In some embodiments, the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence, such as, for example a regulatory sequence e.g., promoter sequence) or a sequence encoding signal peptide. In some embodiments, the heterologous nucleic acid sequence comprises or is a nucleic acid control element. Nonlimiting examples of nucleic acid control elements suitable for the compositions and methods of the disclosure include ribosomal binding sites, promoters, enhancer elements, activator elements, translational start sequences, translational termination sequences, transcription start sequences, transcription termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bpAttorney Docket No.: 048536-807001 WO poly(A) tract, a 325 bp poly(A) tract, replication elements, RNA processing and export elements, transposon sequences, transposase sequences, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, and 3’UTRs. Additional nucleic acid control elements suitable for the compositions and methods of the disclosure include, but are not limited to, mRNA 3’ end processing sequences, boundary elements, locus control regions (LCR), matrix attachment regions (MAR), recombination or cassette exchange sequences, linker sequences, cleavable linker sequences, secretion signals, resistance markers, anchoring peptides, localization signals, fusion tags, affinity tags, chaperonins, and proteases.

[0124] In some embodiments, a nucleic acid encoding a recombinant polypeptide further comprises or is operably linked to a promoter that regulates expression of the recombinant polypeptide in a cell. In some embodiments, the promoter is a heterologous promoter that is part of the same recombinant nucleic acid molecule (e.g., within a plasmid, viral vector, or linear expression cassette) and is selected to drive constitutive or inducible expression of the recombinant polypeptide. Thus, in some embodiments, the recombinant nucleic acid itself includes a promoter sequence operably linked to the coding sequence of the recombinant polypeptide. In some embodiments, the promoter is constitutive and drives continuous expression of the recombinant polypeptide, whereas in other embodiments the promoter is inducible or regulatable. Examples of inducible or regulatable promoters include, for example, promoters responsive to ligand-inducible receptors, small molecules, optogenetic inputs, transcription factor signals, inflammatory or activation-dependent transcription factors, or combinations thereof. In some embodiments, the promoter is an endogenous promoter of an endogenous genomic locus, such that the nucleic acid encoding the recombinant polypeptide is integrated into that locus and brought under control of the native transcriptional regulatory elements of the cell. In such embodiments, the recombinant nucleic acid does not include a promoter but is integrated into an endogenous locus such that an existing endogenous promoter (for example, a T cell-specific or activation-dependent promoter) is operably linked to and drives expression of the inserted recombinant polypeptide coding sequence.

[0125] In some embodiments, the heterologous nucleic acid sequence comprises or is a promoter. Non-limiting examples of promoters suitable for the compositions and methods of the disclosure include EFla promoter, CMV promoter, CAG promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actinAttorney Docket No.: 048536-807001 WO promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD1 la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD 161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL 17 A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, and CD 132 promoter. Additional promoters suitable for the compositions and methods of the disclosure include, but are not limited to, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)- related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, and NR4A3 promoter. In some embodiments, the promoter is an endogenous promoter. Suitable endogenous promoters include, but are not limited to, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, CARD9 promoter, CARDIO promoter, CARD 11 promoter, CARD 14 promoter, PIK3R3 promoter, B2 microglobulin (B2M) promoter, and class II transactivator (CIITA) promoter. In some embodiments, the promoter is a synthetic promoter.

[0126] Additional promoters of a heterologous nucleic acid sequence include, but are not limited to, an MND promoter, a short elongation factor 1 alpha (sEFl) promoter, a gamma- retroviral long terminal repeat (LTR) promoter, a TRAC promoter, a TRBC 1 promoter, a TRBC2 promoter, a CD3a promoter, a CD3P promoter, an albumin promoter, an F3 (CD 142) promoter, a MICA promoter, a MICB promoter, an LRP1 (CD91) promoter, an HMGB1Attorney Docket No.: 048536-807001 WO promoter, an ABO promoter, an RHD promoter, a FUT1 promoter, a KDM5D (HY) promoter, a PDGFRa promoter, an OLIG2 promoter, a GFAP promoter, an immunoglobulin promoter, a heat-shock promoter, a human Ubiquitin C (UBC) promoter, a ubiquitin / S27a promoter, a simian virus 40 (SV40) early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, a Rous Sarcoma Virus (RSV) long terminal repeat promoter, a mouse mammary tumor virus (MMTV) promoter, a Moloney murine leukemia virus (MLV) long terminal repeat promoter, and promoters derived from a polyoma virus, a fowlpox virus, a bovine papilloma virus, an avian sarcoma virus, a cytomegalovirus, a retrovirus, or a hepatitis B virus.

[0127] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a regulatable promoter, e.g., an inducible promoter or a repressible promoter. In some embodiments, the regulatable promoter is a TCRa-promoter or an EF la-promoter.

[0128] In some embodiments, the promoter comprises or is an EFla promoter, an MND promoter, CMV promoter, CAG promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD Ila promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD 161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL 17 A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NFAT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter,Attorney Docket No.: 048536-807001 WOTIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, or NR4A3 promoter.

[0129] Examples of endogenous promoters include, for example, a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, a CD3z promoter, a B2 microglobulin (B2M) promoter, or a class II transactivator (CIITA) promoter

[0130] In some embodiments, when a nucleic acid encoding the recombinant polypeptide is inserted into an endogenous genomic locus and is controlled by an endogenous promoter, the recombinant polypeptide coding sequence is linked to an endogenous coding sequence by a self-cleaving peptide or ribosomal skipping sequence. In some embodiments, the nucleic acid comprises an open reading frame encoding an endogenous protein followed by a selfcleaving peptide sequence (for example, a 2A peptide) and the recombinant polypeptide, such that both the endogenous protein and the recombinant polypeptide are translated from a single transcript and produced as separate polypeptides. In some embodiments, the selfcleaving or ribosomal skipping sequence is selected from P2A, F2A, E2A, T2A, or other viral 2A-like sequences, or functionally similar sequences that mediate co-translational “ribosome skipping.” In other embodiments, the nucleic acid encoding the recombinant polypeptide replaces all or part of the endogenous coding sequence such that the endogenous promoter drives expression of the recombinant polypeptide alone, without a self-cleaving or ribosome skipping sequence.

[0131] In some embodiments, the one or more heterologous nucleic acid sequences comprises or is a coding sequence for a polypeptide of interest. Non-limiting examples of suitable polypeptide of interest include peptide linker sequences, cleavable linker sequences, secretion signals, resistance markers, anchoring peptides, localization signals, fusion tags, affinity tags, chaperonins, protease, or any combination thereof.

[0132] In some embodiments, the recombinant nucleic acids of the disclosure further include a barcode molecule that identifies that recombinant polypeptide. In some embodiments, the barcode can be a nucleic acid sequence incorporated within the P2A sequence upstream of the transgene that corresponds to the identity of the transgene (e.g., identifies the transgene). In some embodiments, the barcodes vary as a combination of synonymous codons for the P2A sequence, such that every barcode encodes the same amino acids.Attorney Docket No.: 048536-807001 WO

[0133] In some embodiments, the recombinant nucleic acids of the disclosure further include nucleic acid sequences encoding for a “self-cleaving” peptide sequence, e.g., ribosomal skipping peptide sequences capable of mediating ribosome-skipping events, thereby enabling the generation of two or more separate peptide products from one mRNA containing the self-cleaving peptide sequence. In some embodiments, the ribosomal skipping peptide sequence is a 2A peptide. Non-limiting examples of ribosomal skipping peptide sequences suitable for the compositions and methods of the disclosure include ribosomal skipping peptide sequences from porcine teschovirus- 1 2 A (P2A), calcium-dependent serine endoprotease (furin), foot-and-mouth disease virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), and Flacherie Virus 2A (BmIFV2A). In some embodiments, the ribosomal skipping peptide sequence includes a ribosomal skipping peptide sequence from a porcine teschovirus- 1 2 A (P2A).

[0134] In some embodiments, the recombinant nucleic acids of the disclosure further may include a second nucleic acid sequence encoding: (a) an engineered antigen-specific receptor having specificity for a target ligand; and / or (b) a T cell receptor (TCR) having specificity for a target ligand. Examples of recombinant antigen receptors include any as described herein, such as those described in connection with additional recombinant nucleic acids that can be present in a recombinant cell. A recombinant nucleic acid encoding an antigen receptor that is present in any of the recombinant cells described herein may be provided on the same recombinant nucleic acid molecule as a nucleic acid encoding a recombinant polypeptide, or on a different recombinant nucleic acid molecule. In some embodiments, a single recombinant nucleic acid comprises a first nucleic acid sequence encoding the recombinant polypeptide and a second nucleic acid sequence encoding the antigen receptor. The first and second nucleic acid sequences may be part of a single expression cassette under the control of a shared promoter (e.g., arranged as a multicistronic transcript separated by a self-cleaving peptide sequence such as a 2A peptide and / or an internal ribosome entry site (IRES)), or may each be operably linked to its own promoter within the same vector. In other embodiments, the recombinant polypeptide and the antigen receptor are encoded by separate expression cassettes located on distinct recombinant nucleic acids (e.g., separate viral vectors, plasmids, or genomic integration events), each operably linked to one or more promoters suitable for expression in the recombinant cell.

[0135] In some embodiments, the second nucleic acid sequence encodes a T-cell receptorAttorney Docket No.: 048536-807001 WO(TCR). A TCR generally includes two polypeptides (e.g., polypeptide chains), such as an oc- chain of a TCR, a P-chain of a TCR, a y-chain of a TCR, a 8-chain of a TCR, or a combination thereof. Such polypeptide chains of TCRs are known in the art. The antigenspecific TCR can include any amino acid sequence, provided that the TCR can specifically bind to and / or immunologically recognize an antigen, such as a cancer antigen or epitope thereof. In some embodiments, the TCR is an endogenous TCR, e.g., a TCR that is endogenous or native (naturally-occurring) to the T cell. In such a case, the T cell expressing the endogenous TCR can be a T cell that was isolated from a mammal which is known to express the particular cancer antigen. For example, in some embodiments, the T cell is a primary T cell isolated from a mammal having a cancer. In some embodiments, the T cell is a TIL or a T cell isolated from a human cancer patient.

[0136] In some embodiments, the engineered antigen receptor comprises or is a chimeric antigen receptor (CAR). Generally, the term CAR refers to a polypeptide construct comprising at least an extracellular antigen-binding domain, a TMD and a cytoplasmic signaling domain (also referred to as “an intracellular signaling domain” or ICD). In some cases, the cytoplasmic signaling domain includes a functional signaling domain derived from a stimulatory molecule. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. Optionally, the ICD can further include one or more functional signaling domains derived from at least one costimulatory molecule.

[0137] Generally, the CARs of the disclosure include an ectodomain and an endodomain each as defined by the host plasma membrane. In this regard, the terms “ectodomain” or “extracellular domain” generally refer to the portion of the CAR polypeptide outside of the cell or exterior to the membranous lipid bilayer, which may include one or more antigen recognition binding domains, an optional hinge domain, and any spacer domains exterior to the amino acid residues physically spanning the membrane. In some embodiments, the ectodomain of the CARs provided herein further include a signal peptide. Conversely, the terms “endodomain” or “intracellular domain” generally refer to the portion of the CAR polypeptide inside the cell or interior to the membranous lipid bilayer, which may also include any spacer domains interior to the amino acid residues physically spanning the membrane, as well as the ICD, which comprises one or more costimulatory signaling domains (e.g., ITAM-containing sequences, costimulatory domains, etc.).

[0138] In some embodiments, the recombinant nucleic acids further include a response element operably incorporated upstream of the coding sequence of the recombinantAttorney Docket No.: 048536-807001 WO polypeptide, wherein the response element includes: (a) a cognate target sequence to which a transcriptional regulator binds; and / or an engineered responsive promoter operably linked to the cognate target sequence. In some embodiments, the transcriptional regulator is incorporated in the ICD of a Hybrid-R receptor as described herein. In some embodiments, the Hybrid-R of the disclosure is designed to combine fast time-scale intracellular signal transduction and long time-scale transcription regulation. For example, in some experiments, the Hybrid-R of the disclosure incorporates (i) costimulatory domains and stimulatory domains of a CAR, for example, the cytoplasmic tail of the CD3zeta chain, and a transcriptional regulator. The architecture of the cytoplasmic tail of a typical Hybrid-R (costimulatory domain, CD3zeta, transcription factor) can be configured in various ways to reliably induce proximal T-cell receptor costimulatory signals and gene regulation in a target cell type, such as, primary human T cells. For example, the Hybrid-R can be configured to simultaneously stimulate (i) fast time-scale (e.g., from seconds to minutes) proximal signaling and (ii) long-time scale transcriptional regulation that usually takes hours to induce to sufficient levels to observe cellular state changes. More information regarding Hybrid-R can be found in, for example, PCT Publication No. WO 2022 / 204324, which is incorporated herein by reference in its entirety.

[0139] In some embodiments, the recombinant nucleic acids of the disclosure may be further configured as (e.g., incorporated into) an expression cassette or a vector. In some embodiments, the vector is an expression vector.

[0140] Accordingly, some embodiments disclosed herein relate to expression cassettes and vectors (e.g., expression vectors) including a recombinant nucleic acid molecule as disclosed herein. As used herein, the term “expression cassette” refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or into a subject. As such, the term expression cassette may be used to refer to an expression construct.

[0141] Also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acid molecules encoding any of the recombinant polypeptides disclosed herein. The nucleic acid molecules described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the artAttorney Docket No.: 048536-807001 WO and are commercially available, or readily prepared by a skilled artisan. Additional vectors can also be found in, for example, Ausubel, F. M., et al. (2014, supra) and Sambrook et al. (2012, supra).

[0142] It should be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow the DNA encoding the recombinant polypeptides of the present disclosure to be amplified in copy number. Such amplifiable vectors are known in the art.

[0143] Accordingly, in some embodiments, the recombinant polypeptides of the present disclosure can be expressed from vectors, generally expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) are also included.

[0144] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operably linked to the nucleic acid sequence to be expressed. DNA vector can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0145] Non-limiting examples of the vectors suitable for the compositions and methods of the disclosure include plasmids, synthetic DNA vectors, linear DNA vectors, closed linear DNA vectors, RNA vectors, mRNA vectors, phagemid vectors, viral vectors, self-replicating RNA viruses, mRNA-packaging virus-like particles, and RNP-packaging virus-like particles.Attorney Docket No.: 048536-807001 WOIn some embodiments, the viral vector may be a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector. In some embodiments, the vector may be formulated in a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a protein nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, a fusosome, an enveloped delivery vehicle, or an immune stimulating complex (ISCOM). In some embodiments, the LNP may be a selective organ targeting LNP or an antibody-targeted LNP.

[0146] The nucleic acid sequences encoding the recombinant polypeptides of the present disclosure can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon optimization are known in the art. Codon usages within the coding sequence of the recombinant polypeptides disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0147] Vectors suitable for use include T7-based vectors for use in bacteria, the pMSXND expression vector for use in mammalian cells, and baculovirus-derived vectors for use in insect cells. In some embodiments, nucleic acid inserts, which encode the subject recombinant polypeptides in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.

[0148] In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject recombinant polypeptide, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this disclosure, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.

[0149] Within these parameters one of skill in the art may select various vector / expression control sequence / host combinations that will express the desired DNA sequences on fermentation or in large scale animal cell culture, for example, using CHO cells or COS 7 cells.

[0150] The choice of expression control sequence and expression vector, in someAttorney Docket No.: 048536-807001 WO embodiments, will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Non-limiting examples of useful expression vectors for eukaryotic hosts, include, for example, vectors with expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Non-limiting examples of useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including col El, pCRI, pER32z, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Non-limiting examples of useful expression vectors for yeast cells include the 2p plasmid and derivatives thereof. Non-limiting examples of useful vectors for insect cells include pVL 941 and pFastBac™ 1.

[0151] A T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue- specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body. Skilled artisans will readily appreciate numerous promoters and other regulatory elements which can be used to direct expression of nucleic acids.

[0152] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resistance (neoR) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells. Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0153] Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), lentivirus, and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0154] As described in greater detail below, prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a subject recombinant polypeptide disclosed herein are also features of the disclosure. A cell of the disclosure is a transfected cell, e.g., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding a recombinant polypeptide of the disclosure, has been introduced by means of recombinantAttorney Docket No.: 048536-807001 WODNA techniques. The progeny of such a cell are also considered within the scope of the disclosure.

[0155] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). For example, a recombinant polypeptide as disclosed herein can be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell (e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, care should be taken to ensure that the components are compatible with one another. Artisans of ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0156] In some embodiments, the vector is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a RNA vector, a mRNA vector, a phagemid vector, a viral vector, a self-replicating RNA virus, a mRNA-packaging virus-like particle, or a Repackaging virus-like particle.

[0157] In some embodiments, the vector is suitable for in vivo delivery of a recombinant nucleic acid encoding a recombinant polypeptide as described herein, and optionally further encoding an antigen receptor (e.g., a chimeric antigen receptor (CAR) or other chimeric receptor) to a target cell such as a T cell. The vectors may be formulated in any suitable delivery vehicle. Non-limiting examples include liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, protein nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, fusosomes, enveloped delivery vehicles, and immune- stimulating complexes (ISCOMs). In some embodiments, the vector is a viral vector, such as a lentiviral vector that is pseudotyped for cell targeting in vivo. In some embodiments, the recombinant nucleic acid is present as an mRNA, a DNA expression cassette, or a viral genome packaged within such a delivery vehicle. The vectors and formulations described herein can be administered to a subject in a pharmaceutically acceptable carrier in an amount effective to deliver the recombinant nucleic acid to target cells (e.g., T cells) in vivo, thereby generatingAttorney Docket No.: 048536-807001 WO recombinant cells in situ that express the recombinant polypeptide and, optionally, the antigen receptor.

[0158] In some embodiments, the vector is formulated in an LNP. The LNP may be a configured with a targeting moiety to preferentially deliver the recombinant nucleic acid to a particular organ or tissue (e.g., lymphoid tissue) or cell type, such as an antibody-targeted LNP that comprises an antibody or antigen-binding fragment thereof on its surface to direct delivery to a desired cell type (e.g., a T cell). In certain embodiments, the antibody-targeted LNP displays an antibody specific for a T cell surface antigen (e.g., CD3, CD2, CD7), thereby promoting in vivo uptake of the recombinant nucleic acid by T cells following administration.

[0159] In other embodiments, the vector is a viral vector, such as a lend viral vector, that packages a recombinant nucleic acid encoding the recombinant polypeptide and, in some embodiments, an antigen receptor (e.g., CAR) within a viral particle suitable for in vivo administration. In some embodiments, the lentiviral vector is pseudotyped with a heterologous envelope glycoprotein. Exemplary heterologous envelope glycoproteins include vesicular stomatitis virus G (VSV-G) glycoprotein and Cocal glycoprotein. In certain embodiments, the heterologous envelope glycoprotein is fused to an antibody or antigenbinding fragment thereof to redirect the lentiviral vector to a target cell type. For example, the envelope glycoprotein may be fused to an anti-CD3 antibody, an anti-CD2 antibody, or an anti-CD7 antibody to facilitate binding to and entry into T cells in vivo.

[0160] The expressed recombinant polypeptides can be purified from the expression system using routine biochemical procedures, and can be used, e.g., as therapeutic agents, as described herein.

[0161] In addition or alternatively, another exemplary method of constructing a DNA sequence encoding the recombinant polypeptides of the disclosure is by chemical synthesis. This includes direct synthesis of a peptide by chemical means of the protein sequence encoding for a recombinant polypeptide exhibiting the properties described. This method can incorporate both natural and unnatural amino acids at positions that affect the functionality of the recombinant polypeptide. Alternatively, a gene which encodes the desired recombinant polypeptide can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired recombinant polypeptide, and suitably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced. In this regard, it is well recognized in the art that the genetic code is degenerate-that an amino acid may be coded for by more thanAttorney Docket No.: 048536-807001 WO one codon. Accordingly, it will be appreciated by those skilled in the art that for a given DNA sequence encoding a particular recombinant polypeptide, there will be many DNA degenerate sequences that will code for that recombinant polypeptide. For example, it will be appreciated that in addition to the DNA sequences for recombinant polypeptides provided in the specification, there will be many degenerate DNA sequences that code for the recombinant polypeptides disclosed herein. These degenerate DNA sequences are considered within the scope of this disclosure. Therefore, “degenerate variants thereof’ in the context of this disclosure means all DNA sequences that code for and thereby enable expression of a particular recombinant polypeptide.

[0162] The DNA sequence encoding the subject recombinant polypeptide, whether prepared by site directed mutagenesis, chemical synthesis or other methods, can also include DNA sequences that encode a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the recombinant polypeptide. It can be prokaryotic, eukaryotic or a combination of the two. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be included.

[0163] The recombinant nucleic acid molecules provided herein can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (e.g., either a sense or an antisense strand).

[0164] In some embodiments, recombinant nucleic acid molecules provided herein include a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 174-286, 306, and 308.

[0165] The nucleic acids of the disclosure are not limited to sequences that encode recombinant polypeptides; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of a recombinant polypeptide) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or byAttorney Docket No.: 048536-807001 WO performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.

[0166] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found as such in the natural state. Thus, this disclosure encompasses recombinant molecules, such as those in which a nucleic acid sequence (for example, a sequence encoding a recombinant polypeptide of the disclosure) is incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a recombinant cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).RECOMBINANT CELLS AND CELL CULTURES

[0167] The nucleic acids and / or vectors of the present disclosure can be introduced into a host cell, such as a human T cell or cancer cell, to produce a recombinant cell containing the nucleic acids and / or vectors. Accordingly, some embodiments of the disclosure relate to methods for preparing / making a recombinant cell, wherein the methods include (a) providing a host cell capable of protein expression; and transducing the provided host cell with a recombinant nucleic acid or a vector of the disclosure to produce a recombinant cell. Some other embodiments of the disclosure related to methods for preparing / making a recombinant cell, wherein the methods include expressing in the T cell one or more of the following: (a) a recombinant polypeptide as described herein; (b) a recombinant nucleic acid as described herein; and (c) a vector as described herein. In some embodiments, the methods of preparing T cells as disclosed herein further include genetically modifying the T cell for expression of the recombinant polypeptide.

[0168] In some embodiments, the methods further include introducing to the T cell a recombinant nucleic acid as described herein, or a vector as described herein. Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0169] Accordingly, in some embodiments, the nucleic acid molecules can be introduced into a host cell by viral or non-viral delivery vehicles known in the art to produce a recombinant cell. For example, the nucleic acid molecule can be stably integrated in the hostAttorney Docket No.: 048536-807001 WO genome, or can be episomally replicating, or present in the recombinant host cell as a minicircle expression vector for a stable or transient expression. Accordingly, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using zinc-finger proteins (ZNF), guide RNA directed CRISPR / Cas enzyme (such as CRISPR / Cas9, CRISPR / Casl2a, or other CRISPR / Cas enzyme), DNA-guided endonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nucleases).

[0170] In some embodiments, the methods of preparing / making recombinant cells, e.g., recombinant T cells, of the disclosure one or more of the following: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, an RNA-guided nuclease including a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex including a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR- associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In some embodiments, the T cells are made using homology-directed repair (HDR) -mediated insertion. In some embodiments, the T cells are made in vivo, ex vivo, or in vitro.

[0171] The recombinant nucleic acid molecules can be encapsulated in a viral capsid, or a lipid nanoparticle, or a capsid whose components are derived from genes of viral origin, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In a non-limiting example, baculoviral virus or adeno-associated virus (AAV) can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses. In some embodiments, AAV may be used to site- specifically introduce gene cargo at the site of a genomic DNA break.Attorney Docket No.: 048536-807001 WO

[0172] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as genedelivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0173] In some embodiments, host cells can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0174] In some embodiments, the host cell is obtained by leukapheresis performed on a sample obtained from a subject, and the cell is transduced ex vivo. In some embodiments, the recombinant nucleic acid or vector is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the methods further include isolating and / or purifying the produced cells. Accordingly, the recombinant cells produced by the methods disclosed herein are also within the scope of the disclosure. In some embodiments, the recombinant cells of the disclosure include one or more of the following: (a) a recombinant polypeptide as described herein; (b) a recombinant nucleic acid as described herein; and (c) a vector as described herein.

[0175] In some embodiments, the recombinant cell is a prokaryotic cell. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a mouse cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the recombinant cell is an immune system cell, e.g., a B cell, a monocyte, a NK cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (TH), aAttorney Docket No.: 048536-807001 WO cytotoxic T cell (TCTL), a memory T cell, a gamma delta (y5) T cell, another T cell, a hematopoietic stem cell, or a hematopoietic stem cell progenitor.

[0176] In some embodiments, the recombinant cell is a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a gamma delta T cell (y5T), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, an engineered T cell including a transcriptional receptor, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, a naive T (TN) cell, a hematopoietic stem cell, or a progenitor cell of the lymphoid lineage. In some embodiments, the recombinant cell is a stem cell derived cell. In some embodiments, the recombinant cell has reduced / eliminated cell surface TCR expression, MHC-I expression, and / or MHC-II expression.

[0177] In some embodiments, the immune system cell is a lymphocyte. In some embodiments, the lymphocyte is a T lymphocyte. In some embodiments, the lymphocyte is a T lymphocyte progenitor. In some embodiments, the T lymphocyte is a CD4+ T cell or a CD8+ T cell. In some embodiments, the T lymphocyte is a CD8+ T cytotoxic lymphocyte cell. Non-limiting examples of CD8+ T cytotoxic lymphocyte cell suitable for the compositions and methods disclosed herein include naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, the T lymphocyte is a CD4+ T helper lymphocyte cell. Suitable CD4+ T helper lymphocyte cells include, but are not limited to, naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

[0178] In some embodiments of the disclosure, the recombinant cell (e.g., recombinant T cell) further includes an additional recombinant nucleic acid molecule encoding: (a) an engineered antigen receptor having specificity for a target ligand; and / or (b) a T cell receptor (TCR) having specificity for a target ligand. In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR).

[0179] In some embodiments, the additional recombinant nucleic acid encodes a recombinant antigen receptor that provides antigen specificity, such as chimeric antigenAttorney Docket No.: 048536-807001 WO receptor (CAR) or a TCR-enabled architecture. In some embodiments, a CAR comprises, in N- to C-terminal direction, an extracellular antigen-binding domain directed against a target ligand (e.g., an scFv, VHH, ligand, receptor, or peptide), an optional spacer or hinge region, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the intracellular signaling domains comprise a primary activation domain (e.g., CD3Q and one or more costimulatory domains (e.g., CD28, 4-1BB, 0X40, ICOS), which together drive T cell activation, proliferation, cytokine production, and cytotoxicity upon engagement of the target antigen. In some embodiments, the CAR is a single-chain CAR in which all domains are encoded in a single polypeptide, or a multi-chain CAR in which antigen binding and signaling functions are distributed across two or more chains (e.g., DAPbased or Fc receptor-based CAR architectures).

[0180] In some embodiments, CARs may include single-targeted CARs, multi-targeted CARs, bivalent tandem CARs, bivalent loop CARs, multicistronic or bicistronic CAR constructs, synNotch-CAR circuits, synthetic intramembrane proteolysis receptors (SNIPRs), multi-chain DAP-CARs, TREM1 / DAP12 CARs, or DAP12 / TREM1 CARs.

[0181] In some embodiments, the CAR is specific for CD19, as in autologous CD19- directed therapies such as tisagenlecleucel (Kymriah®), axicabtagene ciloleucel (Yescarta®), brexucabtagene autoleucel (Tecartus®), lisocabtagene maraleucel (Breyanzi®), and obecabtagene autoleucel (Aucatzyl®). In some embodiments, the CAR is specific for B-cell maturation antigen (BCMA), as in BCMA-directed products such as idecabtagene vicleucel (Abecma®) and ciltacabtagene autoleucel (Carvykti®). In some embodiments, the CAR targets additional antigens, including CD20, CD22, CD123, CD7, GPRC5D, and CLDN18.2, either as single targets or in dual- or multi-target configurations (for example, CD19 / CD22, CD19 / CD20, BCMA / CD19, or BCMA / CD38 / CD19). In some embodiments, a recombinant fusion polypeptide described herein is co-expressed with any of these antigen-specific CARs to enhance antigen sensitivity, effector function, and / or durability of the engineered T cells.

[0182] In some embodiments, the recombinant antigen receptor is a TCR-enabled architecture. In some embodiments, a TCR-enabled architecture comprises one or more native or engineered TCR chains and associated CD3 signaling components, optionally fused or linked to heterologous binding or regulatory domains. In some embodiments, the TCR- enabled receptor is a conventional a[3 TCR recognizing peptide-MHC, a T cell receptor fusion construct (TruC) in which an scFv or other binding domain is fused to a CD3 subunit, an HLA-independent T cell (HIT) receptor that uses TCR / CD3 signaling modules coupled toAttorney Docket No.: 048536-807001 WO non-MHC binding domains, or a synthetic T cell receptor and antigen receptor (STAR) construct in which TCR constant and CD3 signaling components are combined with a nonnative antigen-binding domain. In some embodiments, the TCR-enabled architecture is regulatable, for example a rapamycin-inducible TCR or a rapamycin-inducible Fc receptor, in which split receptor components dimerize or assemble in the presence of a small-molecule dimerizer. In some embodiments, the TCR-enabled architecture is a constitutive TCR-like receptor in which an antibody-like or ligand-binding domain is grafted onto a TCR / CD3 signaling scaffold.

[0183] In some embodiments, the recombinant antigen receptor is a dimerizing-agent regulated immune-receptor complex (DARIC) or an antibody tethered orthogonal multiplexing compatible (ATOMIC) receptor system, in which receptor activity is controlled by a controllable dimerizing or bridging agent. In some embodiments, the antigen receptor is provided as part of a synNotch-CAR circuit or SNIPR architecture, in which binding of a first antigen by a synNotch or SNIPR receptor induces transcriptional activation of a CAR or TCR-enabled receptor specific for a second antigen, thereby implementing logic-gated recognition. In some embodiments, a provided recombinant polypeptide is placed under the control of the same inducible circuits, such that CAR or TCR engagement leads to coordinated up-regulation of the fusion polypeptide, enhancing signaling potency in a spatially and temporally restricted manner. Embodiments of inducible system are further described below.

[0184] In some embodiments, the CAR or TCR-enabled architectures described herein are combined with a provided recombinant polypeptide to reprogram T cell signaling in response to antigen encounter. In some embodiments, antigen recognition via CAR or TCR leads to formation of receptor-proximal signaling complexes (e.g., LAT-SLP-76 signalosomes), to which a provided recombinant fusion polypeptide may be recruited, thereby amplifying or reshaping downstream kinase cascades such as Syk / ZAP70, AKT, PI3K, and Ras-Raf-MEK- ERK. In some embodiments, this combination of engineered antigen receptors and recombinant polypeptides as provided yields T cells with enhanced antigen sensitivity, improved tumor control, and favorable proliferative and functional profiles in vitro and in vivo, as described elsewhere herein.

[0185] In some embodiments, a nucleic acid encoding a provided recombinant polypeptide is operably linked to a promoter that controls expression of the recombinant polypeptide in the cell. In some embodiments, the promoter is an exogenous promoter, which may beAttorney Docket No.: 048536-807001 WO constitutively active or inducibly regulated. In some embodiments, the exogenous promoter comprises a viral, cellular, synthetic, or hybrid promoter (for example, an EFla, PGK, MND, CMV, or CAG promoter), configured to drive expression of the recombinant polypeptide at a desired basal level in the engineered cell. In some embodiments, the promoter is an endogenous promoter of an endogenous locus in the cell, such that insertion of the nucleic acid encoding the recombinant polypeptide into that locus places expression under the control of the native transcriptional regulatory elements of the cell.

[0186] In some embodiments, expression of the recombinant polypeptide is constitutive in the cell. For example, in some embodiments, the recombinant polypeptide is expressed under the control of a constitutive exogenous promoter in a viral vector or non- viral vector, or is integrated into an endogenous locus whose promoter confers stable expression in a given cell type (e.g., a T cell-specific promoter). In such embodiments, the recombinant polypeptide is present in the cell before and during antigen encounter and can be continuously recruited to receptor-proximal signaling complexes upon activation of an engineered antigen receptor.

[0187] In some embodiments, expression of the recombinant polypeptide is inducible in the cell. In some embodiments, inducible expression of a recombinant polypeptide may be desirable where the activity of the recombinant polypeptide is sufficiently potent that, when expressed constitutively, it has the potential to cause excessive activation and toxicity in vivo. In some embodiments, such toxicity may manifest as weight loss, elevated clinical scores, cytokine release-like syndromes, organ infiltration by hyperactivated T cells, or early mortality. In some embodiments, inducible expression is a strategy to mitigate such toxicity while retaining the functional benefits of the recombinant fusion polypeptide. In some embodiments, expression of the recombinant fusion polypeptide is made inducible or controlled, for example by placing the coding sequence of the recombinant polypeptide under the control of a ligand-inducible receptor (such as a synthetic Notch (synNotch) receptor, a Hybrid-R or a Synthetic Intramembrane Proteolysis Receptor (SNIPR)), a small-molecule- regulated transcription factor system, an optogenetic transcription factor system, or an inducible CRISPR-based transcriptional activator system as described herein. In some embodiments, expression of the recombinant polypeptide is induced preferentially following antigen engagement by a CAR or TCR-enabled receptor, thereby spatially and temporally restricting activity to tumor sites and reducing off-target toxicity.

[0188] In some embodiments, the chimeric receptor used to control expression of the recombinant polypeptide (for example, a synNotch receptor, a SNIPR, or a Hybrid-RAttorney Docket No.: 048536-807001 WO receptor) comprises: (a) an extracellular ligand-binding domain that recognizes a cell-surface antigen or other target ligand; (b) a transmembrane domain; and (c) an intracellular domain comprising a transcriptional regulator, and optionally an intracellular signaling domain comprising a primary activation domain and a costimulatory signaling domain. In some embodiments, upon binding of the ligand to the extracellular ligand-binding domain, the receptor undergoes intramembrane proteolysis, releasing the intracellular domain and thereby liberating the transcriptional regulator to translocate to the nucleus and bind a response element within or proximal to a promoter that is operably linked to the nucleic acid encoding the recombinant polypeptide. In some embodiments, such binding induces or enhances transcription of the recombinant polypeptide in response to target recognition, thereby providing spatially and temporally restricted expression of the recombinant polypeptide in antigen-engaged cells.

[0189] In some embodiments, the engineered antigen receptor is a hybrid-CAR receptor (Hybrid-R) comprising an intracellular domain (ICD) which comprises a transcriptional regulator, wherein binding of Hybrid-R to the target ligand results in cleavage of a ligandinducible proteolytic cleavage site and releases of the ICD. In some embodiments, the release of the ICD results in binding of the transcriptional regulator of the released ICD to a cognate target sequence that is operably incorporated upstream of the first recombinant nucleic acid molecule, wherein the binding of the transcriptional regulator to the cognate target sequence modulates transcription of the recombinant polypeptide.

[0190] In some embodiments, the effects of inducible or regulated expression of a provided recombinant polypeptide may be confirmed in vitro and in vivo. In some embodiments, inducible systems are tested in co-culture assays in which the presence or absence of the inducing ligand or receptor engagement modulates expression of the fusion protein, and functional readouts (e.g., IL-2 production, cytotoxicity, proliferation) are compared under induced versus uninduced conditions. In some embodiments, in vivo studies in tumor-bearing mice demonstrate that inducible expression of the recombinant fusion polypeptide preserves or enhances tumor control while reducing systemic toxicity, as evidenced by improved tolerability (e.g., stable body weight, lower clinical scores), preserved survival, and absence of lethal toxicity in the setting of effective tumor clearance.

[0191] In some embodiments, the target ligand (e.g., antigen) of any of the described recombinant antigen receptors (e.g., CAR or TCR architecture) is expressed on a cell associated with or involved in a health condition, such as a disease. In some embodiments,Attorney Docket No.: 048536-807001 WO the health condition is a cancer and the cell is a tumor cell. In some embodiments, the health condition is an autoimmune disease and the cell is an autoreactive cell, such as an autoreactive B cell or an autoreactive T cell.

[0192] In some embodiments, the target ligand (e.g., antigen) of any of the described recombinant antigen receptors (e.g., CAR or TCR architecture) is expressed on a tumor cell. Non-limiting examples of target ligands suitable for the compositions and methods of the disclosure include CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3e, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), and CD340 (HER2). Additional target ligands suitable for the compositions and methods of the disclosure include, but are not limited to, CDH17, CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, ROR1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPCAM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFRp, SSEA-4, and folate receptor alpha. Other target ligands suitable for the compositions and methods of the disclosure include, but are not limited to, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, ROPN1, GPRC5D, CXORF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, and telomerase. Other suitable target ligands for the compositions and methods of the disclosure include, but are not limited to, PCTA-Attorney Docket No.: 048536-807001 WO1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML- IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, and the extracellular portion of the APRIL protein.

[0193] In some embodiments, the target ligand is B7H3, BCMA, CD19, CD20, CD22, CD70, CD79a, CD79b, CDH17, CLDN6, CLDN18.2, DLL3, GD2, GD3, GPC3, GPRC5D, GPA33, GUCY2C (GCC), KLK2, LY6G6D, p53R175H, PRAME, ROPN1, STEAP1, or STEAP2.

[0194] In some embodiments, the cell further comprises or expresses a safety switch. In some embodiments, the safety switch expressed in recombinant cells comprising a provided recombinant polypeptide is configured to permit selective depletion or inactivation of the cell in a subject. In some embodiments, the safety switch provides a pharmacologically controllable mechanism to terminate or attenuate activity of the engineered cell population in the event of excessive toxicity, off-target effects, or other adverse events, while leaving nonengineered cells substantially unaffected. In some embodiments, the safety switch is encoded on the same vector as the recombinant polypeptide or on a separate vector, and may be expressed constitutively or under control of a promoter selected as described herein.

[0195] In some embodiments, the safety switch comprises a safety switch enzyme that is activatable by an exogenously administered agent to promote apoptosis or cytotoxicity of the recombinant cell. In some embodiments, the safety switch enzyme is a prodrug-activating kinase, such as a herpesvirus thymidine kinase (HSV-tk), that phosphorylates a nontoxic prodrug (e.g., ganciclovir or a pharmaceutically acceptable derivative thereof) to generate toxic nucleotide analogs, which incorporate into DNA and cause chain termination and cell death. In some embodiments, administration of the prodrug to a subject selectively kills HSV-tk-expressing engineered cells while sparing cells lacking the safety switch. In some embodiments, the safety switch enzyme is a prodrug-activating deaminase, such as an Escherichia coli cytosine deaminase (EC-CD), that converts a relatively non-toxic prodrug (e.g., 5-fluorocytosine or a pharmaceutically acceptable derivative thereof) into a cytotoxic antimetabolite (e.g., 5-fluorouracil), thereby promoting death of the recombinant cell. In some embodiments, the safety switch enzyme is a dimerization-inducible caspase, such asAttorney Docket No.: 048536-807001 WO inducible caspase-9 (iCasp9), comprising a dimerization domain fused to a caspase-9 protein, where administration of a small-molecule dimerizer that binds the dimerization domain induces dimerization and activation of caspase-9 and initiates rapid apoptosis of the engineered cells. In some embodiments, the exogenously administered agent is a prodrug in the case of prodrug-activating enzymes, or a dimerization agent in the case of inducible caspase switches.

[0196] In some cases, the safety switch is or comprises a protease. In some cases, the protease is a kinase, such as a herpesvirus thymidine kinase (HSV-tk) gene. In some cases, the safety switch is a deaminase, such as an Escherichia coli cytosine deaminase (ECCD). In some cases, the safety switch is a nuclease, such as an inducible Caspase9 (iCasp9) protein.

[0197] In some embodiments, the safety switch comprises a depletion tag expressed on the surface of the recombinant cell, wherein the depletion tag comprises a protein that is specifically bound by an exogenously administered antibody, thereby enabling antibody- mediated depletion of the engineered cell population. In some embodiments, the depletion tag is a full-length or truncated form of a native cell-surface protein selected from CCR4, CD 16, CD 19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, or RQR8. In some embodiments, the full-length or truncated form comprises an extracellular domain that includes an epitope recognized by a suitable antibody, such as a therapeutic or clinically approved antibody, and optionally lacks all or part of an intracellular or signaling domain to minimize unintended signaling through the depletion tag. In some embodiments, administration of an antibody specific for the depletion tag (e.g., an anti-CD19, anti-CD20, anti-EGFR, anti-HER2, or anti-CCR4 antibody) results in complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and / or other immune mechanisms that selectively remove the engineered cells expressing the depletion tag.

[0198] In some embodiments, the safety switch comprises a tolerogenic protein expressed on the surface of the recombinant cell and configured to deliver an inhibitory signal to an immune effector cell, thereby protecting the engineered cell from immune-mediated clearance under baseline conditions. In some embodiments, the tolerogenic protein is CD47, which interacts with signal regulatory protein a (SIRPa) on macrophages and other phagocytes to provide a “don’t-eat-me” signal. In some embodiments, an exogenously administered agent, such as an anti-CD47 antibody or other CD47-binding molecule, binds the tolerogenic protein and reduces or blocks the inhibitory signal. In some embodiments,Attorney Docket No.: 048536-807001 WO blockade of CD47 on the engineered cells permits or enhances immune-mediated clearance of the cells (e.g., by macrophages, dendritic cells, or NK cells), thereby functioning as a safety switch that can be actuated by administration of the anti-CD47 agent.

[0199] In some embodiments, a recombinant cell comprises one or more safety switches in combination with the recombinant polypeptide and a recombinant antigen receptor (e.g., a CAR or TCR-enabled architecture). In some embodiments, enzyme-based safety switches (e.g., HSV-tk, EC-CD, or iCasp9), depletion tag-based switches (e.g., CD19, CD20, EGFR, or RQR8), and tolerogenic protein-based switches (e.g., CD47) are used singly or in combination to provide layered control over the activity and persistence of the engineered cells in vivo. In some embodiments, such safety switches enable administration of potent recombinant polypeptide fusion constructs at doses sufficient to achieve robust antitumor responses while preserving the ability to selectively deplete or inactivate the engineered cells if unacceptable toxicity or off-target effects arise.

[0200] Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. For example, DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection. In some embodiments, the nucleic acid molecule is introduced into a host cell by a transduction procedure, electroporation procedure, or a biolistic procedure.

[0201] In some embodiments, one or more recombinant nucleic acids of the disclosure and / or the additional recombinant nucleic acid molecule may be inserted in a specific site(s) in the genome of the recombinant cell. Non-limiting examples of suitable sites in the cell genome include a T-cell receptor (TCR) locus, a CD3 locus, a B2 microglobulin (B2M) locus, a class II transactivator (CIITA) locus, and a safe harbor locus. In some embodiments, the TCR locus is a TRAC locus, a TRBC1 locus, a TRBC2 locus, or a TRAJ locus. In some embodiments, one or more recombinant nucleic acids of the disclosure may be inserted in a safe harbor locus. Examples of safe harbor loci suitable for the compositions and methods of the disclosure include, but are not limited to, AAVS1 locus, ABO locus, CCR5 locus,Attorney Docket No.: 048536-807001 WOCLYBL locus, CXCR4 locus, F3 locus, FUT1 locus, HMGB1 locus, KDM5D locus, LRP1 locus, MICA locus, MICB locus, RHD locus, ROSA26 locus, and SHS231 locus.

[0202] In some embodiments, the recombinant nucleic acid encoding the recombinant polypeptide is inserted into an endogenous genomic locus that encodes a fusion partner of the recombinant polypeptide. In some embodiments, the insertion is configured such that the coding sequence of the recombinant polypeptide, or a portion thereof (for example, an MLM, an SEM, or an MLM-SEM fragment), is placed in-frame with an endogenous coding sequence at the locus. In some embodiments, transcription from the endogenous promoter at that locus produces a single transcript encoding an endogenous protein sequence fused to all or part of the recombinant polypeptide, such that expression of the recombinant polypeptide or portion thereof occurs as a fusion with the endogenous protein. In some embodiments, the endogenous promoter, untranslated regions, introns, and / or regulatory elements at the locus are preserved, thereby maintaining physiological control of expression while adding the endogenous segment as a C-terminal or N-terminal fusion to the endogenous protein.

[0203] In some embodiments, the endogenous genomic locus into which the recombinant nucleic acid is inserted is selected from loci encoding signaling proteins that naturally participate in the kinase cascades modulated by the MLM-SEM fusion architecture. In some embodiments, the endogenous genomic locus is a Tec family kinase locus, such as an ITK locus; a transmembrane adaptor protein (TRAP) locus, such as a LAT locus; a locus of a kinase in a Syk / ZAP70 non-receptor tyrosine kinase cascade, such as a SYK locus or a ZAP70 locus; a locus of a kinase in an AKT serine / threonine kinase cascade, such as an AKT1 locus; a locus of a kinase or regulatory subunit of a class IA phosphoinositide 3-kinase (PI3K) lipid-kinase cascade, such as a PDK1 locus or a PIK3R1 locus; or a locus of an adaptor protein of a Ras-Raf-MEK-ERK cascade, such as a GRB2 locus. In some embodiments, insertion at these loci yields fusion proteins that couple endogenous signaling scaffolds or enzymes to MLM and / or SEM domains described herein, thereby enhancing or rewiring receptor-proximal signaling in a pathway-specific manner.

[0204] In some embodiments, the endogenous genomic locus is selected from loci encoding additional kinases, adaptors, or regulatory proteins that function in T cell receptor and related signaling pathways. In some embodiments, the endogenous genomic locus is selected from the group consisting of an LCK locus, a FYN locus, a GADS locus, a S0S1 locus, a SLP76 locus, an ADAP locus, a PLCyl locus, a VAV1 locus, an NCK1 locus, a BTK locus, a MAP4K3 locus, a PKC theta locus, a SHP1 locus, a c-Cbl locus, and an HPK1 locus. In someAttorney Docket No.: 048536-807001 WO embodiments, insertion of the recombinant nucleic acid at any of these loci produces an endogenous fusion partner-recombinant polypeptide fusion that localizes the MLM and / or SEM to defined receptor-proximal complexes and signaling hubs, while allowing expression to be governed by the endogenous transcriptional program of the selected locus.

[0205] In some embodiments, the expression of the recombinant polypeptide in the recombinant cells (e.g., recombinant T cells) results in changes in one or more functionalities and / or characteristics of the T cells comprising the recombinant polypeptide. Non-limiting examples of T cell functionalities include (i) increasing proliferative capacity, (ii) enhancing antigen sensitivity, (iii) enhancing anti-tumor effect, (iv) altering T cell signaling through NF AT, NF-KB and / or AP-1 pathways, (v) altering cytokine or chemokine production, (vi) altering JAK / STAT signaling in T cells, (vii) altering co-stimulatory molecule signaling in T cells, (viii) altering RAS / MEK / ERK signaling in T cells, (ix) altering phospholipase C gamma signaling, (x) altering a transcription factor activity in T cells, and / or (xi) altering or enhancing in vivo persistence in tumors of the T cells including the recombinant polypeptide. In some embodiments, the expression of the recombinant polypeptide in a T cell promotes its proliferation in vivo. In some embodiments, the recombinant T cell comprising the recombinant polypeptide has one or more of the following characteristics: increased proliferative capacity, enhanced antigen sensitivity, enhanced anti-tumor effect, enhanced replicative lifespan, reduced exhaustion, decreased replicative senescence, reduced dysfunction, enhanced persistence, and increase intratumoral presence in vivo as compared to a reference T cell that does not include the recombinant polypeptide.

[0206] In some embodiments, provided recombinant fusion polypeptides confer enhanced functional properties on T cells in which they are expressed, particularly when co-expressed with a recombinant antigen receptor such as a chimeric antigen receptor (CAR) or a TCR- enabled architecture (e.g., TCR, TruC, HIT, STAR, or TCR-like receptors as described herein). In some embodiments, T cells expressing the recombinant fusion polypeptides exhibit increased antigen sensitivity, improved tumor control in vivo, enhanced proliferative and persistence properties, and augmented cytokine secretion relative to control T cells lacking the recombinant fusion polypeptide.

[0207] In some embodiments, assays used to assess the functional impact of the recombinant fusion polypeptides on T cells include one or more of: (i) short-term co-culture assays with antigen-positive targets to measure cytokine production (e.g., IL-2, IFN-y, TNF- a) and activation markers (e.g., CD69, CD25); (ii) cytotoxicity assays (e.g., luciferase-basedAttorney Docket No.: 048536-807001 WO killing, impedance-based assays, flow-based killing assays, or chromium release assays); (iii) proliferation and persistence assays (e.g., cell counting, dye dilution, longitudinal flow cytometric tracking, or serial restimulation experiments); (iv) phenotypic analysis of differentiation and exhaustion markers (e.g., memory subset markers and inhibitory receptors); and (v) in vivo tumor burden and survival studies in mouse models using bioluminescence imaging and Kaplan-Meier survival analysis. In some embodiments, T cells expressing a provided recombinant polypeptide exhibit improvements in one or more of these parameters relative to appropriate control T cells, thereby demonstrating a proliferative and functional advantage that is associated with enhanced tumor control and survival. In particular embodiments, the improvements are observed without unacceptable toxicity.

[0208] In some embodiments, enhanced antigen sensitivity is observed in short-term coculture assays in which engineered T cells are cultured with target cells expressing a cognate antigen for the CAR or TCR-enabled receptor. In some embodiments, T cells comprising both a recombinant antigen receptor and a provided recombinant polypeptide respond to lower densities of antigen on target cells, or to lower ratios of target cells to effector T cells, compared to otherwise identical T cells lacking the recombinant polypeptide. In some embodiments, antigen sensitivity is quantified by measuring IL-2 secretion in supernatants after a defined co-culture interval (for example, about 16-48 hours) using ELISA, bead-based multiplex assays, or other immunoassays. In some embodiments, additional cytokines such as IFN-y, TNF-a, IL-6, IL- 10, or GM-CSF are measured in parallel. In some embodiments, T cells expressing the recombinant polypeptide show increased IL-2 and / or other cytokines at a given antigen dose, or show a left-shifted antigen dose-response curve relative to control T cells. In some embodiments, antigen sensitivity is further assessed by measuring early activation markers such as CD69 and CD25, or by using reporter constructs (e.g., NFAT-, NF-KB-, or AP-l-driven fluorescent or luciferase reporters).

[0209] In some embodiments, the recombinant fusion polypeptides confer a proliferative and / or persistence advantage on engineered T cells. In some embodiments, T cells expressing a recombinant polypeptide are co-cultured with antigen-positive target cells for multiple days, and T cell expansion is assessed by direct cell counting, dye dilution (e.g., CFSE, CellTrace), or flow cytometric tracking of genetically marked T cells. In some embodiments, T cells expressing the recombinant polypeptide exhibit increased proliferation, greater accumulation, and / or improved survival and persistence relative to control T cells. In some embodiments, persistence is assessed in longer-term culture or in serial restimulation assays in which T cellsAttorney Docket No.: 048536-807001 WO are repeatedly exposed to fresh antigen-positive targets. In some embodiments, T cells comprising the recombinant polypeptide maintain cytokine production and cytolytic activity over multiple rounds of stimulation more effectively than control T cells. In some embodiments, phenotypic analysis (e.g., by flow cytometry) may be used to assess frequencies of memory-like or stem-like subsets (e.g., TCM, TSCM, or stem-like memory T cells) and / or reduced markers of dysfunction or exhaustion under defined stimulation conditions.

[0210] In some embodiments, the activity of the recombinant fusion polypeptides is evaluated in cytotoxicity assays. In some embodiments, T cells expressing an provided recombinant polypeptide and a recombinant antigen receptor are co-cultured with luciferase- labeled or otherwise traceable tumor cells at a range of effector-to-target (E:T) ratios, and tumor cell killing is measured by loss of luciferase signal, release of cytosolic enzymes (e.g., LDH), impedance-based readouts, or flow cytometry. In some embodiments, T cells expressing a provided recombinant polypeptide display enhanced cytotoxicity at a given E:T ratio and / or achieve comparable levels of target cell killing at lower E:T ratios relative to control T cells. In some embodiments, cytotoxicity assays are performed under stress conditions (e.g., low antigen density, presence of immunosuppressive cytokines, or repeated stimulation), and enhanced function of the T cells expressing the recombinant polypeptide is observed under such conditions.

[0211] In some embodiments, the functional impact of the recombinant fusion polypeptides is assessed in vivo in tumor models. In some embodiments, tumor cells expressing a luciferase reporter are engrafted into immunodeficient mice, such as NOD / SCID / yc / (NSG) mice, and engineered T cells are adoptively transferred after tumor establishment. In some embodiments, tumor burden is monitored over time by bioluminescence imaging, for example one or two times per week. In some embodiments, at each imaging session, mice are injected intraperitoneally with a luciferin substrate, anesthetized using isoflurane, and imaged on both front and back using an in vivo imaging system; the average radiance from the front and back images is quantified to provide a measure of whole-body tumor burden. In some embodiments, mice treated with T cells expressing the recombinant polypeptide show reduced tumor biolumine scent signal, delayed tumor progression, or complete tumor regression relative to mice treated with control T cells. In some embodiments, these improvements translate into prolonged survival as assessed by Kaplan-Meier analysis, and / or reduced clinical signs of disease.Attorney Docket No.: 048536-807001 WO

[0212] In a related aspect, some embodiments of the disclosure relate to a cell culture including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any one of suitable culture media for the cell cultures described herein. In some embodiments, the recombinant cell expresses a recombinant polypeptide as described herein. In some embodiments, the recombinant cell further expresses an engineered immune receptor that binds to a target ligand. Accordingly, cell cultures including at least one recombinant cell, and optionally an engineered immune receptor, as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.PHARMACEUTICAL COMPOSITIONS

[0213] The recombinant polypeptides, nucleic acids, vectors, recombinant cells, and / or cell cultures of the disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include the recombinant polypeptides, nucleic acids, vectors, recombinant cells, and / or cell cultures as described herein and a pharmaceutically acceptable carrier. Accordingly, in one aspect, some embodiments of the disclosure relate to pharmaceutical compositions for treating, preventing, ameliorating, reducing or delaying the onset of health condition, for example a proliferative disease (e.g., cancer). In some embodiments, the pharmaceutical composition includes at least one recombinant polypeptide, nucleic acid, vector, recombinant cell, and / or cell culture as disclosed herein, in an admixture with a pharmaceutically acceptable carrier, some embodiments of the disclosure relate to a pharmaceutical composition including a pharmaceutically acceptable carrier and one or more of the following: (a) a recombinant polypeptide of the disclosure; (b) a recombinant nucleic acid of the disclosure; (c) a vector of the disclosure; and (d) a recombinant cell of the disclosure. In some embodiments, the composition includes a recombinant nucleic acid of the disclosure and a pharmaceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the composition includes a recombinant cell, e.g., a recombinant immune cell expressing a CAR, of the disclosure and a pharmaceutically acceptable carrier.

[0214] In certain embodiments, the pharmaceutical compositions in accordance with some embodiments disclosed herein include cell cultures that can be washed, treated, combined, supplemented, or otherwise altered prior to administration to an individual in need thereof. Furthermore, administration can be at varied doses, time intervals or in multipleAttorney Docket No.: 048536-807001 WO administrations.

[0215] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to an individual. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin.

[0216] Formulations of the cells suitable for administration to a subject or individual (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 subject or individual according to a selected route of administration. Accordingly, in some embodiments, the pharmaceutical composition of the disclosure is sterilely formulated for administration into an individual. In some embodiments, the individual is a human. One of ordinary skilled in the art will appreciate that the formulation should suit the mode of administration.

[0217] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to an individual. For example, the pharmaceutical composition may be formulated to be suitable for one or more of the following administration routes: intranasal administration, transdermal administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration. In some particular embodiments of the disclosure, the composition is formulated intramuscular administration.

[0218] Pharmaceutical compositions that include the recombinant polypeptides, nucleic acids, vectors, recombinant cells, and cell cultures of the present disclosure may be preparedAttorney Docket No.: 048536-807001 WO by mixing the recombinant polypeptides, nucleic acids, vectors, recombinant cells, and cell cultures having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0219] An aqueous formulation of the recombinant polypeptides, nucleic acids, vectors, recombinant cells, and cell cultures may be prepared in a pH-buffered solution, e.g., at pH ranging from about 7.0 to 8.0, 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.

[0220] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Exemplary 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 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.

[0221] In some embodiments, a surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduceAttorney Docket No.: 048536-807001 WO adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactant may range from about 0.001% to about 1% w / v.

[0222] In some instances, the pharmaceutical composition includes the recombinant polypeptides, nucleic acids, vectors, recombinant cells, and cell cultures 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, m-cresol, p-chloro-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).METHODS OF THE DISCLOSUREMethods for preparing recombinant T cells for use in cell therapies

[0223] As discussed above, some embodiments of the disclosure relate to methods for preparing / making a recombinant cell, the method including expressing in the T cell one or more of the following: (a) a recombinant polypeptide as described herein; (b) a recombinant nucleic acid as described herein; and (c) a vector as described herein. In some embodiments, the methods of preparing T cells as disclosed herein further include genetically modifying the T cell for expression of the recombinant polypeptide. In some embodiments, the methods further include introducing to the T cell a recombinant nucleic acid as described herein, or a vector as described herein. In some embodiments, the method includes one or more of the following: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, an RNA-guided nuclease including a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex including a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-Attorney Docket No.: 048536-807001 WO deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR- associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In some embodiments, the T cell is made using homology-directed repair (HDR) -mediated insertion. In some embodiments, the T cell is made in vivo, ex vivo, or in vitro. In some embodiments, the methods further include expressing in the T cell an engineered immune receptor that binds to a target ligand.Methods for preparing recombinant T cells for use in cell therapies and methods of administration and use

[0224] Administration of any one of the therapeutic compositions described herein, e.g., recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used in the prevention or treatment of relevant conditions, such as proliferative disorders e.g., cancers), infectious diseases, autoimmune diseases, and inflammatory diseases. In some embodiments, the recombinant polypeptides, nucleic acids, vectors, recombinant cells, and / or pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of preventing and / or treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more health conditions, such as such as proliferative disorders (e.g., cancers), infectious diseases, autoimmune diseases, and inflammatory diseases. In some embodiments, the individual is a patient under the care of a physician. In some embodiments, the proliferative disorder is a cancer.

[0225] In various aspects, the disclosure provides both ex vivo and in vivo therapeutic modalities. In some embodiments, a therapeutic cell composition comprising a recombinant cell of the disclosure (e.g., a cell expressing a recombinant polypeptide and optionally an antigen receptor, such as a CAR or other chimeric receptor) is administered to a subject. In other embodiments, administration is by in vivo delivery of a therapeutic composition comprising one or more nucleic acids, vectors, or other delivery vehicles configured to introduce a nucleic acid encoding a recombinant polypeptide and, in some cases, an antigen receptor into target cells (e.g., T cells) within the subject. In some embodiments, the therapeutic composition comprises one or more recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions comprising any of the foregoing and one or more pharmaceutically acceptable excipients. In some embodiments, the subject has a cancer or an autoimmune disease.

[0226] In one aspect, the disclosure provides methods of treating a subject, comprisingAttorney Docket No.: 048536-807001 WO administering to the subject a therapeutic composition described herein. In some embodiments, the therapeutic composition comprises a recombinant cell of the disclosure or a cell prepared by a method of the disclosure. In some such embodiments, the therapeutic composition is a cell composition formulated for administration (e.g., intravenous, intratumoral, intralymphatic, subcutaneous, or other suitable route). In some embodiments, the therapeutic composition comprises a vector or delivery vehicle configured for in vivo delivery of a nucleic acid encoding a recombinant polypeptide and optionally an antigen receptor to cells of the subject. In some such embodiments, administration is by in vivo gene delivery using a viral or non-viral vector, a lipid-based nanoparticle, or another delivery system configured to deliver the nucleic acid to target cells (e.g., T cells or other immune cells). In some embodiments, the disclosure provides uses of such therapeutic compositions for the manufacture of a medicament for the treatment of a disease. In some embodiments, the disease is a cancer or an autoimmune disease.

[0227] In some embodiments, the autoimmune disease is lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, Crohn’s disease, ulcerative colitis, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, or celiac disease.

[0228] In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is myeloid neoplasm, myelodysplastic syndromes (MDS), myeloproliferative / myelodysplastic syndromes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), blast crisis chronic myelogenous leukemia (bcCML), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), T-cell lymphoma, or B-cell lymphoma.

[0229] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, nonsmall cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head & neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

[0230] In some embodiments, therapeutic agents described herein, e.g., recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceuticalAttorney Docket No.: 048536-807001 WO compositions, can be used in methods of preventing and / or treating individual who have, who are suspected of having, or who may be at high risk for developing a cancer expressing one or more target ligands. Non-limiting examples of target ligands suitable for the compositions and methods disclosed herein include CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, Claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD72, CD97, CDH17, CEA, Claudin 6(CLDN6), CLECL1, CS-1, and DLL-3. Additional target ligands suitable for the compositions and methods disclosed herein include, but are not limited to, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GCC, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-P, PRAME, PRSS21, PSCA, PSMA, R0R1, SIRPa, SSEA-4, STEAP2, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Ax.

[0231] In some embodiments, therapeutic agents described herein, e.g., recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used in methods of preventing and / or treating individual who have, who are suspected of having, or who may be at high risk for developing a cancer which may be small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, or esophageal cancer. Additional cancers suitable for the compositions and methods disclosed herein include, but are not limited to, liver cancer, kidney cancer, head and neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

[0232] Administration of therapeutic agents described herein, e.g., recombinant polypeptides, nucleic acids, vectors, recombinant cells, and / or pharmaceutical compositions, can be used in the stimulation of an immune response. In some embodiments, recombinant polypeptides, nucleic acids, vectors, recombinant cells, and / or pharmaceutical compositions as described herein are administered to an individual after induction of remission of cancer with chemotherapy, or after autologous or allogeneic hematopoietic stem cell transplantation.

[0233] In some embodiments, therapeutic agents described herein include a therapeutically effective amount of recombinant cells as disclosed herein. In some embodiments, the recombinant cells are allogeneic relative to the subject. In some embodiments, theAttorney Docket No.: 048536-807001 WO recombinant cells are autologous relative to the subject.

[0234] An effective amount of the therapeutic agents described herein, e.g., recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions, is determined based on the intended goal, for example tumor regression. For example, where existing cancer is being treated, the amount of a therapeutic agent disclosed herein to be administered may be greater than where administration of the therapeutic agent is for prevention of cancer. One of ordinary skill in the art would be able to determine the amount of a therapeutic agent to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the individual to be treated, the state of the individual, and the protection desired. Precise amounts of the therapeutic agent also depend on the judgment of the practitioner and are peculiar to each individual. Frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner.

[0235] Longer intervals between administration and lower amounts of therapeutic agents may be employed where the goal is prevention. For instance, amounts of therapeutic agents administered per dose may be 50% of the dose administered in treatment of active disease, and administration may be at weekly intervals. One of ordinary skill in the art, in light of this disclosure, would be able to determine an effective amount of therapeutic agents and frequency of administration. This determination would, in part, be dependent on the particular clinical circumstances that are present e.g., type of cancer, severity of cancer).

[0236] In certain embodiments, it may be desirable to provide a continuous supply of the therapeutic agents to the subject to be treated, e.g., a patient. In some embodiments, continuous perfusion of the region of interest (such as the tumor) may be suitable. The time period for perfusion would be selected by the clinician for the particular subject and situation, but times could range from about 1-2 hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2 weeks or longer. Generally, the dose of the therapeutic agent via continuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the doses are administered.

[0237] One of ordinary skill in the art would be familiar with techniques for administering therapeutic agents to an individual. Furthermore, one of ordinary skill in the art would be familiar with techniques and pharmaceutical reagents necessary for preparation of these therapeutic agents prior to administration to an individual.Attorney Docket No.: 048536-807001 WO

[0238] In certain embodiments of the present disclosure, the therapeutic agents will be an aqueous composition that includes the recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein. Aqueous compositions of the present disclosure contain an effective amount of a therapeutic agent disclosed herein in a pharmaceutically acceptable carrier or aqueous medium. Thus, the “pharmaceutical preparation” or “pharmaceutical composition” of the disclosure can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the recombinant cells disclosed herein, its use in the manufacture of the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Center for Biologies.

[0239] One of ordinary skill in the art would appreciate that biological materials should be extensively dialyzed to remove undesired small molecular weight molecules and / or lyophilized for more ready formulation into a desired vehicle, where appropriate. The therapeutic agents described herein, e.g., recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions, will then generally be formulated for administration by any known route, such as parenteral administration. Determination of the amount of therapeutic agents to be administered will be made by one of skill in the art, and will in part be dependent on the extent and severity of cancer, and whether the recombinant cells are being administered for treatment of existing cancer or prevention of cancer. The preparation of the therapeutic agents containing the recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions of the disclosure will be known to those of skill in the art in light of the present disclosure.

[0240] Upon formulation, therapeutic agents will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The therapeutic agents can be administered in a variety of dosage forms, such as the type of injectable solutions described above. For parenteral administration, the therapeutic agents disclosed herein should be suitably buffered. As discussed in greater detail below, the therapeutic agents as described herein may be administered with other therapeutic agents that are part of the therapeutic regiment of the individual, such as other immunotherapy orAttorney Docket No.: 048536-807001 WO chemotherapy.

[0241] The recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to cure established tumors, inhibit tumor growth or metastasis of the cancer in the treated subject relative to the tumor growth or metastasis in subjects who have not been administered one of the therapeutic compositions disclosed herein. In some embodiments, the recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to stimulate proliferation and / or killing capacity of CAR T-cells in the treated subject relative to subjects who have not been administered one of the therapeutic compositions disclosed herein.Administration of recombinant cells to a subject

[0242] In some embodiments, the methods of the disclosure involve administering an effective amount of pharmaceutical compositions or an effective number of the recombinant cells, e.g., recombinant immune cells comprising a CAR, provided herein to a subject in need thereof for the prevention and / or treatment of health conditions. Methods for administering pharmaceutical compositions comprising recombinant cells, e.g., CAR-expressing immune cells, for the treatment of health conditions, e.g., cancers, are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described in US 2003 / 0170238; US 4690915; S.A. Rosenberg, Nat Rev Clin Oncol (2011) 8(10):577-85. See also M. Themeli etal., Nat Biotechnol (2013)31 ( 10):928-33; and T. Tsukahara et al., Biochem Biophys Res Commun (2013) 438(l):84-89.

[0243] In some embodiments, this administering step can be accomplished using any method of implantation delivery in the art. For example, the recombinant cells can be infused directly in the subject’s bloodstream or otherwise administered to the subject.

[0244] In some embodiments, the methods disclosed herein include administering, which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” recombinant cells into an individual, by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is / are produced. The recombinant cells or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of theAttorney Docket No.: 048536-807001 WO individual, i.e., long-term engraftment.

[0245] When provided prophylactically, the recombinant cells described herein can be administered to a subject in advance of any symptom of a disease or condition to be treated. Accordingly, in some embodiments the prophylactic administration of a recombinant cell population prevents the occurrence of symptoms of the disease or condition.

[0246] When provided therapeutically in some embodiments, recombinant cells are provided at (or after) the onset of a symptom or indication of a disease or condition, e.g., upon the onset of disease or condition.

[0247] For use in the various embodiments described herein, an effective amount of recombinant cells as disclosed herein, can be at least 102cells, at least 5 x 102cells, at least 103cells, at least 5 x 103cells, at least 104cells, at least 5 x 104cells, at least 105cells, at least 2 x 105cells, at least 3 x 105cells, at least 4 x 105cells, at least 5 x 105cells, at least 6 x 105cells, at least 7 x 105cells, at least 8 x 105cells, at least 9 x 105cells, at least 1 x 106cells, at least 2 x 106cells, at least 3 x 106cells, at least 4 x 106cells, at least 5 x 106cells, at least 6 x 106cells, at least 7 x 106cells, at least 8 x 106cells, at least 9 x 106cells, or multiples thereof. The recombinant cells can be derived from one or more donors (e.g., they are allogeneic relative to the subject) or can be obtained from an autologous source. In some embodiments, the recombinant cells are expanded in culture prior to administration to a subject in need thereof.

[0248] In some embodiments, the delivery of a recombinant cell composition e.g., a composition including a plurality of recombinant cells according to any of the cells described herein) into a subject by a method or route results in at least partial localization of the cell composition at a desired site. A composition including recombinant cells can be administered by any appropriate route that results in effective treatment in the subject, e.g., administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, e.g., at least 1 x 104cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, and instillation. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracap sular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, delivery by injection or infusion is a standard mode of administration.Attorney Docket No.: 048536-807001 WO

[0249] In some embodiments, the recombinant cells are administered systemically, e.g., via infusion or injection.

[0250] The efficacy of a treatment including any of the compositions provided herein for the treatment of a disease or condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by decreased hospitalization or need for medical interventions e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in a subject or an animal (some nonlimiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0251] Measurement of the degree of efficacy is based on parameters selected with regard to the health condition, e.g., disease, being treated and the symptoms experienced. In general, a parameter is selected that is known or accepted as correlating with the degree or severity of the disease, such as a parameter accepted or used in the medical community. For example, in the treatment of a solid cancer, suitable parameters can include reduction in the number and / or size of metastases, number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, the reduction in diagnostic biomarkers (for example without limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like), and combinations thereof. It will be understood that the effective dose and the degree of efficacy will generally be determined with relation to a single subject and / or a group or population of subjects. Therapeutic methods of the disclosure reduce symptoms and / or disease severity and / or disease biomarkers by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% as compared to a reference subject that has not been administered with the recombinant immune cells or pharmaceutical compositions of the disclosure.

[0252] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular beneficial effect when administered to a subject, such as one who has, is suspected of having, or is at risk for aAttorney Docket No.: 048536-807001 WO disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.Additional therapies

[0253] As discussed supra, any one of the recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be administered to a subject in need thereof as a sole therapy (e.g., monotherapy). In addition or alternatively, in some embodiments of the disclosure, the recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be administered to a subject in combination with one or more additional therapeutic agents, e.g., at least one, two, three, four, or five additional therapies. Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. Other suitable therapies include chemotherapeutic s, anti-cancer agents, and anti-cancer therapies.

[0254] Administration “in combination with” one or more additional therapeutic includes simultaneous (e.g., concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.

[0255] Accordingly, in some embodiments, the methods of the disclosure include administration of a composition disclosed herein to a subject individually as a sole therapy (e.g., monotherapy). In some embodiments, a composition of the disclosure is administered to a subject as a first therapy in combination with a second therapy, such as an anti-cancer agent, a chemotherapeutic, or an anti-cancer therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapyAttorney Docket No.: 048536-807001 WO and the second therapy are administered in rotation. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.KITS

[0256] Also provided herein are various kits for the practice of a method described herein. In particular, some embodiments of the disclosure provide kits for the prevention of a health condition in a subject in need thereof. Some other embodiments relate to kits for methods of treating a health condition in a subject in need thereof. For example, provided herein, in some embodiments, are kits that include one or more of the recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures and / or pharmaceutical compositions as provided and described herein, as well as written instructions for making and using the same.

[0257] In some embodiments, the kits of the disclosure further include one or more means useful for the administration of any one of the provided recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures or pharmaceutical compositions to an individual. For example, in some embodiments, the kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided recombinant polypeptides, nucleic acids, vectors, recombinant cells, cell cultures, or pharmaceutical compositions to an individual. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for preventing and / or treating a health condition in a subject in need thereof.

[0258] Any of the above-described kits can further include one or more additional reagents, where such additional reagents can be one or more of the following: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents suitable for in vitro or ex vivo production of the CARs.

[0259] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container.

[0260] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods disclosed herein. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit orAttorney Docket No.: 048536-807001 WO components thereof (e.g., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.EXEMPLARY EMBODIMENTS

[0261] Among the provided embodiments are:

[0262] 1. A recombinant cell comprising a recombinant polypeptide, wherein the recombinant polypeptide comprises, in N- to C-terminal direction: (a) a first polypeptide sequence that is a membrane-localization module (MLM) comprising a domain that mediates localization of the recombinant polypeptide to at least one of (i) a plasma membrane of the cell, or (ii) a receptor-proximal region at the plasma membrane that forms in response to engagement of the cell with its cognate ligand; and (b) a second polypeptide sequence that is a signaling-effector module (SEM) that, when localized to the plasma membrane by the MLM, potentiates a kinase signaling cascade in the cell.

[0263] 2. The recombinant cell of embodiment 1, wherein the MLM mediates inducible recruitment of the recombinant polypeptide to a receptor-proximal signaling complex that assembles at the plasma membrane in response to engagement of the receptor with its cognate ligand.

[0264] 3. The recombinant cell of embodiment 2, wherein the receptor-proximal signaling complex comprises a LAT-SLP-76 signalosome.

[0265] 4. The recombinant cell of embodiment 3, wherein the cell is a T cell and the receptor is a T cell receptor (TCR), and the LAT-SLP-76 signalosome is induced by engagement of the TCR with its cognate ligand.

[0266] 5. The recombinant cell of embodiment 1, wherein the MLM lacks a functional enzymatic domain.

[0267] 6. The recombinant cell of any one of embodiments 1-5, wherein the MLM is a domain capable of localizing the recombinant polypeptide to a plasma membrane of the cell.

[0268] 7. The recombinant cell of any one of embodiments 1-6, wherein the domainAttorney Docket No.: 048536-807001 WO capable of localizing the recombinant polypeptide to the plasma membrane is a lipid-binding pleckstrin homology (PH) domain.

[0269] 8. The recombinant cell of any one of embodiments 1-7, wherein the MLM comprises a pleckstrin homology (PH) domain, optionally wherein the MLM further comprises an SH3 and / or SH2 domain.

[0270] 9. The recombinant cell of any one of embodiments 1-7, wherein the MLM comprises a pleckstrin homology (PH) domain and a Tec-homology (TH) region of a Tec-family kinase, optionally wherein the MLM further comprises a SH3 and / or SH2 domain.

[0271] 10. The recombinant cell of embodiment 9, wherein the PH region is derived from ITK, BTK, TEC, or BMX, or is a variant thereof capable of localizing to a plasma membrane.

[0272] 11. The recombinant cell of embodiment 9, wherein the PH-TH region is derived from ITK, BTK, TEC, or BMX, or is a variant thereof capable of localizing to a plasma membrane.

[0273] 12. The recombinant cell of any one of embodiments 1-5, wherein the MLM comprises a transmembrane domain that spans the plasma membrane of the cell.

[0274] 13. The recombinant cell of embodiment 1 or embodiment 5 or embodiment 12, wherein the MLM comprises a single-pass transmembrane anchor from a transmembrane adaptor protein (TRAP).

[0275] 14. The recombinant cell of embodiment 13, wherein the MLM is characterized by a transmembrane domain, and (ii) a cytosolic juxtamembrane palmitoylation motif comprising 2 or more cysteines.

[0276] 15. The recombinant cell of embodiment 14, wherein the MLM further comprises a luminal N-terminal segment of 20 amino acids or less.

[0277] 16. The recombinant cell of embodiment 13 or embodiment 14, wherein the TRAP is selected from LAT, PAG / Cbp, LIME, SIT, TRIM, LAX, SCIMP, PRR7, LST1 / A, or a variant of any of the foregoing that retains localization to the membrane by palmitoylation-dependent membrane anchoring.

[0278] 17. The recombinant cell of any one of embodiments 1-16, wherein the signaling cascade potentiated by the SEM is (a) a Syk / ZAP-70 non-receptor tyrosine kinase cascade; (b) an AKT (serine / threonine kinase) cascade, (c) a class IA phosphoinositide-3-kinase (PI3K) lipid-kinase cascade; or (d) a Ras-Raf-MEK-ERK cascade.

[0279] 18. The recombinant cell of any one of embodiments 1-17, wherein the SEMAttorney Docket No.: 048536-807001 WO comprises a Syk / ZAP70-family tyrosine kinase, an AKT serine / threonine kinase, or a PDK1 serine / threonine kinase or a portion thereof comprising a kinase domain.

[0280] 19. The recombinant cell of any one of embodiments 1-17, wherein the SEM comprises a class IA PI3K regulatory subunit or a portion comprising nSH2-iSH2-cSH2 domains.

[0281] 20. The recombinant cell of any one of embodiments 1-19, wherein the SEM comprises a GRB2 or a portion comprising the N-terminal SH3 domain and optionally an SH2 domain and / or C-terminal SH3 domain.

[0282] 21. A recombinant cell comprising a recombinant polypeptide, wherein the recombinant polypeptide comprises in N- to C-terminal direction: (a) a first polypeptide sequence that is a membrane-localization module (MLM) selected from (i) a pleckstrin-homology (PH) domain, optionally further comprising an SH3 and / or SH2 domain, (ii) a pleckstrin-homology (PH) domain and Tec -homology (TH) region of a Tec -family kinase, optionally further comprising an SH3 and / or SH2 domain, or (iii) a single-pass transmembrane sequence of LAT, wherein the MLM lacks functional enzymatic domain; and (b) a second polypeptide sequence that is a signaling-effector module (SEM) selected from (i) a Syk / Zap70-family non-receptor tyrosine kinase or a portion thereof comprising the kinase domain and, optionally, interdomain B, (ii) an AKT serine / threonine kinase or a portion thereof comprising the kinase domain, (iii) a PDK1 serine / threonine kinase or a portion thereof comprising the kinase domain; (iv) a class IA PI3K regulatory subunit (PIK3R1 alpha) or a portion comprising nSH2-iSH2-cSH2 domains; and (v) a GRB2 or a portion comprising the N-SH3 domain and optionally an SH2 domain and / or C-SH3 domain.

[0283] 22. The recombinant cell of any one of embodiments 1-11 and 17-21, wherein the MLM comprises a PH region from ITK.

[0284] 23. The recombinant cell of any one of embodiments 1-11 and 17-21, wherein the MLM comprises a PH and TH region from ITK.

[0285] 24. The recombinant cell of any one of embodiments 1-11 and 17-23, wherein the MLM is selected from: ITKPH’THor ITKPH’TH’SH2-SH3.

[0286] 25. The recombinant cell of any one of embodiments 1-11 and 17-24, wherein the MLM is or comprises the sequence of amino acids set forth in SEQ ID NO: 287, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 287, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 287.

[0287] 26. The recombinant cell of any one of embodiments 1-11 and 17-24, wherein theAttorney Docket No.: 048536-807001 WOMLM is or comprises the sequence of amino acids set forth in SEQ ID NO: 288, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 288, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 288.

[0288] 27. The recombinant cell of any one of embodiments 1, 5, and 13-21, wherein the MLM comprises the transmembrane domain of LAT (LAT™D).

[0289] 28. The recombinant cell of any one of embodiments 1, 5, 13-21, and 27, wherein the MLM is or comprises the sequence of amino acids set forth in SEQ ID NO: 289, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 289, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 289.

[0290] 29. The recombinant cell of any one of embodiments 1-28, wherein the SEM comprises a ZAP70 or a portion thereof comprising a kinase domain, and optionally interdomain B.

[0291] 30. The recombinant cell of any one of embodiments 1-29, wherein the SEM is ZAP70 ZAP70KDZAP70intB-KDor AP7O^' '2-1I1,A-SI ^-iniB-uiisonieretn-Ki)

[0292] 31. The recombinant cell of any one of embodiments 1-30, wherein the SEM is or comprises: (i) the sequence of amino acids set forth in SEQ ID NO: 3, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 3; (ii) the sequence of amino acids set forth in SEQ ID NO: 290, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 290, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 290; (iii) the sequence of amino acids set forth in SEQ ID NO: 291, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 291, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 291; or (iv) the sequence of amino acids set forth in SEQ ID NO: 292, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 292, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 292.

[0293] 32. The recombinant cell of any one of embodiments 1-28, wherein the SEM comprises a SYK or a portion thereof comprising a kinase domain, and optionally interdomain B.Attorney Docket No.: 048536-807001 WO

[0294] 33. The recombinant cell of any one of embodiments 1-28 and 32, wherein the SEM is SYK, SYKKD, or SYKlntB’KD.

[0295] 34. The recombinant cell of any one of embodiments 1-28, 32, and 33, wherein the SEM is or comprises: (i) the sequence of amino acids set forth in SEQ ID NO: 4, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 4; (ii) the sequence of amino acids set forth in SEQ ID NO:293, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 293, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 293; or (iii) the sequence of amino acids set forth in SEQ ID NO: 294, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:294, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 294.

[0296] 35. The recombinant cell of any one of embodiments 1-28, wherein the SEM comprises AKT1 or a portion thereof comprising a kinase domain.

[0297] 36. The recombinant cell of embodiment 35, wherein the SEM is a truncated AKT1 comprising the kinase domain and lacking the PH domain.

[0298] 37. The recombinant cell of any one of embodiments 1-28, 35, and 36, wherein the SEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 295, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 295, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 295.

[0299] 38. The recombinant cell of any one of embodiments 1-28, wherein the SEM comprises PDK1 or a portion thereof comprising a kinase domain.

[0300] 39. The recombinant cell of embodiment 38, wherein the SEM is a truncated PDK1 comprising the kinase domain and lacking the PH domain.

[0301] 40. The recombinant cell of any one of embodiments 1-28, 38, and 39, wherein the SEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 296, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 296, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 296.

[0302] 41. The recombinant cell of any one of embodiments 1-28, wherein the SEM comprises PIK3R1 alpha or a fragment comprising nSH2-iSH2-cSH2 domains.

[0303] 42. The recombinant cell of any one of embodiments 1-28 and 41, wherein theAttorney Docket No.: 048536-807001 WOSEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 19, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 19.

[0304] 43. The recombinant cell of any one of embodiments 1-28, wherein the SEM comprises a GRB2 or a portion comprising the N-SH3 domain and optionally an SH2 domain and / or C-SH3 domain.

[0305] 44. The recombinant cell of any one of embodiments 1-28 and 43, wherein the SEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 6, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 6.

[0306] 45. The recombinant cell of any one of embodiments 1-39, wherein the MLM and the SEM are heterologous to one another, each derived from a different protein.

[0307] 46. A recombinant cell comprising a recombinant polypeptide, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in any one of SEQ ID NOs: 31-143, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 31-143, 305, and 307.

[0308] 47. The recombinant cell of embodiment 46, wherein the recombinant polypeptide is or comprises the sequence of amino acids set forth in any one of SEQ ID NOs: 31-143, 305, and 307.

[0309] 48. The recombinant cell of embodiment 46 or embodiment 47, wherein the recombinant polypeptide is encoded by a nucleic acid sequence of any one of SEQ ID NOS: 174-286, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 174-286.

[0310] 49. The recombinant cell of any one of embodiments 46-48, wherein the recombinant polypeptide is encoded by a nucleic acid sequence of any one of SEQ ID NOS: 174-286.

[0311] 50. The recombinant cell of any one of embodiments 1-49, wherein the recombinant polypeptide is selected from: (a) ITKPH’TH’SH2-SH3_ZAP70KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 33 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Attorney Docket No.: 048536-807001 WO94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33; (b) ITKPH TH" sH2-sH3-z P70lntB-KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 305 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 305; (c) ITKPH-TH-SYKlntB’KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 307 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 307; (d) ITKPH TH-SYK, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 69 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 69; (e) LAT™D-AKTltrunc, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 60 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 60; (f) LATTMD- SYK, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 50 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 50; (g) LAT™D-PIK3Rla, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 64 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 64; (h) ITKPH TH-PIK3Rla, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 76 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 76; (i) LAT™D-GRB2, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 51 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 51; or j) LAT™D- PDK1, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 63 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 63.

[0312] 51. The recombinant cell of any one of embodiments 1-50, wherein the recombinant polypeptide is ITKPH TH SH2-SH3-ZAP70KDcomprising the sequence of amino acids set forth in SEQ ID NO: 33 or a sequence that has at least about 85%, 86%, 87%, 88%,Attorney Docket No.: 048536-807001 WO89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33;

[0313] 52. The recombinant cell of any one of embodiments 1-50, wherein the recombinant polypeptide is ITKPH TH SH2-SH3-zAP70lntB KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 305 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 305.

[0314] 53. The recombinant cell of any one of embodiments 1-52, provided that the polypeptide is not a fusion arising from a somatic chromosomal translocation in a mammalian cell, such as ITK-SYK.

[0315] 54. The recombinant cell of any one of embodiments 1-49, wherein the recombinant polypeptide is selected from: (a) ADAP-12234valency-lncreased, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 87 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 87; (b) GADSSH2“SLP76dlsordered, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 91 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 91; (c) HPKldlsordered-SLP76SH2, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 94 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 94; (d) NCK iSH3-SH3-SH3-SLP76dlsordered, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 105 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 105; (e) LCKCD4-CD8-lnteractors’ su3-sH2-j pzsH3-sH2,p-jg recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 111 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 111; and (f) LAT™D-HPKldlsordered-SLP76dlsorderedA, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 137 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 137.

[0316] 55. The recombinant cell of any one of embodiments 1-54, wherein a nucleic acid encoding the recombinant polypeptide is operably linked to a promoter, and wherein theAttorney Docket No.: 048536-807001 WO promoter is: (i) an exogenous promoter, optionally a promoter that is constitutively active or inducibly regulated; or (ii) an endogenous promoter of an endogenous locus in the cell.

[0317] 56. The recombinant cell of any one of embodiments 1-55, wherein expression of the recombinant polypeptide is expressed constitutively in the cell.

[0318] 57. The recombinant cell of any one of embodiments 1-55, wherein expression of the recombinant polypeptide is inducible in the cell.

[0319] 58. The recombinant cell of any one of embodiments 1-54, 56, and 57, wherein expression of the recombinant polypeptide is controlled by an inducible system selected from the group consisting of: (i) a chimeric receptor that is a ligand- inducible intramembrane proteolysis receptor; (ii) a small-molecule-inducible transcription factor system; (iii) an optogenetic transcription factor system; and (iv) an inducible CRISPR-based transcriptional activator system.

[0320] 59. The recombinant cell of embodiment 58, wherein the recombinant cell comprises the chimeric receptor of (i) and the chimeric receptor is a synthetic Notch (synNotch) receptor or a Synthetic Intramembrane Proteolysis Receptor (SNIPR).

[0321] 60. The recombinant cell of embodiment 58 or embodiment 59, wherein the chimeric receptor comprises: (a) an extracellular ligand-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a transcriptional regulator, and optionally an intracellular signaling domain comprising a primary activation domain and a costimulatory signaling domain, wherein, upon binding of a ligand to the extracellular ligand-binding domain, the transcriptional regulator is released from the plasma membrane and binds a response element within or proximal to a promoter that is operably linked to the nucleic acid encoding the recombinant polypeptide, thereby inducing expression of the recombinant polypeptide.

[0322] 61. The recombinant cell of any one of embodiments 1-60, comprising a recombinant antigen receptor having specificity for a target ligand, optionally wherein the recombinant antigen receptor is a CAR or TCR enabled architecture.

[0323] 62. The recombinant cell of embodiment 61, wherein the recombinant antigen receptor is a chimeric antigen receptor (CAR).

[0324] 63. The recombinant cell of embodiment 61 or embodiment 62, wherein the CAR is a single-chain CAR, a multi-chain CAR, a single-targeted CAR, a multi-targeted CAR, a bivalent tandem CAR, a bivalent loop CAR, a multicistronic CAR, a bicistronic CAR, a synNotch-CAR circuit, a synthetic intramembrane proteolysis receptor (SNIPR), a multi-chain DAP-CAR, a TREM1 / DAP12 CAR, or a DAP12 / TREM1 CAR.

[0325] 64. The recombinant cell of embodiment 63, wherein the recombinant antigenAttorney Docket No.: 048536-807001 WO receptor is a TCR enabled architecture.

[0326] 65. The recombinant cell of embodiment 61 or embodiment 64, wherein the TCR enabled architecture is a TCR, a T cell receptor fusion construct (TruC), an HLA-independent T cell (HIT) receptor, a synthetic T cell receptor and antigen receptor (STAR), a rapamycin inducible TCR, a rapamycin inducible Fc receptor, or a constitutive TCR-like receptor.

[0327] 66. The recombinant cell of embodiment 61, wherein the recombinant antigen receptor is a dimerizing agent regulated immune-receptor complex (DARIC), an antibody tethered orthogonal multiplexing compatible (ATOMIC), a synNotch-CAR circuit, a synthetic intramembrane proteolysis receptor (SNIPR), a rapamycin inducible Fc receptor, a multi-chain DAP-CAR, a TREM1 / DAP12 CAR, or a DAP12 / TREM1 CAR.

[0328] 67. The recombinant cell of any one of embodiments 1-66, further comprising a safety switch configured to permit selective depletion or inactivation of the cell in a subject.

[0329] 68. The recombinant cell of embodiment 67, wherein the safety switch comprises a safety switch enzyme that is activatable by an exogenously administered agent to promote apoptosis or cytotoxicity of the cell, wherein the safety switch enzyme is selected from the group consisting of a prodrug-activating kinase, a prodrug-activating deaminase, and a dimerization-inducible caspase, and wherein the exogenously administered agent comprises a prodrug or a dimerization agent.

[0330] 69. The recombinant cell of embodiment 68, wherein the safety switch enzyme comprises: (i) a prodrug-activating kinase that comprises a herpesvirus thymidine kinase (HSV-tk) and is activatable by a prodrug, optionally wherein the prodrug comprises ganciclovir or a pharmaceutically acceptable derivative thereof; (ii) a prodrug-activating deaminase that comprises an Escherichia coli cytosine deaminase (EC-CD) and is activatable by a prodrug, optionally wherein the prodrug comprises 5-fluorocytosine or a pharmaceutically acceptable derivative thereof; or (iii) a dimerization-inducible caspase that comprises an inducible caspase-9 (iCasp9) comprising a dimerization domain fused to a caspase-9 protein and is activatable by dimerization agent, optionally wherein the dimerization agent is a smallmolecule dimerizer that binds the dimerization domain.

[0331] 70. The recombinant cell of embodiment 67, wherein the safety switch comprises a depletion tag comprising a protein that is bound by an exogenously administered antibody.

[0332] 71. The recombinant cell of embodiment 70, wherein the protein is a full-length or truncated form of a protein selected from the group consisting of CCR4, CD 16, CD 19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8, wherein the full-length or truncated form comprises an extracellular domain that includes an epitope bound by theAttorney Docket No.: 048536-807001 WO antibody and optionally lacks all or part of an intracellular or signaling domain.

[0333] 72. The recombinant cell of embodiment 67, wherein the safety switch comprises a tolerogenic protein expressed on a surface of the recombinant cell and configured to deliver an inhibitory signal to an immune effector cell, and wherein binding of an exogenously administered agent to the tolerogenic protein reduces or blocks the inhibitory signal, thereby permitting immune-mediated clearance of the recombinant cell.

[0334] 73. The recombinant cell of embodiment 72, wherein the tolerogenic protein comprises CD47 and the exogenously administered agent comprises an anti-CD47 antibody.

[0335] 74. The recombinant cell of any one of embodiments 1-73, wherein the recombinant polypeptide is inserted into an endogenous genomic locus of the cell.

[0336] 75. The recombinant cell of embodiment 74, wherein the endogenous genomic locus is selected from the group consisting of a T-cell receptor (TCR) locus, a CD3 locus, a P2-microglobulin (B2M) locus, a class II transactivator (CIITA) locus, and a safe harbor locus.

[0337] 76. The recombinant cell of embodiment 75, wherein the TCR locus is selected from the group consisting of a TRAC locus, a TRBC1 locus, a TRBC2 locus, and a TRAJ locus.

[0338] 77. The recombinant cell of embodiment 75, wherein the safe harbor locus is an AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, or SHS231 locus.

[0339] 78. The recombinant cell of embodiment 74, wherein the endogenous genomic locus encodes a fusion partner of the recombinant polypeptide, such that insertion of the nucleic acid encoding the recombinant polypeptide or a portion thereof into the endogenous genomic locus results in expression of the recombinant polypeptide or the portion thereof as a fusion with an endogenous protein encoded at the locus.

[0340] 79. The recombinant cell of embodiment 65 or embodiment 69, wherein the endogenous genomic locus is selected from the group consisting of: (i) a Tec family kinase locus, optionally an ITK locus; (ii) a transmembrane adaptor protein (TRAP) locus, optionally a LAT locus; (iii) a locus of a kinase of a Syk / ZAP70 non-receptor tyrosine kinase cascade, optionally a SYK locus or a ZAP70 locus; (iv) a locus of a kinase of an AKT serine / threonine kinase cascade, optionally an AKT1 locus; (v) a locus of a kinase or regulatory subunit of a class IA phosphoinositide 3-kinase (PI3K) lipid-kinase cascade, optionally a PDK1 locus or a PIK3R1 locus; and (vi) a locus of an adaptor protein of a Ras-Raf-MEK-ERK cascade, optionally a GRB2 locus.

[0341] 80. The recombinant cell of embodiment 74 or embodiment 78, wherein theAttorney Docket No.: 048536-807001 WO endogenous genomic locus is selected from the group consisting of: a LCK locus, a FYN locus, a GADS locus, a SOS1 locus, a SLP76 locus, a ADAP locus, a PLCgl locus, a VAV1 locus, a NCK1 locus, a BTK locus, a MAP4K3 locus, a PKC theta locus, a SHP1 locus, a c-Cbl locus, and a HPK1 locus.

[0342] 81. A recombinant polypeptide comprising, in N- to C-terminal direction: (a) a first polypeptide sequence that is a membrane-localization module (MLM) comprising a domain that mediates localization of the recombinant polypeptide to at least one of (i) a plasma membrane of a cell, or (ii) a receptor-proximal region at the plasma membrane that forms in response to engagement of a cell with its cognate ligand; and (b) a second polypeptide sequence that is a signaling-effector module (SEM) that, when localized to the plasma membrane by the MLM, potentiates a kinase signaling cascade in the cell.

[0343] 82. The recombinant polypeptide of embodiment 81, wherein the MLM mediates inducible recruitment of the recombinant polypeptide to a receptor-proximal signaling complex that assembles at the plasma membrane in response to engagement of the receptor with its cognate ligand.

[0344] 83. The recombinant polypeptide of embodiment 82, wherein the receptor-proximal signaling complex comprises a LAT-SLP-76 signalosome.

[0345] 84. The recombinant polypeptide of embodiment 83, wherein the cell is a T cell and the receptor is a T cell receptor (TCR), and the LAT-SLP-76 signalosome is induced by engagement of the TCR with its cognate ligand.

[0346] 85. The recombinant polypeptide of embodiment 81, wherein the MLM lacks a functional enzymatic domain.

[0347] 86. The recombinant polypeptide of any one of embodiments 81-85, wherein the MLM is a domain capable of localizing the recombinant polypeptide to a plasma membrane of the cell.

[0348] 87. The recombinant polypeptide of embodiment 81 or embodiment 85, wherein the domain capable of localizing the recombinant polypeptide to the plasma membrane is a lipid- binding pleckstrin homology (PH) domain.

[0349] 88. The recombinant polypeptide of any one of embodiments 81-87, wherein the MLM comprises a pleckstrin homology (PH) domain, optionally wherein the MLM further comprises an SH3 and / or SH2 domain.

[0350] 89. The recombinant polypeptide of any one of embodiments 81-87, wherein the MLM comprises a pleckstrin homology (PH) domain and a Tec-homology (TH) region of a Tec-family kinase, optionally wherein the MLM further comprises an SH3 and / or SH2 domain.Attorney Docket No.: 048536-807001 WO

[0351] 90. The recombinant polypeptide of embodiment 89, wherein the PH region is derived from ITK, BTK, TEC, or BMX, or is a variant thereof capable of localizing to a plasma membrane.

[0352] 91. The recombinant polypeptide of embodiment 89, wherein the PH-TH region is derived from ITK, BTK, TEC, or BMX, or is a variant thereof capable of localizing to a plasma membrane.

[0353] 92. The recombinant polypeptide of any one of embodiments 81-85, wherein the MLM comprises a transmembrane domain that spans the plasma membrane of the cell.

[0354] 93. The recombinant polypeptide of embodiment 81 or embodiment 85 or embodiment 91, wherein the MLM comprises a single-pass transmembrane anchor from a transmembrane adaptor protein (TRAP).

[0355] 94. The recombinant polypeptide of embodiment 93, wherein the MLM is characterized by a transmembrane domain, and (ii) a cytosolic juxtamembrane palmitoylation motif comprising 2 or more cysteines.

[0356] 95. The recombinant polypeptide of embodiment 94, wherein the MLM further comprises a luminal N-terminal segment of 20 amino acids or less.

[0357] 96. The recombinant polypeptide of embodiment 93 or embodiment 94, wherein the TRAP is selected from LAT, PAG / Cbp, LIME, SIT, TRIM, LAX, SCIMP, PRR7, LST1 / A, or a variant of any of the foregoing that retains localization to the membrane by palmitoylation-dependent membrane anchoring.

[0358] 97. The recombinant polypeptide of any one of embodiments 81-96, wherein the signaling cascade potentiated by the SEM is (a) a Syk / ZAP-70 non-receptor tyrosine kinase cascade; (b) an AKT (serine / threonine kinase) cascade, (c) a class IA phosphoinositide-3-kinase (PI3K) lipid-kinase cascade; or (d) a Ras-Raf-MEK-ERK cascade.

[0359] 98. The recombinant polypeptide of any one of embodiments 81-97, wherein the SEM comprises a Syk / ZAP70-family tyrosine kinase, an AKT serine / threonine kinase, or a PDK1 serine / threonine kinase or a portion thereof comprising a kinase domain.

[0360] 99. The recombinant polypeptide of any one of embodiments 84-97, wherein the SEM comprises a class IA PI3K regulatory subunit or a portion comprising nSH2-iSH2-cSH2 domains.

[0361] 100. The recombinant polypeptide of any one of embodiments 81-99, wherein the SEM comprises a GRB2 or a portion comprising the N-terminal SH3 domain and optionally an SH2 domain and / or C-terminal SH3 domain.

[0362] 101. A recombinant polypeptide comprising in N- to C-terminal direction: (a) a firstAttorney Docket No.: 048536-807001 WO polypeptide sequence that is a membrane-localization module (MLM) selected from (i) a pleckstrin-homology (PH) domain, optionally further comprising an SH3 and / or SH2 domain, (ii) a pleckstrin-homology (PH) domain and Tec -homology (TH) region of a Tec -family kinase, optionally further comprising an SH3 and / or SH2 domain, or (iii) a single-pass transmembrane sequence of LAT, wherein the MLM lacks functional enzymatic domain; and (b) a second polypeptide sequence that is a signaling-effector module (SEM) selected from (i) a Syk / Zap70-family non-receptor tyrosine kinase or a portion thereof comprising the kinase domain and, optionally, interdomain B, (ii) an AKT serine / threonine kinase or a portion thereof comprising the kinase domain, (iii) a PDK1 serine / threonine kinase or a portion thereof comprising the kinase domain; (iv) a class IA PI3K regulatory subunit (PIK3R1 alpha) or a portion comprising nSH2-iSH2-cSH2 domains; and (v) a GRB2 or a portion comprising the N-SH3 domain and optionally an SH2 domain and / or C-SH3 domain.

[0363] 102. The recombinant polypeptide of any one of embodiments 81-79 and 97-101, wherein the MLM comprises a PH region from ITK.

[0364] 103. The recombinant polypeptide of any one of embodiments 81-91 and 97-101, wherein the MLM comprises a PH and TH region from ITK.

[0365] 104. The recombinant polypeptide of any one of embodiments 81-91 and 97-103, wherein the MLM is selected from: ITKPH-THor ITKPH’TH’SH2-SH3.

[0366] 105. The recombinant polypeptide of any one of embodiments 81-91 and 97-104, wherein the MLM is or comprises the sequence of amino acids set forth in SEQ ID NO: 287, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 287 , optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 287.

[0367] 106. The recombinant polypeptide of any one of embodiments 81-91 and 97-104, wherein the MLM is or comprises the sequence of amino acids set forth in SEQ ID NO: 288, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 288, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 288.

[0368] 107. The recombinant polypeptide of any one of embodiments 81, 85, and 93-101, wherein the MLM comprises the transmembrane domain of LAT (LAT™D).

[0369] 108. The recombinant polypeptide of any one of embodiments 81, 85, and 93-101, wherein the MLM is or comprises the sequence of amino acids set forth in SEQ ID NO: 289, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 289, optionally wherein theAttorney Docket No.: 048536-807001 WOMLM is set forth by the sequence in SEQ ID NO: 289.

[0370] 109. The recombinant polypeptide of any one of embodiments 81-89, wherein the SEM comprises a ZAP70 or a portion thereof comprising a kinase domain, and optionally interdomain B.

[0371] 110. The recombinant polypeptide of any one of embodiments 81-90, wherein the SEM is ZAP70, ZAP70KD, ZAP70lntB’KD, or ZAP70SH2’lntA’SH2’lntB’(dlsordered)’KD.

[0372] 111. The recombinant polypeptide of any one of embodiments 81-91, wherein the SEM is or comprises: (i) the sequence of amino acids set forth in SEQ ID NO: 3, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 3; (ii) the sequence of amino acids set forth in SEQ ID NO:290, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 290, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 290; (iii) the sequence of amino acids set forth in SEQ ID NO: 291, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:291, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 291; or (iv) the sequence of amino acids set forth in SEQ ID NO: 292, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 292, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 292.

[0373] 112. The recombinant polypeptide of any one of embodiments 81-89, wherein the SEM comprises a SYK or a portion thereof comprising a kinase domain, and optionally interdomain B.

[0374] 113. The recombinant polypeptide of any one of embodiments 81-89 and 93, wherein the SEM is SYK, SYKKD, or SYKlntB KD.

[0375] 114. The recombinant polypeptide of any one of embodiments 81-108, 112, and 113, wherein the SEM is or comprises: (i) the sequence of amino acids set forth in SEQ ID NO: 4, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 4; (ii) the sequence of amino acids set forth in SEQ ID NO: 293, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 293, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 293; (iii) the sequenceAttorney Docket No.: 048536-807001 WO of amino acids set forth in SEQ ID NO: 294, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 294, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 294; or

[0376] 115. The recombinant polypeptide of any one of embodiments 81-108, wherein the SEM comprises AKT1 or a portion thereof comprising a kinase domain.

[0377] 116. The recombinant polypeptide of embodiment 115, wherein the SEM is a truncated AKT1 comprising the kinase domain and lacking the PH domain.

[0378] 117. The recombinant polypeptide of any one of embodiments 81-108, 115, and 117, wherein the SEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 295, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 295, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 295.

[0379] 118. The recombinant polypeptide of any one of embodiments 81-108, wherein the SEM comprises PDK1 or a portion thereof comprising a kinase domain.

[0380] 119. The recombinant polypeptide of embodiment 118, wherein the SEM is a truncated PDK1 comprising the kinase domain and lacking the PH domain.

[0381] 120. The recombinant polypeptide of any one of embodiments 81-108, 118, and 119, wherein the SEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 296, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 296, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 296.

[0382] 121. The recombinant polypeptide of any one of embodiments 81-89, wherein the SEM comprises PIK3R1 alpha or a fragment comprising nSH2-iSH2-cSH2 domains.

[0383] 122. The recombinant polypeptide of any one of embodiments 81-89 and 102, wherein the SEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 19, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 19.

[0384] 123. The recombinant polypeptide of any one of embodiments 81-89, wherein the SEM comprises a GRB2 or a portion comprising the N-SH3 domain and optionally an SH2 domain and / or C-SH3 domain.

[0385] 124. The recombinant polypeptide of any one of embodiments 81-108 and 123, wherein the SEM is or comprises the sequence of amino acids set forth in SEQ ID NO: 6, or aAttorney Docket No.: 048536-807001 WO sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6, optionally wherein the MLM is set forth by the sequence in SEQ ID NO: 6.

[0386] 125. The recombinant polypeptide of any one of embodiments 81-119, wherein the MLM and the SEM are heterologous to one another, each derived from a different protein.

[0387] 126. A recombinant polypeptide comprising the sequence of amino acids set forth in any one of SEQ ID NOs: 31-143, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 31-143.

[0388] 127. The recombinant polypeptide of embodiment 126, wherein the recombinant polypeptide is or comprises the sequence of amino acids set forth in any one of SEQ ID NOs: 31-143.

[0389] 128. The recombinant polypeptide of embodiment 126 or embodiment 127, wherein the recombinant polypeptide is encoded by a nucleic acid sequence of any one of SEQ ID NOS: 174-286, or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NO: 174-286.

[0390] 129. The recombinant polypeptide of any one of embodiments 126-128, wherein the recombinant polypeptide is encoded by a nucleic acid sequence of any one of SEQ ID NOS: 174-286.

[0391] 130. The recombinant polypeptide of any one of embodiments 81-129, wherein the recombinant polypeptide is selected from: (a) ITKPH’TH’SH2-SH3_ZAP70KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 33 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33; (b) ITKPH TH" sH2-sH3-z P70lntB-KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 305 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 305; (c) ITKPH-TH-SYKlntB’KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 307 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 307; (d) ITKPH TH-SYK, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 69 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,Attorney Docket No.: 048536-807001 WO91%, 98% or 99% sequence identity to SEQ ID NO: 69; (e) LAT™D-AKTltrunc, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 60 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 60; (f) LATTMD- SYK, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 50 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 50; (g) LAT™D-PIK3Rla, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 64 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 64; (h) ITKPH TH-PIK3Rla, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 76 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 76; (i) LAT™D-GRB2, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 51 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 51; or j) LAT™D- PDK1, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 63 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 63.

[0392] 131. The recombinant polypeptide of any one of embodiments 81-130, wherein the recombinant polypeptide is ITKPH TH SH2-SH3-ZAP70KDcomprising the sequence of amino acids set forth in SEQ ID NO: 33 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33;

[0393] 132. The recombinant polypeptide of any one of embodiments 81-130, wherein the recombinant polypeptide is ITKPH TH SH2-SH3-zAP70lntB KD, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 305 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 305.

[0394] 133. The recombinant polypeptide of any one of embodiments 81-132, provided that the polypeptide is not a fusion arising from a somatic chromosomal translocation in a mammalian cell, such as ITK-SYK.Attorney Docket No.: 048536-807001 WO

[0395] 134. The recombinant polypeptide of any one of embodiments 81-129, wherein the recombinant polypeptide is selected from: (a) ADAP-12234valency-lncreased, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 87 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 87; (b) GADSSH2“SLP76dlsordered, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 91 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 91; (c) HPKldlsordered-SLP76SH2, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 94 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 94; (d) NCK iSH3-SH3-SH3-SLP76dlsordered, wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 105 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 105; (e) LCKCD4-CD8-lnteractors’ su3-sH2-j pzsH3-sH2,p-jg recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 111 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 111; and (f) LAT™D-HPKldlsordered-SLP76dlsorderedA, optionally wherein the recombinant polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 137 or a sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 137.

[0396] 135. A recombinant nucleic acid comprising a nucleic acid sequence encoding the recombinant polypeptide of any one of embodiments 81-134.

[0397] 136. The recombinant nucleic acid of embodiment 135, wherein the nucleic acid sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 174-286.

[0398] 137. The recombinant nucleic acid of any one of embodiments 135-136, wherein the nucleic acid sequence is operably linked to one or more heterologous nucleic acid sequence.

[0399] 138. The recombinant nucleic acid of embodiment 137, wherein the one or more heterologous nucleic acid sequences comprises or is a nucleic acid control element.

[0400] 139. The recombinant nucleic acid of embodiment 138, wherein the nucleic acid control element is selected from the group consisting of self-cleaving peptide sequences, ribosomal binding sites, promoters, enhancer elements, activator elements, translational startAttorney Docket No.: 048536-807001 WO sequences, translational termination sequences, transcription start sequences, transcription termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, replication elements, RNA processing and export elements, transposon sequences, transposase sequences, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences, boundary elements, locus control regions (LCR), matrix attachment regions (MAR), recombination or cassette exchange sequences, or any combination thereof.

[0401] 140. The recombinant nucleic acid of any one of embodiment 137-139, wherein the heterologous nucleic acid sequence comprises or is a promoter.

[0402] 141. The recombinant cell of any one of embodiments 137-140, wherein the promoter is selected from the group consisting of: an MND promoter; an elongation factor 1 alpha (EFla) promoter, including a short EFla (sEFl) promoter; a gamma-retroviral long terminal repeat (LTR) promoter; a TRAC promoter; a TRBC1 promoter; a TRBC2 promoter; a TCRa promoter; a TCRP promoter; a CD3a promoter; a CD3P promoter; a CD3y promoter; a CD36 promoter; a CD3e promoter; a CD3(^ promoter; a CD4 promoter; a CD8a promoter; a CD8P promoter; a CD25 promoter; an IL2 promoter; an IL7 promoter; an IL 15 promoter; a KLRG-1 promoter; an HLA-DR promoter; a CD38 promoter; a CD69 promoter; a Ki-67 promoter; a CD I la promoter; a CD58 promoter; a CD99 promoter; a CD62L promoter; a CD 103 promoter; a CCR4 promoter; a CCR5 promoter; a CCR6 promoter; a CCR7 promoter; a CCR9 promoter; a CCR10 promoter; a CXCR3 promoter; a CXCR4 promoter; a cutaneous lymphocyte antigen (CLA) promoter; a Granzyme A promoter; a Granzyme B (GzmB) promoter; a Perforin promoter; a CD57 promoter; a CD 161 promoter; an IL-18Ra promoter; a T-bet promoter; an IFN-y promoter; a TIM-3 promoter; an IL1 promoter; an IL4 promoter; an IL5 promoter; an IL6 promoter; an IL7 promoter; an IL 10 promoter; an IL 13 promoter; an IL 15 promoter; an IL17A promoter; an IL21 promoter; an IL23R promoter; a FoxP3 promoter; a GATA3 promoter; a CTLA4 promoter; a PD- 1 promoter; a CD45RA promoter; a CD45RO promoter; a CD27 promoter; a CD28 promoter; a CD95 promoter; a CD 127 promoter; a CD 122 promoter; a CD 132 promoter; a c-Kit promoter; a lymphocyte-activation protein 3 (LAG-3) promoter; a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter; a B- and T-lymphocyte attenuator (BTLA) promoter; a Fas ligand (FasL) promoter; a TIGIT promoter; a transforming growth factor-P (TGF-P) promoter; an Eomes promoter; a 2B4 promoter; a Type I interferon alpha promoter; a Type I interferon beta promoter; an IRF3 promoter; an IRF7 promoter; a nuclear factor of activated T cells (NF AT) promoter; an NF-KBAttorney Docket No.: 048536-807001 WO promoter; an AP-1 promoter; a TNF-a promoter; a CD 130 promoter; an NR4A1 promoter; an NR4A2 promoter; an NR4A3 promoter; a B2M promoter; a CIITA promoter; an albumin promoter; an F3 (CD142) promoter; a MICA promoter; a MICB promoter; an LRP1 (CD91) promoter; an HMGB1 promoter; an ABO promoter; an RHD promoter; a FUT1 promoter; a KDM5D (HY) promoter; a PDGFRa promoter; an OLIG2 promoter; a GFAP promoter; a minimal TATA promoter; a phosphoglycerate kinase (PGK) promoter; an actin promoter; an immunoglobulin

[0403] promoter; a heat-shock promoter; a human Ubiquitin C (UBC) promoter; a ubiquitin / S27a promoter; a CAG promoter; a simian virus 40 (SV40) early promoter; an adenovirus major late promoter; a mouse metallothionein-I promoter; a Rous Sarcoma Virus (RSV) long terminal repeat promoter; a mouse mammary tumor virus (MMTV) promoter; a Moloney murine leukemia virus long terminal repeat promoter; and a promoter derived from a polyoma virus, a fowlpox virus, a bovine papilloma virus, an avian sarcoma virus, a cytomegalovirus, a retrovirus, or a hepatitis B virus.

[0404] 142. The recombinant nucleic acid of embodiment 140 or embodiment 141, wherein the promoter comprises or is an EFla promoter, an MND promoter, CMV promoter, CAG promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG- 1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CDlla promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD 161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69Attorney Docket No.: 048536-807001 WO promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, or NR4A3 promoter.

[0405] 143. The recombinant nucleic acid of any one of embodiments 140-142, wherein the promoter is an endogenous promoter.

[0406] 144. The recombinant nucleic acid of embodiment 143, wherein the endogenous promoter comprises or is a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, a CD3z promoter, a B2 microglobulin (B2M) promoter, or a class II transactivator (CIITA) promoter.

[0407] 145. The recombinant nucleic acid of embodiment 140, wherein the promoter is a synthetic promoter.

[0408] 146. The recombinant nucleic acid of embodiment 135 or embodiment 136, wherein the nucleic acid does not comprise a promoter.

[0409] 147. The recombinant nucleic acid of any one of embodiments 137-146, wherein the one or more heterologous nucleic acid sequences comprises or is a coding sequence for a polypeptide.

[0410] 148. The recombinant nucleic acid of embodiment 147, wherein the polypeptide comprises or is a peptide linker sequence, a cleavable linker sequence, a secretion signal, a resistance marker, an anchoring peptide, a localization signal, a fusion tag, an affinity tag, a chaperonin, a protease, or any combination thereof.

[0411] 149. The recombinant nucleic acid of any one of embodiments 135-148, wherein the polypeptide comprises a peptide linker sequence that is a ribosomal skipping peptide sequence.

[0412] 150. The recombinant nucleic acid of embodiment 149, wherein the ribosomal skipping peptide sequence is a 2A peptide.

[0413] 151. The recombinant nucleic acid of embodiment 149 or embodiment 151, wherein the ribosomal skipping peptide comprises one or more ribosomal skipping peptide sequences from a porcine teschovirus-1 2A (P2A), a calcium-dependent serine endoprotease (furin), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.

[0414] 152. The recombinant nucleic acid of any one of embodiments 135-151, further comprising a barcode molecule that identifies that recombinant polypeptide.Attorney Docket No.: 048536-807001 WO

[0415] 153. The recombinant nucleic acid of any one of embodiments 135-151, further comprising a nucleic acid sequence encoding an inducible system configured to regulate expression of the recombinant polypeptide, optionally wherein the nucleic acid sequence encoding the recombinant polypeptide and the nucleic acid sequence encoding the inducible system are present as one or more expression cassettes.

[0416] 154. The recombinant nucleic acid of embodiment 153, wherein the inducible system is selected from the group consisting of: (i) a chimeric receptor that is a ligand-inducible intramembrane proteolysis receptor; (ii) a small-molecule-inducible transcription factor system; (iii) an optogenetic transcription factor system; and (iv) an inducible CRISPR-based transcriptional activator system.

[0417] 155. The recombinant nucleic acid of embodiment 154, wherein the inducible system comprises a nucleic acid encoding the chimeric receptor of item (i), and wherein the chimeric receptor is a synthetic Notch (synNotch) receptor or a Synthetic Intramembrane Proteolysis Receptor (SNIPR).

[0418] 156. The recombinant nucleic acid of embodiment 154 or embodiment 155, wherein the recombinant nucleic acid comprises: (a) a first expression cassette comprises a promoter operably linked to a nucleic acid encoding the recombinant polypeptide; and (b) a second expression cassette comprises a nucleic acid encoding the chimeric receptor, wherein the chimeric receptor comprises: (i) an extracellular ligand-binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a transcriptional regulator and optionally an intracellular signaling domain comprising a primary activation domain and a costimulatory signaling domain; and wherein, upon binding of a ligand to the extracellular ligand-binding domain, the transcriptional regulator is released from the plasma membrane and binds a response element within or proximal to the promoter of the first expression cassette, thereby inducing expression of the recombinant polypeptide.

[0419] 157. The recombinant nucleic acid of any one of embodiments 135-156, further comprising a nucleic acid encoding a recombinant antigen receptor having specificity for a target ligand, optionally wherein the nucleic acid sequence encoding the recombinant polypeptide and the nucleic acid sequence encoding the recombinant antigen receptor are present as one or more expression cassettes.

[0420] 158. The recombinant nucleic acid of embodiment 158, wherein the recombinant antigen receptor is a CAR or a TCR-enabled architecture.

[0421] 159. The recombinant nucleic acid of embodiment 158 or embodiment 158, wherein the recombinant antigen receptor is a chimeric antigen receptor (CAR).Attorney Docket No.: 048536-807001 WO

[0422] 160. The recombinant nucleic acid of embodiment 158 or embodiment 159, wherein the CAR is selected from the group consisting of a single-chain CAR, a multi-chain CAR, a single-targeted CAR, a multi-targeted CAR, a bivalent tandem CAR, a bivalent loop CAR, a multicistronic CAR, a bicistronic CAR, a synNotch-CAR circuit, a synthetic intramembrane proteolysis receptor (SNIPR), a multi-chain DAP-CAR, a TREM1 / DAP12 CAR, and a DAP12 / TREM1 CAR.

[0423] 161. The recombinant nucleic acid of embodiment 157 or embodiment 158, wherein the recombinant antigen receptor is a TCR-enabled architecture.

[0424] 162. The recombinant nucleic acid of embodiment 161, wherein the TCR-enabled architecture is selected from the group consisting of a T cell receptor (TCR), a T cell receptor fusion construct (TruC), an HLA-independent T cell (HIT) receptor, a synthetic T cell receptor and antigen receptor (STAR), a rapamycin-inducible TCR, a rapamycin-inducible Fc receptor, and a constitutive TCR-like receptor.

[0425] 163. The recombinant nucleic acid of embodiment 157 or embodiment 158, wherein the recombinant antigen receptor is selected from the group consisting of a dimerizing agent regulated immune-receptor complex (DARIC), an antibody tethered orthogonal multiplexing compatible (ATOMIC) receptor, a synNotch-CAR circuit, a synthetic intramembrane proteolysis receptor (SNIPR), a rapamycin- inducible Fc receptor, a multi-chain DAP-CAR, a TREM1 / DAP12 CAR, and a DAP12 / TREM1 CAR.

[0426] 164. The recombinant nucleic acid of any one of embodiments 135-163, further comprising a nucleic acid sequence encoding a safety switch configured to permit selective depletion or inactivation of a cell comprising the recombinant nucleic acid in a subject, optionally wherein the nucleic acid sequence encoding the recombinant polypeptide and the nucleic acid sequence encoding the safety switch are present as one or more expression cassettes.

[0427] 165. The recombinant nucleic acid of embodiment 164, wherein the safety switch comprises a safety switch enzyme that is activatable by an exogenously administered agent to promote apoptosis or cytotoxicity of the cell, wherein the safety switch enzyme is selected from the group consisting of a prodrug-activating kinase, a prodrug-activating deaminase, and a dimerization-inducible caspase, and wherein the exogenously administered agent comprises a prodrug or a dimerization agent.

[0428] 166. The recombinant nucleic acid of embodiment 165, wherein the safety switch enzyme comprises one or more of: (i) a prodrug-activating kinase that comprises a herpesvirus thymidine kinase (HSV-tk) and is activatable by a prodrug, optionally wherein the prodrugAttorney Docket No.: 048536-807001 WO comprises ganciclovir or a pharmaceutically acceptable derivative thereof; (ii) a prodrugactivating deaminase that comprises an Escherichia coli cytosine deaminase (EC-CD) and is activatable by a prodrug, optionally wherein the prodrug comprises 5-fluorocytosine or a pharmaceutically acceptable derivative thereof; and (iii) a dimerization-inducible caspase that comprises an inducible caspase-9 (iCasp9) comprising a dimerization domain fused to a caspase-9 protein and is activatable by a dimerization agent, optionally wherein the dimerization agent is a small-molecule dimerizer that binds the dimerization domain.

[0429] 167. The recombinant nucleic acid of embodiment 164, wherein the safety switch comprises a nucleic acid sequence encoding a depletion tag comprising a protein that is bound by an exogenously administered antibody.

[0430] 168. The recombinant nucleic acid of embodiment 167, wherein the protein is a full- length or truncated form of a protein selected from the group consisting of CCR4, CD 16, CD 19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8, wherein the full-length or truncated form comprises an extracellular domain that includes an epitope bound by the antibody and optionally lacks all or part of an intracellular or signaling domain.

[0431] 169. The recombinant nucleic acid of embodiment 164, wherein the safety switch comprises a nucleic acid sequence encoding a tolerogenic protein expressed on a surface of the cell and configured to deliver an inhibitory signal to an immune effector cell, and wherein binding of an exogenously administered agent to the tolerogenic protein reduces or blocks the inhibitory signal, thereby permitting immune-mediated clearance of the cell.

[0432] 170. The recombinant nucleic acid of embodiment 169, wherein the tolerogenic protein comprises CD47 and the exogenously administered agent comprises an anti-CD47 antibody.

[0433] 171. A vector comprising the recombinant nucleic acid of any one of embodiments 135-170.

[0434] 172. The vector of embodiment 171, wherein the vector is an expression vector.

[0435] 173. The vector of any one of embodiments 171-172, wherein the vector is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a RNA vector, a mRNA vector, a phagemid vector, a viral vector, a self-replicating RNA virus, a mRNA-packaging virus-like particle, or a RNP-packaging virus-like particle.

[0436] 174. The vector of embodiment 173, wherein the viral vector is a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector.

[0437] 175. The vector of any one of embodiments 171-174, wherein the vector is formulated in a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a proteinAttorney Docket No.: 048536-807001 WO nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, a fusosome, an enveloped delivery vehicle, or an immune stimulating complex (ISCOM).

[0438] 176. The vector of embodiment 175, wherein the LNP is a selective organ targeting LNP or an antibody-targeted LNP.

[0439] 177. The vector of embodiment 174, wherein the viral vector is a lentiviral vector.

[0440] 178. The vector of embodiment 177, wherein the lentiviral vector is pseudotyped with a heterologous envelope glycoprotein.

[0441] 179. The vector of embodiment 178, wherein the heterologous envelope glycoprotein is a vesicular stomatitis virus G (VSV-G) glycoprotein or a Cocal glycoprotein.

[0442] 180. The vector of any one of embodiments 177-179, wherein the heterologous envelope glycoprotein is fused to an antibody to redirect the lentiviral vector to a target cell, wherein the target cell is a T cell, optionally wherein the antibody is an anti-CD3 antibody, an anti-CD2 antibody, or an anti-CD7 antibody.

[0443] 181. A recombinant cell comprising one or more of the following: (a) a recombinant polypeptide according to any one of embodiments 81-134; (b) a recombinant nucleic acid according to any one of embodiments 135-170; and (c) a vector according to any one of embodiments 171-180.

[0444] 182. The recombinant cell of any one of embodiments 1-79 and 181, wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell.

[0445] 183. The recombinant cell of any one of embodiments 1-79, 181 and 182, wherein the recombinant cell is a eukaryotic cell.

[0446] 184. The recombinant cell of embodiment 183, wherein the eukaryotic cell is a mammalian cell.

[0447] 185. The recombinant cell of embodiment 184, wherein the mammalian cell is a human cell.

[0448] 186. The recombinant cell of any one of embodiments 1-79 and 181-185, wherein the recombinant cell is a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a gamma delta T cell (y5T), an invariant natural ki...

Claims

Attorney Docket No.: 048536-807001 WOCLAIMSWHAT IS CLAIMED IS:

1. A recombinant polypeptide comprising, in N- to C-terminal direction:(a) a first portion comprising a first polypeptide sequence derived from a protein selected from the group consisting of ITK, SHP1, c-Cbl, LCK, FYN, ZAP70, and SYK; and(b) a second portion comprising a second polypeptide sequence derived from a protein selected from the group consisting of SYK, LCK, FYN, ZAP70, LCK, and HPK1, wherein the first and the second polypeptide sequences are heterologous to one another.

2. The recombinant polypeptide of claim 1, wherein (i) the first polypeptide sequence is derived from ITK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, ZAP70, PKC theta, LCK, and FYN.

3. The recombinant polypeptide of claim 1, wherein (i) the first polypeptide sequence is derived from SHP1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, LCK, FYN, and ZAP70.

4. The recombinant polypeptide of claim 1, wherein (i) the first polypeptide sequence is derived from c-Cbl and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of ZAP70, LCK, FYN, and SYK.

5. The recombinant polypeptide of claim 1, wherein (i) the first polypeptide sequence is derived from a protein selected from the group consisting of LCK, FYN, ZAP70, and SYK; and (ii) the second polypeptide sequence is derived from HPK1.

6. The recombinant polypeptide of any one of claims 1-5, wherein the recombinant polypeptide comprises a kinase activity.

7. The recombinant polypeptide of any one of claims 1-6, wherein the recombinant polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 31-46, 305, and 307.Attorney Docket No.: 048536-807001 WO8. A recombinant polypeptide comprising, in N- to C-terminal direction:(a) a first portion comprising a first polypeptide sequence derived from a protein selected from the group consisting of LAT and ITK; and(b) a second portion comprising a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, PKC theta, ZAP70, LCK, FYN, SYK, GRB2, GADS, SOS1, ADAP, PLCgl, ITK, VAV1, NCK1, AKT1, BTK, MAP4K3, PDK1, and PIK3R1 alpha, wherein the first and the second polypeptide sequences are heterologous to one another.

9. The recombinant polypeptide of claim 8, wherein (i) the first polypeptide sequence is derived from LAT and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, PKC theta, ZAP70, LCK, FYN, , SYK, GRB2, GADS, SOS1, ADAP, PLCgl, ITK, VAV1, NCK1, AKT1, BTK, MAP4K3, PDK1, and PIK3R1 alpha.

10. The recombinant polypeptide of claim 8, wherein (i) the first polypeptide sequence is derived from ITK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SYK, PKC theta, ZAP70, LCK, FYN, GRB2, GADS, SLP76, ADAP, NCK1, MAP4K3, and PIK3R1 alpha.

11. The recombinant polypeptide of any one of claims 8-10, wherein the recombinant polypeptide comprises a transmembrane domain or a domain capable of tethering the recombinant polypeptide to a plasma membrane.

12. The recombinant polypeptide of any one of claims 8-11, wherein the recombinant polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 47-77.

13. A recombinant polypeptide comprising, in N- to C-terminal direction:(a) a first portion comprising a first polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP; and(b) a second portion comprising a second polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP, wherein the first and the second polypeptide sequences are heterologous to one another.Attorney Docket No.: 048536-807001 WO14. The recombinant polypeptide of claim 13, further comprising a third polypeptide sequence derived from a protein selected from the group consisting of LAT, SLP76, and ADAP.

15. The recombinant polypeptide of any one of claims 13-14, wherein the recombinant polypeptide comprises one or more additional domains capable of mediating protein-protein interaction (e.g., thereby increasing valency).

16. The recombinant polypeptide of any one of claims 13-15, wherein the recombinant polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 78-89.

17. A recombinant polypeptide comprising, in N- to C-terminal direction:(a) a first portion comprising a first polypeptide sequence derived from a protein selected from the group consisting of GADS, MAP4K1, GRB2, SOS1, PLCgl, V AVI, and NCK1; and(b) a second portion comprising a second polypeptide sequence derived from a protein selected from the group consisting of SLP76, ITK, MAP4K1, NCK1, SOS 1, and VAV1, wherein the first and the second polypeptide sequences are heterologous to one another.

18. The recombinant polypeptide of claim 17, wherein (i) the first polypeptide sequence is derived from GADS and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, ITK, and MAP4K1.

19. The recombinant polypeptide of claim 17, wherein (i) the first polypeptide sequence is derived from MAP4K1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of MAP4K1, SOS1, and VAV1.

20. The recombinant polypeptide of claim 17, wherein (i) the first polypeptide sequence is derived from SOS1 and (ii) the second polypeptide sequence is derived from NCK1.

21. The recombinant polypeptide of claim 17, wherein (i) the first polypeptide sequence is derived from GRB2 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76 and NCK1.Attorney Docket No.: 048536-807001 WO22. The recombinant polypeptide of claim 17, wherein (i) the first polypeptide sequence is derived from PLCgl and (ii) the second polypeptide sequence is derived from SLP76.

23. The recombinant polypeptide of claim 17, wherein (i) the first polypeptide sequence is derived from VAV1 and (ii) the second polypeptide sequence is derived from SLP76.

24. The recombinant polypeptide of claim 17, wherein (i) the first polypeptide sequence is derived from NCK1 and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of SLP76, MAP4K1, and SOS1.

25. The recombinant polypeptide of any one of claims 17-24, wherein the recombinant polypeptide comprises a first additional domain capable of binding to protein(s) in the LAT / SLP76 signalosome (e.g., thereby conferring an “intra-signalosome adaptor activity”).

26. The recombinant polypeptide of any one of claims 17-25, wherein the recombinant polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 90-107.

27. The recombinant polypeptide of any one of claims 1-26, wherein the recombinant polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 41, 46, 54, 65, 68, 69, 77, 103, and 109.

28. A recombinant polypeptide comprising, in N- to C-terminal direction:(a) a first portion comprising a first polypeptide sequence derived from a protein selected from the group consisting of LCK and ZAP70; and(b) a second portion comprising a second polypeptide sequence derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, SOS1, VAV1, PLCgl, and NCK1, wherein the first and the second polypeptide sequences are heterologous to one another.

29. The recombinant polypeptide of claim 28, wherein (i) the first polypeptide sequence is derived from LCK and (ii) the second polypeptide sequence is derived from a protein selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, SOS1, VAV1, PLCgl, and NCK1.

30. The recombinant polypeptide of claim 28, wherein (i) the first polypeptide sequence is derived from ZAP70 and (ii) the second polypeptide sequence is derived from a proteinAttorney Docket No.: 048536-807001 WO selected from the group consisting of GADS, SLP76, ITK, MAP4K1, GRB2, SOS1, VAV1, PLCgl, and NCK1.

31. The recombinant polypeptide of any one of claims 28-30, wherein the recombinant polypeptide comprises ((i) a first additional domain capable of binding to protein(s) in the LAT / SLP76 signalosome, and (ii) a second additional domain capable of binding to protein(s) outside of the LAT / SLP76 signalosome (e.g., thereby conferring an “extra- signalosome adaptor” activity).

32. The recombinant polypeptide of any one of claims 28-31, wherein the recombinant polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 108-131.

33. A recombinant polypeptide comprising, in N- to C-terminal direction:(a) a first portion comprising a first polypeptide sequence derived from LAT; and(b) a second portion comprising a second polypeptide sequence derived from a protein selected from the group consisting of LP76, SOS1, MAP4K1, and ADAP, wherein the first and the second polypeptide sequences are heterologous to one another.

34. The recombinant polypeptide of claim 33, further comprising a third polypeptide sequence derived from a protein selected from the group consisting of MAP4K1, ADAP, SLP76, and SOS1.

35. The recombinant polypeptide of any one of claims 33-34, wherein the recombinant polypeptide comprises (i) a transmembrane domain, and (ii) one or more intrinsically disordered regions (IDRs).

36. The recombinant polypeptide of any one of claims 33-35, wherein the recombinant polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 132-143.

37. The recombinant polypeptide of any one of claims 1-36, further comprising an amino acid sequence for the 6th Ankyrin Repeat (AR6) of the protein IKBOI, or a functional variant thereof, wherein the AR6 sequence or functional variant thereof mediates a targeted ubiquitin-independent degradation.Attorney Docket No.: 048536-807001 WO38. The recombinant polypeptide of claim 37, wherein the AR6 sequence comprises or is a sequence selected from the group consisting of SEQ ID NOS: 297-299.

39. A recombinant nucleic acid comprising a nucleic acid sequence encoding a recombinant polypeptide of any one of claims 1-38.

40. The recombinant nucleic acid of claim 39, wherein the nucleic acid sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 174-286, 306, and 308.

41. The recombinant nucleic acid of any one of claims 39-40, wherein the nucleic acid sequence is operably linked to one or more heterologous nucleic acid sequence.

42. The recombinant nucleic acid of claim 41, wherein the one or more heterologous nucleic acid sequences comprises or is a nucleic acid control element.

43. The recombinant nucleic acid of claim 42, wherein the nucleic acid control element is selected from the group consisting of ribosomal binding sites, promoters, enhancer elements, activator elements, translational start sequences, translational termination sequences, transcription start sequences, transcription termination sequences, poly adenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, replication elements, RNA processing and export elements, transposon sequences, transposase sequences, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences, boundary elements, locus control regions (LCR), matrix attachment regions (MAR), recombination or cassette exchange sequences, or any combination thereof.

44. The recombinant nucleic acid of any one of claim 41-43, wherein the heterologous nucleic acid sequence comprises or is a promoter.

45. The recombinant nucleic acid of claim 44, wherein the promoter comprises or is an EFla promoter, CMV promoter, CAG promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CDl la promoter, CD58Attorney Docket No.: 048536-807001 WO promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD 161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T- bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL 17 A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte- activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, or NR4A3 promoter.

46. The recombinant nucleic acid of any one of claims 43-45, wherein the promoter is an endogenous promoter.

47. The recombinant nucleic acid of claim 46, wherein the endogenous promoter comprises or is a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, a CD3z promoter, a CARD9 promoter, a CARDIO promoter, a CARD 11 promoter, a CARD 14 promoter, or a PIK3R3 promoter, a B2 microglobulin (B2M) promoter, or a class II transactivator (CIITA) promoter.

48. The recombinant nucleic acid of claim 44, wherein the promoter is a synthetic promoter.

49. The recombinant nucleic acid of any one of claims 44-47, wherein the promoter is a constitutive promoter.Attorney Docket No.: 048536-807001 WO50. The recombinant nucleic acid of any one of claims 44-47, wherein the promoter is a regulatable promoter.

51. The recombinant nucleic acid of any one of claims 41-50, the one or more heterologous nucleic acid sequences comprises or is a coding sequence for a polypeptide of interest.

52. The recombinant nucleic acid of claim 51, wherein the polypeptide of interest comprises or is a peptide linker sequence, a cleavable linker sequence, a secretion signal, a resistance marker, an anchoring peptide, a localization signal, a fusion tag, an affinity tag, a chaperonin, a protease, or any combination thereof.

53. The recombinant nucleic acid of any one of claims 39-52, further comprising a barcode molecule that identifies that recombinant polypeptide.

54. The recombinant nucleic acid of any one of claims 39-53, further comprising nucleic acid sequences encoding for a ribosomal skipping peptide sequence.

55. The recombinant nucleic acid of claim 54, wherein the ribosomal skipping peptide comprises one or more ribosomal skipping peptide sequences from a porcine teschovirus- 1 2A (P2A), a calcium-dependent serine endoprotease (furin), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.

56. The recombinant nucleic acid of any one of claims 39-55, further comprising a second nucleic acid sequence encoding:(a) an engineered antigen receptor having specificity for a target ligand; and / or(b) a T cell receptor (TCR) having specificity for a target ligand.

57. The recombinant nucleic acid of claim 56, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR).

58. The recombinant nucleic acid of any one of claims 39-55, further comprising a response element operably incorporated upstream of the coding sequence of the recombinant polypeptide, wherein the response element comprises: a) a cognate target sequence to which a transcriptional regulator binds; and / or b) an engineered responsive promoter operably linked to the cognate target sequence.Attorney Docket No.: 048536-807001 WO59. The recombinant nucleic acid of any one of claims 39-57, wherein the recombinant nucleic acid is incorporated into an expression cassette or an expression vector.

60. A vector comprising the recombinant nucleic acid of any one of claims 39-59.

61. The vector of claim 60, wherein the vector is an expression vector.

62. The vector of any one of claims 60-61, wherein the vector is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a RNA vector, a mRNA vector, a phagemid vector, a viral vector, a self-replicating RNA virus, a mRNA-packaging virus-like particle, or a RNP-packaging virus-like particle.

63. The vector of claim 62, wherein the viral vector is a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector.

64. The vector of any one of claims 60-63, wherein the vector is formulated in a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a protein nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, a fusosome, an enveloped delivery vehicle, or an immune stimulating complex (ISCOM).

65. The vector of claim 64, wherein the LNP is a selective organ-targ eting LNP or an antibody-targeted LNP.

66. A recombinant cell comprising one or more of the following:(a) a recombinant polypeptide according to any one of claims 1-38;(b) a recombinant nucleic acid according to any one of claims 39-59; and(c) a vector according to any one of claims 60-65.

67. The recombinant cell of claim 66, wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell.

68. The recombinant cell of claim 67, wherein the recombinant cell is a eukaryotic cell.

69. The recombinant cell of claim 68, wherein the eukaryotic cell is a mammalian cell.

70. The recombinant cell of claim 69, wherein the mammalian cell is a human cell.

71. The recombinant cell of any one of claims 69-70, wherein the recombinant cell is a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a gamma delta T cell (y5T), anAttorney Docket No.: 048536-807001 WO invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, an engineered T cell comprising a transcriptional receptor, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, a naive T (TN) cell, a hematopoietic stem cell, or a progenitor cell of the lymphoid lineage.

72. The recombinant cell of any one of claims 69-71, wherein the recombinant cell is a stem cell derived cell.

73. The recombinant cell of any one of claims 69-72, wherein the recombinant cell has reduced / eliminated cell surface TCR expression, MHC-I expression, and / or MHC-II expression.

74. The recombinant cell of any one of claims 71-73, wherein the recombinant cell is a T cell.

75. The recombinant cell of claim 74, wherein the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell.

76. The recombinant cell of claim 75, wherein the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, bulk CD8+ T cells.

77. The recombinant cell of claim 75, wherein the CD4+ T helper lymphocyte cell is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

78. The recombinant cell of any one of claims 66-77, wherein the recombinant cell further comprises an additional recombinant nucleic acid molecule encoding:(a) an engineered antigen receptor having specificity for a target ligand; and / or.Attorney Docket No.: 048536-807001 WO(b) a T cell receptor (TCR) having specificity for a target ligand.

79. The recombinant cell of claim 78, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR).

80. The recombinant cell of claim 78, wherein the engineered antigen receptor is a hybrid- CAR receptor (Hybrid-R) comprising an intracellular domain (ICD) which comprises a transcriptional regulator, wherein binding of Hybrid-R to the target ligand results in cleavage of a ligand-inducible proteolytic cleavage site and releases of the ICD.

81. The recombinant cell of claim 78, wherein the release of the intracellular domain results in binding of the transcriptional regulator of the released intracellular domain to a cognate target sequence that is operably incorporated upstream of the first recombinant nucleic acid molecule, wherein the binding of the transcriptional regulator to the cognate target sequence modulates transcription of the recombinant polypeptide.

82. The recombinant cell of any one of claims 78-79, wherein the target ligand is expressed on a tumor cell.

83. The recombinant cell of any one of claims 78-82, wherein the target ligand is CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3e, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CDH17, CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B- cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, ROR1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPCAM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFRp, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2,Attorney Docket No.: 048536-807001 WO gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, R0PN1, GPRC5D, CX0RF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT- 1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NAU, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

84. The recombinant cell of any one of claims 71-79, wherein the recombinant nucleic acid and / or the additional recombinant nucleic acid molecule are / is inserted in a specific sites in the cell genome.

85. The recombinant cell of claim 84, wherein the specific sites in the cell genome is / are independently selected from a T-cell receptor (TCR) locus, a CD3 locus, a B2 microglobulin (B2M) locus, a class II transactivator (CIITA) locus, a TRAC locus, and a safe harbor locus.

86. The recombinant cell of claim 85, wherein the safe harbor locus is AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, or SHS231 locus.

87. The recombinant cell of any one of claims 71-79, wherein the expression of the recombinant polypeptide results in (i) increasing proliferative capacity, (ii) enhancing antigen- sensitivity, (iii) enhancing anti-tumor effect, (iv) altering T cell signaling through NF AT, NF-KB and / or AP-1 pathways, (v) altering cytokine or chemokine production, (vi) altering JAK / STAT signaling in T cells, (vii) altering co-stimulatory molecule signaling in T cells, (viii) altering RAS / MEK / ERK signaling in T cells, (ix) altering phospholipase C gamma signaling, (x) altering a transcription factor activity in T cells, and / or (xi) altering or enhancing in vivo persistence in tumors of the T cells comprising the recombinant polypeptide.Attorney Docket No.: 048536-807001 WO88. The recombinant cell of claim 87, wherein the expression of the recombinant polypeptide in a T cell promotes the cell’s in vivo proliferation.

89. The recombinant cell of any one of claims 71-79, wherein the recombinant T cell has increased proliferative capacity, enhanced antigen sensitivity, enhanced anti-tumor effect, enhanced replicative lifespan, reduced exhaustion, decreased replicative senescence, reduced dysfunction, enhanced persistence, and / or increase intratumoral presence in vivo.

90. A cell culture comprising at least one recombinant cell according to any one of claims 66-89 and a culture medium.

91. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following:(a) a recombinant polypeptide according to any one of claims 1-38;(b) a recombinant nucleic acid according to any one of claims 39-59;(c) a vector according to any one of claims 60-65; and(d) a recombinant cell according to any one of claims 66-89.

92. The pharmaceutical composition of claim 91, wherein the composition comprises a recombinant cell according to any one of claims 66-79, a pharmaceutically acceptable carrier.

93. A method of preparing / making a T cell for use in a cell therapy, the method comprising expressing in the T cell one or more of the following:(a) a recombinant polypeptide according to any one of claims 1-38;(b) a recombinant nucleic acid according to any one of claims 39-59; and(c) a vector according to any one of claims 60-65.

94. The method of claim 93, comprising genetically modifying the T cell for expression of the recombinant polypeptide.

95. The method of any one of claims 93-94, comprising introducing to the T cell a recombinant nucleic acid according to any one of claims 39-59, or a vector according to any one of claims 60-63.

96. The method of any one of claims 93-95, wherein the method comprises one or more of the following: a sequence specific nuclease, a nucleic acid programmable DNA bindingAttorney Docket No.: 048536-807001 WO protein, an RNA guided nuclease, an RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease- deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR- associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof.

97. The method of any one of claims 93-96, wherein the T cell is made using homology- directed repair (HDR) -mediated insertion.

98. The method of any one of claims 93-96, wherein the T cell is made in vivo, ex vivo, or in vitro.

99. The method of any one of claims 93-98, further comprising expressing in the T cell an engineered immune receptor that binds to a target ligand.

100. A method of preventing and / or treating a health condition in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the recombinant cells according to any one of claims 66-89.

101. The method of claim 100, wherein the recombinant cells are allogeneic relative to the subject.

102. The method of claim 100, wherein the recombinant cells are autologous relative to the subject.

103. The method of any one of claims 100-102, wherein the health condition is a proliferative disorder (e.g., cancers), an infectious disease, an autoimmune disease, and / or inflammatory disease.

104. The method of claim 103, wherein the proliferative disorder is a cancer.

105. The method of claim 104, wherein the cancer expresses a ligand selected from the group consisting of CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, Claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD72, CD97, CEA, Claudin 6(CLDN6), CLECL1, CS-1,Attorney Docket No.: 048536-807001 WODLL-3, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GCC, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-P, PRAME, PRSS21, PSCA, PSMA, R0R1, SIRPa, SSEA-4, STEAP2, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Ax.

106. The method of any one of claims 104-105, wherein the cancer is small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head and neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

107. A kit for (i) preparing / making a T cell for use in cell therapy or (ii) for preventing and / or treating a health condition in a subject in need thereof, the kit comprising:(a) a recombinant polypeptide according to any one of claims 1-38;(b) a recombinant nucleic acid according to any one of claims 39-59;(c) a vector according to any one of claims 60-65; and(d) a recombinant cell according to any one of claims 66-89.

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