Engineered immune proteins and uses thereof

Abstract

The present disclosure provides compositions and methods for improving cellular immunotherapy, such as against cancer, infection, autoimmune disease, or neurodegenerative disease. Disclosed embodiments include proteins for initiating, providing, boosting, and / or improving signaling in a host (e.g., T) cell that further expresses a target (e.g., antigen)-binding protein when the target-binding protein encounters the target. Some embodiments provide CD3 / costimulatory domain fusion proteins. In some embodiments, a fusion protein is provided that comprises a CD3, or a functional portion or variant thereof, and a costimulatory domain or a functional portion or variant thereof. Other embodiments comprise engineered CD3 polypeptides. Also provided are polynucleotides that encode a disclosed CD3 polypeptide or an engineered CD3 polypeptide, vectors that comprise the polynucleotides, and related host cells, compositions, and uses.

Description

[0001] ENGINEERED IMMUNE PROTEINS AND USES THEREOF

[0002] STATEMENT OF GOVERNMENT INTEREST

[0003] This invention was made with government support under CAI 14536 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (360056-516W0-SL.xml; Size: 647,168 bytes; and Date of Creation: November 5, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Adoptive transfer (ACT) of T cells expressing a Chimeric Antigen Receptor (CAR) has shown success in treating patients with B cell malignancies. However, tumor relapses following CAR-T cell therapy are frequent (Abramson, J. S., et al., Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. The Lancet, 2020396(10254): p. 839-852; Munshi, N. C., et al., Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. New England Journal of Medicine, 2021. 384(8): p. 705-716). Factors associated with tumor recurrence include: outgrowth of cancer cells with low levels or complete absence of the target antigen; CAR-T cell dysfunction or lack of persistence, and a suppressive TME (Cappell, K. M. and J. N.

[0008] Kochenderfer, Long-term outcomes following CAR T cell therapy: what we know so far. Nature Reviews Clinical Oncology, 2023. 20(6): p. 359-371; Lee, H., et al., Mechanisms of antigen escape from BCMA- or GPRC5D-targeted immunotherapies in multiple myeloma. Nature Medicine, 2023. 29(9): p. 2295-2306; Spiegel, J. Y., et al., CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nature Medicine, 2021. 27(8): p. 1419-1431). Chimeric TCRs (chTCRs, also called TCR / CARs) are a new modality for enabling antigen-specific targeting of T cells. Signaling and activation functions of engineered T cells, such as T cells comprising a chTCR, are of interest for therapeutic applications.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figures 1A-1D relate to certain embodiments “chimeric TCR” or “chTCR” (also referred to herein and shown in certain of the figures herein as “TCR / CAR”) constructs of the present disclosure. (A) Top left: schematic of TCR / CAR in "split-scFv" format (schematic of expression vector shown at bottom left) expressed at a cell membrane; top right: schematic of TCR / CAR in "full-scFv" format (schematic of expression vector shown at bottom right) expressed at a cell membrane. A non-limiting example of an expression product of a split-scFv TCR / CAR vector as illustrated is provided in SEQ ID NO.: 1. A non-limiting example of an expression product of a full-scFv TCR / CAR vector as illustrated is provided in SEQ ID NO.:3. As described further herein, TCR / CARs can include a(ny) target-binding domain (e.g., VH, VL, scFv, VHH, ligand, receptor ectodomain, fully synthetic (e.g., designed de novo) binding protein, or the like), such as for example a scFv comprising VH and VL from a tumor antigen-specific antibody.

[0011] In the illustrated "split-scFv" format, the VH and VL are not linked to one another by a peptide linker (as would be in for example a scFv) - though in some embodiments they may share one or more interchain disulfide bond-, but each is fused or linked to one of two TCR constant domains. For example, VH can be fused or linked to a T cell receptor beta-chain constant domain (TRBC) and VL can be fused or linked to a T cell receptor alpha chain-constant domain (TRAC), or VL can be fused or linked to a TRBC domain and VH can be fused or linked to a TRAC domain. Amino acid sequences of non-limiting examples of “split-scFv” expression products (vector-encoded amino acid sequence) are provided in SEQ ID NOs.: 1 (VL-TRBC VH-TRAC) and 2 (VH-TRBC VL-TRAC). In the illustrated “full-scFv” format, a scFv includes a linker and can be in VH-linker-VL or VL-linker-VH orientation, and the scFv can be fused to either TCR constant domain e.g. to TRAC or to TRBC) of a TCR constant domain dimer. Non-limiting examples of “full-scFv” expression products (vector-encoded amino acid sequence) are provided in SEQ ID NOs.:3-6. As shown in, for example, Figure 5C, each TCR constant domain can be fused to a binding domain, such as a scFv. Contemplated embodiments include polypeptide chains comprising two or more binding domains (e.g., scFv-linker-scFv-TCR constant domain). In embodiments comprising two or more scFvs (comprised in one or two polypeptide chains), two or more scFvs can be in the same orientation (e.g., two or more can be VH-linker-VL or VL-linker-VH) or one scFv can be in a VH-linker-VL and another scFv can be in VL-linker-VH. Likewise, a scFab can be VH-CH1 -linker- VL-CL or VL-CL-linker-VH-CH1, and two or more scFabs can be in the same or different orientations.

[0012] In the illustrated expression vectors, a signal peptide (SP; also called a leader sequence) is shown disposed at the N-terminal end of each polypeptide chain, and a furin cleavage sequence (RAKR, SEQ ID NO.: 131) and a Thoseaasigna virus 2 A (T2A) self-cleaving peptide (LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 134) separate the two polypeptides. An alternative 2A peptide sequence, such as a P2A self-cleaving peptide with N-terminal GSG linker (GSGATNFSLLKQAGDVEENPGP; SEQ ID NO.: 133), can be used. It will be understood that other self-cleaving peptides and cleavage (e.g. protease recognition) sequences may be used. Signal peptides are typically removed, in whole or in part, prior to expression of a polypeptide at a cell surface. TCR / CAR constructs can be delivered to host cells using, for example, a viral vector such as a lentiviral vector. A vector can include a promoter, such as, for example, an EFla promoter (SEQ ID NO.:7) or a MNDU3 promoter (SEQ ID NO.:8). (B) Expression of CARs and TCR / CARs in primary T cells with knockout of endogenous TCRa and TCRp. (C) Basal activation of TCRs, CD19 / 28z CAR, CD19 / BBz CAR and TCR / CARs in Jurkat NF AT reporter cells. (D) Lysis of (left) CD19-negative and (right) CD19+ target cells by cells expressing a CAR or TCR / CAR as indicated. Figure 2 shows that R0R1 -specific TCR / CARs exhibit better recognition at low antigen density than a R0R1 -specific CAR.

[0013] Figures 3A-3C relate to certain embodiments of CD226-based or TIGIT-based chimeric costimulatory receptors (“CCRs”) (also referred-to herein as immunomodulatory fusion proteins “IFPs”), which can be co-expressed with TCR / CARs of the present disclosure, or with, for example, TCRs or CARs. (A (left, right)) Schematics showing TCR / CARs and CCRs. The extracellular and optionally the transmembrane components can be from CD226 (left) or TIGIT (right), and the intracellular component can comprise, for example, a mutated CD226 endodomain or portion thereof, a CD2 endodomain or portion thereof (e.g., a truncated CD2 endodomain), a CD28 endodomain or portion thereof, or a 4- IBB endodomain or portion thereof. (B) Co-expression of TCR / CARs and CCRs in primary CD8 T cells with knock-out of endogenous TCRa, TCRP and TIGIT expression. (C) CD226 (left) and TIGIT (right) expression in T cells expressing CD19-specific TCR / CAR alone (WT) or in T cells engineered with TCR / CARs as in (B) and with different CCRs with a CD226 or TIGIT ectodomain.

[0014] Figure 4 shows embodiments of full-design and split-design TCR / CARs in association with CD3 proteins of a TCR complex at a cell membrane. ITAM = Immunoreceptor Tyrosinebased Activation Motif, (left) “Split-scFv” construct with antibody variable domains; (right) “full-scFv” construct. X = immunoreceptor tyrosine-based activation motifs (ITAMs) present in CD3 proteins. Figures 5A-5E relate to certain embodiments of TCR / CAR constructs of the present disclosure. (A) Schematic representation of an example of a “split-scFv” construct as an expression vector (bottom) and TCR / CAR expressed at a cell membrane (top). In this example, VH is comprised in a single chain fusion with the TRAC domain, and VL is comprised in a single chain fusion with the TRBC domain. (B) Schematic representation of an example of a “full-scFv” construct as an expression vector (bottom) and TCR / CAR expressed at a cell membrane (top). In this example, a VL-linker-VH scFv is comprised in a single chain fusion with TRAC. (C) Schematic represenation of an example of a “bi-specific format” construct as an expression vector (bottom) and as TCR / CAR expressed at a cell membrane (top). In this example, a different VL-linker-VH scFv is comprised in a single chain fusion with each of the TRAC and TRBC domains. Alternatively, a scFv can be VH-linker-VL, and different scFvs present in a TCR / CAR can have different orientations. Each scFv can be specific for a different target (e.g. tumor antigen) to recognize cells expressing either or both antigens, or can target the same epitope or can target different epitopes within the same target to provide greater avidity. In other embodiments, a mono-specific TCR / CAR can comprise one (or more) scFv fused or linked to each of two TCR constant domains, wherein the scFvs are the same or bind the same epitope. In other words, a “full-scFv” TCR / CAR can comprise two scFvs while having single-target specificity. A non-limiting example of an expression product according to the illustrated bispecific full-scFv TCR / CAR vector is provided in SEQ ID NO.:9. It will be understood that other binding domains, addtionally or alternatively to scFvs, may be employed in multi-specific (e.g., bispecific) or multivalent (e.g., bivalent) TCR / CARs. Any of the presently disclosed TCR / CAR constructs or IFP constructs can be co-expressed with one another, and / or with a transduction marker; a transduction marker may also function as a suicide switch e.g. targetable by an antibody or antigen-binding fragment that induces cell death of a cell expressing the suicide switch); non-limiting examples of suicide switch transduction markers include tEGFR, tCD19, tNGFR, or the like. (D) Schematic representations of “VHH-based” (also referred-to as “nanobody -based”) TCR / CARs shown with a VHH linked or fused to one, the other, or both TCR constant domains of a TCR constant domain dimer. For example, a VHH can be linked or fused to TRAC (left; e.g. vector-encoded amino acid sequence of SEQ ID NO.: 10, wherein two copies of a VHH are comprised in a polypeptide chain further comprising TRAC) or to TRBC (center; e.g. vector-encoded amino acid sequence of SEQ ID NO.: 11, wherein two copies of a VHH are comprised in a polypeptide chain further comprising TRBC), or a VHH can be linked or fused to each of TRAC and TRBC (right; e.g. vector-encoded amino acid sequence of SEQ ID NO.: 12, wherein two copies of a VHH are comprised in a polypeptide chain further comprising TRAC and two copies of a VHH are comprised in a polypeptide chain further comprising TRBC). In embodiments where two (or more) VHH are present, the two (or more) VHH can be the same or can be different, and if different, may have specificity for different targets or for different epitopes of the same target. (E) Schematic representation of “protein-based” TCR / CARs with a N-terminal HA-tag and Bcl-2 ectodomain linked or fused to TRAC (left; e.g. vector-encoded amino acid sequence of SEQ ID NO.: 13) or TRBC (right; e.g. vector-encoded amino acid sequence of SEQ ID NO.: 14). Not shown: schematic of embodiment wherein a Bcl-2 ectodomain (with N-terminal HA-tag) is linked or fused to each of TRAC and TRBC (e.g., vector-encoded amino acid sequence of SEQ ID NO.: 15). Figure 6 provides non-limiting examples of targets targeted using TCR / CARs of the present disclosure. Figure 7 shows a schematic of certain sequence modifications that can be used to improve chain pairing and stability of TCR / CARs. Shown is an embodiment of a “split-scFv” TCR / CAR with certain amino acid mutations to improve pairing efficiency between the two chains of the construct and to increase its stability when expressed at the cell surface. The upper “C” in TRAC represents a threonine to cysteine mutation at position 48 (T48C), and the lower “C” in TRBC represents a serine to cysteine mutation at postion 57 (S57C). The other “C”s (bottom in TRAC, top in TRBC) are native cysteines. The T48C and S57C mutations permit formation of novel disulfide bonds bewteen TRAC and TRBC. The “LVL” found in the diagram of the plasma membrane represents mutations in the TRAC transmembrane portion that introduce leucine (L), valine (V), and leucine (L) amino acid residues (described further herein). These three hydrophobic residues counterbalance instability. The positions of the LVL mutations within TRAC sequence are known (see Haga-Friedman et al., J Immunol 755:5538-5546 (2012)) and discussed further herein (see SEQ ID NOS.:57 and 63). TCR / CARs comprising TRAC and TRBC domains can include any or all of the above-mentioned pairing efficiency and stabilizing mutations, and / or can include other mutations as described herein. Additionally or alternatively, TCR / CARs may be expressed in T cells in which one or more endogenous TCR locus (e.g., TRAC, TRBC) is knocked-out to prevent mispairing between a TCR / CAR constant domain and a potential cognate endogenous TCR constant domain. Figures 8A-8C relate to certain embodiments in which endogenous TRAC and TRBC genes of T cells were targeted for knockout utilizing either CRISPR or base-editing technologies. (A) Schematic of experimental workflow (see e.g.

[0015] Kluesner et al., Nature Communications 12:2437 (2021)) to generate TCR / CAR T cells. The exemplified strategy includes activating bulk T cells, transducing the activated T cells with lentivirus encoding a TCR / CAR, and knocking out endogenous TRAC and / or TRBC genes using a CRISPR / Cas or base editor system. (B) Results of Base Editor knockout of endogenous TRAC and / or TRBC in primary T cells. sgRNA TRAC-1, sgRNA TRAC-2, sgRNA TRBC-1, and sgRNA TRBC-2 refer to different base editor sgRNAs targeting either the TRAC or TRBC locus. See SEQ ID NOS.:20-23. Each sgRNA was used individually (top row) or in the listed combinations (bottow row). (C) Summarizes efficiency of base-editing knockout (% of edited TCRs) in graphical format. sgRNA TRAC-1 is represented by the symbol al, sgRNA TRAC -2 is represented by the symbol a2, sgRNA TRBC-1 is represented by the symbol 1, and sgRNA TRBC-2 is represented by P2. Figures 9A and 9B relate to experiments for improving T cell transduction and base-editing efficiency. Timing and sequencing (ordering) of lentiviral transduction and base-editing was tested. (A) Six different tranduction and base-editing conditions were tested experimentally. DO, DI, D2, D3, and D6 refer to day zero (the time when T cells were placed into culture), day one, day two, day three, and day six of the culture period, respectively. “Td” represents the time, with reference to D0-D6, at which cells were transduced with lentiviral expression constructs. “BE” represents the time, with reference to D0-D6, at which base-editing was performed. (B) Cellular expression of TCR / CAR and knockout of endogeneous TRAC and / or TRBC measured by flow cytometry. Cells were stimulated on either day zero (top row) or day one (bottom row). Left panels: cells were transduced with lentivirus, rested for six hours, and then base-edited. Center panels: cells were base-edited, rested for six hours, and then transduced with lentivirus. Right panels: cells were transduced with lentivirus on day two and base-edited on day three. Figures 10A-10E relate to certain embodiments of TCR / CARs of the present disclosure. (A) Cell surface expression of CD19-specific CARs (with either CD28 costimulatory domain and CD3(^ effector domain or 4-1BB costimulatory domain and CD3(^ effector domain), a CD19-specific “split-scFv” TCR / CAR, and a CD19-specific “full-scFv” TCR / CAR in primary CD4+ and CD8+ T cells. TCR / CARs are expressed in a similar frequency as CARs in primary T cells. (B) Transduction percentage (top row) and expression (quantified as geometric mean rCD19) (bottom row) of the CAR and TCR / CAR constructs shown in Figure 10A, in primary CD4+ and CD8+ T cells. (C) Cell surface expression of ROR1-specific CARs, a ROR1 -specific “split-scFv” TCR / CAR, and a ROR1 -specific “full-scFv” TCR / CAR in primary CD4+ and CD8+ T cells. (D) Transduction percentage (top row) and expression (quantified as geometric mean rRORl) (bottom row) of the constructs shown in Figure 10C, in primary CD4+ and CD8+ T cells. (E) Cell surface expression of BCMA-specific CARs, a BCMA-specific “split-scFv” TCR / CAR, and a BCMA-specific “full-scFv” TCR / CAR in primary CD4+ and CD8+ T cells. These data show that TCR / CARs specific for different antigens and in either split-scFv or full-scFv format expressed efficiently in primary CD4+ and CD8+ T cells. Figure 11 shows data comparing cell surface expression levels of the indicated TCR / CARs when the encoding lentiviral vector contained either an EFla promoter or a MNDU3 promoter. The MNDU3 promoter provided increased cell surface TCR / CAR expression and frequency of T cells that express the TCR / CAR as compared to the EFla promoter. Figures 12A and 12B relate to certain embodiments of “bi-specific full-scFv” constructs recognizing different multiple myeloma antigens. (A) Schematic representations of “bi-specific full-scFv” constructs (top row). Expression of “bi-specific full-scFv” constructs (bottom row) in primary T cells. (B) Schematic representation of an anti-CD229 x anti-BCMA “bi-specific full-scFv” construct (far left panel), cell surface expression of the anti-CD229 x anti-BCMA “bi-specific full-scFv” construct (center left panel), binding by the anti-CD229 x anti-BCMA “bi-specific full-scFv” TCR / CAR to biotinylated BCMA (center right panel), and binding by the anti-CD229 x anti-BCMA “bi-specific full-scFv” TCR / CAR to biotinylated CD229 (far right panel). These data show that bispecific TCR / CARs, such as in full scFv format, can target different antigens and be expressed in primary T cells. Figure 13 relates to certain embodiments of “VHH-based” and “protein-based” constructs of the present disclosure. Left panel: expression (bottom) of an anti-RORl “VHH-based” TCR / CAR (schematic shown at top) in primary T cells. Right panel: expression of a “protein-based” TCR / CAR (schematic shown at top) in primary T cells. A “protein-based” TCR / CAR includes a TCR / CAR comprising a target-binding portion that is a native protein or portion thereof that is not an antigen-binding domain of an antibody and interacts with its native ligand (target). Figure 14A-14C: (A) Jurkat cells with a triple reporter system NFAT-eGFP, NFkB-CFP, AP-l-mCherry were unstimulated or were stimulated with PMA / Ionomycin demonstrating upregulation of reporter constructs with stimulation. (B) TCR, CAR, “split-scFv” TCR / CAR, and “full-scFv” TCR / CAR constructs were individually transduced into Jurkat NFAT-eGFP, NFkB-CFP, AP-l-mCherry triple reporter cells and the percentage of NF AT -reporter positive cells in unstimulated conditions was measured. TCRs, CARs, and TCR / CARs were specific for the indicated antigen. (C) TCR, CAR, “split-scFv” TCR / CAR, and “full-scFv” TCR / CAR constructs were individually transduced into Jurkat NFAT-eGFP, NFkB-CFP, AP-l-mCherry triple reporter cells and the percentage of NFkB-reporter positive cells in unstimulated conditions was measured. TCRs, CARs, and TCR / CARs were specific for the indicated antigen. These data show that TCR / CAR constructs induce minimal antigen-independent signaling. Figures 15A and 15B show results from western blot experiments. (A) Detection of TRAC or TRBC in cell lysate from R0R1 -specific TCR / CAR-positive T cells. Western blots were performed against TRAC (left) and TRBC (right) using total input samples or immunoprecipitation (IP) samples with biotinylated recombinant R0R1 protein coated on streptavidin-coated beads. (B) Detection of all 4 CD3 sub-unit proteins in cell lysate from R0R1 -specific TCR / CAR+ T cells after immunoprecipitation with biotinylated recombinant R0R1 protein coated on streptavidin-coated beads. These data show that unlike a CAR, TCR / CAR constructs assemble with all CD3 signaling complex proteins. Figure 16 shows cellular Ca2+flux following CD19 antigen stimulation of T cells transduced with a CD19-specific CAR comprising CD28 / CD3(^ or 4-lBB / CD3(^ signaling domains, or with a CD19-specific TCR / CAR of the present disclosure. These data show that T cells expressing TCR / CAR constructs flux Ca2+following antigen-specific stimulation as well as or better than T cells expressing comparator CARS. Figure 17 shows the percentage of killing of cancer cell lines by T cells transduced with CD19-specific CARs or CD19-specific TCR / CARs at various effector to target (E: T) ratios. K562 and Naim 6 refer to CD19-positive cancer cell lines. K562 CD19koand Naim 6 CD19korefer to engineered versions of K562 and Nalm6 cell lines, respectively, in which CD19 has been knocked-out. These data show that T cells expressing TCR / CARs specifically recognize and lyse tumor cells expressing a targeted antigen. Figure 18 shows cytokine production (IL-2; IFN-y) by T cells transduced with a CD19-specific CAR or a CD19-specific TCR / CAR, in response to antigen-positive and antigen-negative cell lines. Figure 19 shows proliferation, by T cells transduced with a CD19-specific CAR or a CD19-specific TCR / CAR, in response to various cell lines. Top, representative histograms from flow cytometry of T cells cultured in media (CTL) or with the indicated cell lines using Cell Trace Violet. Bottom left, percentage of divided T cells; bottom right, Geometric mean of CTV intensity. Figure 20 shows that TCR / CAR+ T cells possess higher functional avidity (measured as percentage of bound T cells) for their target than CAR+ T cells do for their target, as measured by z-movi, which determines the acoustic force required to disrupt binding of T cells to target cells. The tested TCR / CARs and CARs used antigen-binding domains from the same CD19-specific antibody. Figures 21 A and 21B show that TCR / CARs have superior antigen sensitivity as compared to CARs. (A) Response of T cells transduced with CD19-specific TCR / CARs or CD19-specific CARs to increasing concentrations of recombinant CD19 antigen (x-axis). Cellular reponse is monitored by measuring IL-2, TNF-a, and IFN-g cytokine concentration (y-axis). (B) Half-maximal effective concentration (EC50) measurements of CD19-specific CARs and CD19-specific TCR / CARs with respect to IL-2 and IFN-g production in response to antigen. Figure 22 provides a schematic illustrating an in vivo experiment in which CD19+ Raji GFP-FfLuc cells were infused into NSG mice. Seven days after administration of CD19+ Raji GFP-FfLuc cells to mice, equal numbers of CD4+ and CD8+ T cells transduced with CARs or TCR / CARs were infused into the mice. Growth of the Raji GFP-FfLuc cells was measured by bioluminescence (radiance) and the survival of the mice was monitored over time. These data show that TCR / CARs have antitumor efficacy in a NSG mouse model. Figures 23A-23E relate to certain IFPs (also called CCRs, chimeric costimulatory receptors) of the present disclosure. CD226 is an adhesion molecule that binds to CD155 and amplifies T cell functions following receptor triggering. CD226 is expressed on unstimulated T cells and can be gradually lost following chronic antigen stimulation, while TIGIT, which also binds CD155, is expressed following T cell activation and becomes constitutively expressed during T cell exhaustion (see, e.g. Ge etal., Front Immunol 2021;

[0016] doi.org / 10.3389 / fimmu.2021.699895). IFPs leveraging CD226 or TIGIT extracellular domain were constructed to potentially manipulate the PVR / TIGIT / CD226 signaling axis, where the common ligand for TIGIT and CD226, PVR aka CD155, is widely expressed and is often overexpressed by tumor cells. TIGIT has a higher affinity for PVR (approximately 100-fold) than does CD226. These IFPs are also referred-to herein as chimeric costimulatory receptors “CCRs”. Alternatively or additionally, endogenous TIGIT can be knocked-out in the T cell to avoid competition for ligand binding with the CCR. CCRs can be expressed in T cells (e.g. can be encoded by the same vector) in trans with TCR / CARs of the present disclosure, and function to provide a co-stimulatory signal to the cell. (A) Schematic representation of a CCR expressed in trans with a “split-scFv” TCR / CAR (top left) or with a “full-scFv” TCR / CAR (top right); (bottom) schematic representation of expression vector encoding a “split-scFv” TCR / CAR with a CCR (coding sequence shown as “Co-stim”). Tested constructs included an extracellular portion from CD226 or TIGIT, and an intracellular portion from a mutated CD226, or from CD2, truncated CD2, CD28, or 4-1BB. (B) Schematic illustration of a strategy for lentiviral transduction of T cells with expression TCR / CAR + CCR constructs of the present disclosure, along with a CRISPR or Base Editor knockout of endogenous TRAC, TRBC, and TIGIT genes. Eliminating endogenous TIGIT eliminates competition for ligand binding with the CCR and TIGIT-mediated inhibitor signaling, while the CCR provides a positive signal to the T cell. (C) Knockout of TIGIT expression in T cells using a base editor system, (first sheet) Indel frequency analysis in T cells edited for TIGIT using sgRNAs 5 and 7. (second sheet) TIGIT expression in non-edited T cells (non-targeting sgRNA) and T cells edited with the combination of TIGIT-targeting sgRNAs 5 and 7 measured by flow cytometry after overnight stimulation with anti-CD3 antibody. (D) TCR / CAR expression (split-scFv format, top; full-scFv format, bottom) assessed by anti-TCR antibody and in-house labelled recombinant CD19 protein in primary T cells transduced with TCR / CAR alone (leftmost panel, labeled “WT”) or co-transduced with different CCRs (other panels). The “CD226-based” CCRs tested in these experiments comprise a CCR ectodomain and either a: mutated CD226 intracellular domain; truncated CD2 intracellular domain; or 4-1BB intracellular domain, as indicated. The “TIGIT-based” CCRs tested in these experiments comprise a TIGIT ectodomain and either a: truncated CD2 intracellular domain; or 4-1BB intracellular domain, as indicated. (E) Shows expression of CD19-specific “split-scFv” and “full-scFv” TCR / CARs when a CCR is encoded in the same expression vector, as measured by percent positive cells and by geometric mean fluorescence intensity (MFI). Panels: frequency of TCR / CAR+ T cells (split-scFv format, 4 donors) transduced with a vector encoding the TCR / CAR alone or with the indicated CCR; geometric mean of recombinant CD19 protein (labelled with AF647) binding to TCR / CAR+ T cells in T cells co-expressing a CCR or not; frequency of TCR / CAR+ T cells (full-scFv format, 4 donors) transduced with a vector encoding the TCR / CAR alone or along with the indicated CCR; geometric mean of recombinant CD19 protein (labelled with AF647) binding to TCR / CAR+ T cells in T cells co-expressing a CCR or not. (D) and (E) show that TCR / CARs express well when a CCR is encoded in the same vector. Figures 24A-24D: (A) First sheet: Expression of CD226 and TIGIT in T cells transduced with TCR / CAR (split-scFv format) and the indicated CCR using either CD226 or TIGIT as the extracellular binding domain. Controls (two left-most panels) used knockout of endogenous TCR or of endogenous TCR and TIGIT. Second sheet: histograms of TIGIT and CD226 expression showing overexpression of these molecules in T cells transduced with chimeric co-stimulation molecules using CD226 or TIGIT as their extracellular binding domain. (B) Frequency of CD226+ in T cells transduced with a vector encoding the TCR / CAR alone or along with the indicated CCR (4 donors); geometric mean (MFI) of CD226 in TCR / CAR+ T cells in T cells co-expressing the indicated CCR; frequency of TIGIT+ in T cells transduced with a vector encoding the TCR / CAR alone or along with the indicated CCR (4 donors); geometric mean of TIGIT in TCR / CAR+ T cells in T cells co-expressing the indicated CCR. (C) (First sheet) Expression of CD226 and TIGIT in T cells transduced with TCR / CAR (full format) and the CCRs using CD226 or TIGIT ectodomain as their extracellular binding domain. (Second sheet) Histograms of TIGIT and CD226 expression showing overexpression of these molecules in T cells transduced with CCRs using CD226 or TIGIT ectodomain as their extracellular binding domain. (D) Frequency of CD226+ in T cells transduced with a vector encoding the TCR / CAR alone or the TCR / CAR along with the CCR (4 donors); geometric mean of CD226 in TCR / CAR+ T cells in T cells co-expressing the different CCRs; frequency of TIGIT+ in T cells transduced with a vector encoding the TCR / CAR alone or the TCR / CAR along with the CCR (4 donors); geometric mean of TIGIT in TCR / CAR+ T cells in T cells coexpressing the different CCRs. Figure 25 Four panels at left: concentration of IL-2 (top) and IFN-g (bottom) produced by TCR / CAR T cells (split-scFv format) co-cultured with Raji cell line WT (left) or overexpressing CD155 (right). Four panels at right: concentration of IL-2 (top) and IFN-g (bottom) produced by TCR / CAR T cells (full-scFv format) co-cultured with Raji cell line WT (left) or overexpressing CD155 (right). These data show that co-expression with CCRs increases cytokine production by TCR / CAR+ T cells in response to antigen-expressing tumor cells. Figure 26 shows (top) non-limiting examples of hinge sequences (and their substituent components) that can be present in a TCR / CAR of the present disclosure, between the targetbinding portion (e.g. scFv) and the TCR constant domain portion, and (bottom) that including the identified hinge sequence in a TCR / CAR does not compromise expression. “No hinge” refers to a construct without an additional hinge. Additional hinges and are tested and can be selected, combined, or engineered for preferred characteristics such as flexibility and length. Figures 27A-27C relate to experiments knocking-out endogenous T cell coreceptor functions to potentiate TCR / CAR sensitivity. Without wishing to be bound by theory, endogenous CD4 and CD8 co-receptors may sequester Lek. Knocking-out endogenous expression of these coreceptors may “free-up” Lek and enhance TCR / CAR sensitivity. (A) CD4 (left) and CD8 (right) gene editing using base editors along with TRAC and TRBC genes in TCR / CAR T cells. (B) CD8 gene editing using base editors along with TRAC and TRBC genes in T cells expressing TCR / CARs (left and center panels) or a TCR (right panel). TCR / CARs and TCR are specific for NY-ESO-1157-165 HLA-A2. (C) (First sheet) CD19-specific TCR / CAR expression in primary T cells also edited for CD8 co-receptor or not; (second sheet) NY-ESO-1 antigen: HLA tetramer binding in primary T cells expressing TCR / CARs or a TCR specific for NY-ESO-1 antigen: HLA and edited for CD8 co-receptor or not. These data show that modulation of T cell functions in co-receptor-knockout T cells can rely on dependency to MHC. In these experiments, antigenbinding was unaffected in MHC-independent-specific TCR / CARs, but is reduced in CD8-knockout-T cells expressing TCR / CARs and CARs specific for peptide: MHC (these TCR / CARs and CARs comprise a scFv specific for NY-ESO-1: HLA-A2), as it is for “conventional” TCRs (MHC-dependent binding). Figures 28A and 28B: (A) (left) Calcium flux measured by flow cytometry in CD19-specific CAR T cells edited for CD8 or not and stimulated with CD19 recombinant protein; (right) calcium flux measured by flow cytometry in CD19-specific TCR / CAR T cells edited for CD8 or not and stimulated with CD19 recombinant protein.

[0017] Calcium flux increased in T cells expressing CARs or TCR / CARs specific for MHC -independent antigens when the co-receptor was knocked-out. (B) (left) Calcium flux measured by flow cytometry in NY-ESO-1 -specific TCR T cells edited for CD8 and stimulated with NY-ESO-1 dextramer; (right) calcium flux measured by flow cytometry in NY-ESO-1 -specific TCR T cells edited for CD8 and stimulated with 0KT3. Calcium flux was abrogated in TCR T cells that are CD8-knockout when stimulated using MHC-peptide dextramer (MHC-dependent), but increased in TCR T cells that are CD8-knockout when stimulated through CD3 (MHC-independent).

[0018] These data show that T cells expressing a binding protein (TCR / CAR or CAR) with MHC-independent binding to antigen have increased sensitivity to antigen when the endogenous T cell co-receptor is knocked-out. Figures 29A-29Q show amino acid sequences encoded by certain TCR / CAR or TCR / CAR plus CCR-encoding constructs of the present disclosure. Figures 30A and 30B relate to certain embodiments of “bi-specific format” TCR / CAR constructs. (A) Schematic representations of bi-specific constructs as TCR / CARs expressed at a cell membrane. The two scFvs in each TCR / CAR construct have a different specificity. Illustrated “Format #1” and “Format #2” differ in that the scFvs have swapped positions: the scFv that is in a fusion with Ca in Format #1 is in a fusion with CP in Format #2, and the scFv that is in a fusion with CP in Format #1 is in a fusion with Ca in Format #2. In illustrated Formats #1 and #2, both scFvs have a (N-terminal to C-terminal) orientation of VH-linker-VL. In illustrated “Format #3”, both scFvs have a VL-linker-VH orientation. In illustrated “Format #4”, the scFv in a fusion with Ca has a VH-linker-VL oriendation and the scFv in a fusion with CP has a VL-linker-VH orientation. (B) Schematic representation of an expression vector encoding a TCR / CAR according to Format #1, and further encoding a truncated EGFR transduction marker. Figure 31 shows (top row) schematic representations of the bi-specific format TCR / CAR constructs in Figure 30 and expression of the constructs (bottom row) in T cells. Figures 32A and 32B relate to knockout of endogenous SLAMF7 gene in T cells utilizing cytidine base editing. (A) Histograms of SLAMF7 expression following knockout with sgRNA. sgRNAs 1, 2, 3, 4, and 5 are different base editor sgRNAs targeting the SLAMF7 locus. Co sgRNA is a control. (B) Percentage of SLAMF7+ T cells following base editing. Figures 33A and 33B relate to knockout of endogenous CD229 gene in T cells utilizing cytidine base editing. (A) Histograms of CD229 expression following knockout with sgRNA. sgRNAs 1, 2, and 3 are different base editor sgRNAs targeting the CD229 locus. sgRNAs 2+3 were also tested as a combination. (B) Percentage of CD229+ cells following base editing. Data in Figures 32A-33B support that endogenous SLAMF7 and CD229 can be knocked-out along with endogenous TCR chain genes using cytidine base editing to prevent fratricide lysis. Figures 34A and 34B show fold expansion and viability of T cells expressing bi-specific TCR / CAR constructs of the present disclosure. (A) Fold expansion (left) and cell viability (right) of T cells expressing a bi-specific (anti-BCMA x anti-SLAMF7) TCR / CAR as compared to that of T cells expressing a conventional anti-BCMA CAR (“BCMA BBz CAR”) or a conventional anti-SLAMF7 CAR (“SLAMF7 BBz CAR”). (B) Fold expansion (left) and viability (right) of T cells expressing a bi-specific (anti-CD229 x anti-BCMA) TCR / CAR as compared to that of T cells expressing a conventional anti-BCMA CAR (“BCMA BBz CAR”) or a conventional anti-CD229 CAR (“CD229 BBz CAR”). These data show that bispecific TCR / CARs expand as well as conventional CAR T cells and are viable. Figures 35A-35C relate to antigen bi-specificity and reactivity of certain TCR / CAR constructs of the present disclosure. (A) Expression of BCMA and SLAMF7 in INA-6 cell lines with a BCMA knock out (INA-6 BCMAko) or a SLAMF7 knock out (INA-6 SLAMF7ko), and wild-type INA-6 cell line (INA-6WT). (B) Cytokine production (IFN-y) by T cells transduced with a conventional anti-BCMA CAR (“BCMA BBz-CAR"), a conventional anti-SLAMF7 CAR (“SLAMF7 BBz-CAR”), or an anti-SLAMF7 x anti-BCMA bi-specific TCR / CAR, in response to the cell lines described in (A) and an unstimulated control. For each condition, the dots and bars are, from left to right, “BMC A BBz-CAR”, “SLAMF7 BBz-CAR”, “Bi-specific TCR / CAR”. (C) Proliferation, by T cells transduced with a conventional anti-BCMA CAR (“BCMA BBz-CAR"), a conventional anti-SLAMF7 CAR (“SLAMF7 BBz-CAR”), or an anti-SLAMF7 x anti-BCMA bi-specific TCR / CAR, in response to the cell lines described in (A). Top, representative histograms from flow cytometry of T cells. Bottom, Geometric mean of CTV (Cell Trace Violet) intensity. These data show that bispecific TCR / CARs demonstrate antigen bi-specificity and reactivity. For each condition, the dots and bars are, from left to right, “BMC A BBz-CAR”, “SLAMF7 BBz-CAR”, “Bi-specific TCR / CAR”. Figures 36A and 36B relate to antigen bi-specificity and reactivity of certain embodiments of bi-specific TCR / CAR constructs of the present disclosure. (A) Percentage of killing of the INA-6 cell lines depicted in Figure 35A by T cells transduced with a conventional anti-BCMA CAR (“BCMA BBz-CAR"), a conventional anti-SLAMF7 CAR (“SLAMF7 BBz-CAR”), or an anti-SLAMF7 x anti-BCMA TCR / CAR at various effector to target (E: T) ratios. (B) Graph shows percentage of killing of INA-6 cells at various ratios of INA-6 BCMAkoto INA-6 SLAMF7kocells by T cells transduced with a conventional anti-BCMA CAR (“BCMA BBz-CAR"), a conventional anti-SLAMF7 CAR (“SLAMF7 BBz-CAR”), or an anti-SLAMF7 x anti-BCMA TCR / CAR. Figures 37A and 37B relate to antigen sensitivity of certain embodiments of bi-specific TCR / CAR constructs of the present disclosure. (A) Schematic of an in vitro experiment in which various concentrations of recombinant BCMA (rBCMA) and / or recombinant SLAMF7 (rSLAMF7) were incubated (15 minutes) with T cells transduced with a conventional anti-BCMA CAR (“BCMA BBz-CAR"), a conventional anti-SLAMF7 CAR (“SLAMF7 BBz-CAR”), or an anti-SLAMF7 x anti-BCMA TCR / CAR construct (“Bi-specific TCR / CAR”), and intracellular staining for phosphorylated extracellular signal-regulated kinase (“pERK”) was performed. (B) shows the % of pERK+ T cells measured under the indicated conditions. These data show that bi-specific TCR / CARs demonstrate superior antigen sensitivity as compared to conventional CARs. Figures 38A and 38B relate to antigen sensitivity of certain embodiments of bi-specific TCR / CAR constructs of the present disclosure. (A) Schematic of an in vitro experiment in which various concentrations of recombinant BCMA (rBCMA) and / or recombinant SLAMF7 (rSLAMF7) were incubated (24 hours) with T cells transduced with a conventional anti-BCMA CAR (“BCMA BBz-CAR”), a conventional anti-SLAMF7 CAR (“SLAMF7 BBz-CAR”), or an anti-SLAM-F7 x anti-BCMA TCR / CAR construct (“Bi-specific TCR / CAR”), followed by ELISA to measure IFN-y levels. (B) Concentration of IFN-y (left) and % max IFN-y (right) produced by T cells transduced with the indicated conventional CAR or bi-specific TCR / CAR construct in the persence of antigen. These data show that bispecific TCR / CARs demonstrate superior antigen sensitivity as compared to conventional CARs.

[0019] Figures 39A and 39B relate to certain embodiments of chTCRs and associated signaling proteins. (A) Bottom: schematic of a multiprotein complex at a(n, e.g., T) cell membrane. The complex comprises: a chTCR in "split-scFv" format; a CD3(7costimulatory domain fusion protein comprising a full-length CD3(^ and, carboxy-terminal to the CD3(^ intracellular signaling domain, a 4-1BB costimulatory domain (CD3^_4-1BB); and (optionally endogenous) CD3s, CD36, and CD3y proteins. Top: schematic of an expression construct encoding the chTCR and the CD3^_4-1BB costimulatory domain fusion protein. EFla = promoter; P2A = non-limiting example of a self-cleaving peptide. It will be understood that a self-cleaving peptide sequence may be flanked immediately (e.g., at its N-terminal end) by a short linker, such as, for example, GS, GSG, GP, GPP, PGP, AAA, or the like. The short linker may be flanked (e.g., at its N-terminal end) by a protease cleavage site, such as a furin protease cleavage site, e.g, a RAKR sequence. (B) Bottom: schematic of the multiprotein complex as shown in (A), except that instead of the CD3(^ 4- I BB costimulatory domain fusion protein, a full-length CD3(^ (without a costimulatory domain) is present. The CD3(^ may be endogenously expressed by the host cell and / or can be encoded by a polynucleotide that is exogenous to the host cell and / or can comprise a CD3(^ amino acid sequnce that is exogenous to the host cell. In some embodiments, the host cell overexpresses CD3(^, such as by pairing endogenous expression of CD3(^ with exogenous expression of CD3(^, or by exogenous expression of CD3(^ at a level that is higher than the endogenous expression level of CD3(^. Top: schematic of an expression construct encoding the chTCR and the CD3i

[0020] Figures 40A-40B relate to experiments using expression constructs as in Figures 39A-39B, wherein the endogenous CD3(^ gene in a host T cell was knocked out utilizing cytidine base editing or otherwise manipulated. (A) Histograms of CD3(^ expression (BV421-A:: TCR as a proxy) in T cells following knockout using sgRNA; isotype control is shown at top. sgRNAs 1, 3, and 4 are different base editor sgRNAs targeting the CD3(^ locus and "Combo" is a combination of sgRNAs 1, 2, 3, and 4 targeting the CD3(^ locus. SgRNA sequences were:

[0021] (CD3z-l) AGTTCCTGCAGAAGAGGGCG (SEQ ID NO.:248); (CD3z-2) TCTCTGCTAGGAAAGACAAC (SEQ ID NO.:249); (CD3z-3) TTCTCTGCTAGGAAAGACAA (SEQ ID NO.:250; (CD3z-4) CAGGCAcAGUUGCCGAUUAC (SEQ ID NO.:251. "Iso" is an isotype control. "Scramble" refers an irrelevant sgRNA. For subsequent CD3(^ knockout experiments, sgRNA_4 was used.

[0022] (B) Left: schematic as in Figure 39A, wherein the chTCR and the CD3^_4-1BB costimulatory domain fusion protein are expressed in a host T cell in which endogenous CD3(^ has been knocked out. Without wishing to be bound by theory, in this setting, a homodimer of CD3(^_4-1BB costimulatory domain fusion proteins may form. Right: schematic as in the left image, except that in this schematic, endogenous CD3(^ has not been knocked out. Without wishing to be bound by theory, in the absence of knockout of endogenous CD3(^, heterodimers comprised of a CD3^_4-1BB costimulatory domain fusion protein and an endogenous CD3(^ may form (as shown), homodimers of endogenous CD3(^ may form (not shown), and / or homodimers of CD3(^-4- IBB costimulatory domain fusion proteins may form (not shown).

[0023] Figures 41A-41C relate to expression of a chTCR (anti-CD19 in split-scFv format), without or with exogenous CD3(^ or a CDSi^costimulatory domain fusion protein of the present disclosure, in T cells with knockout of endogenous TCR a-chain (TCRa) and TCR P-chain (TCRP), or with knockout of endogenous TCRa, TCRP, and CD3(^. (A) Expression of chTCR with knockout of endogenous TCRa and TCRp. (B) (L-R) Expression of: the chTCR; the chTCR when co-expressed with exogenous CD3(^; the chTCR when co-expressed with a CD3^_4-1BB costimulatory domain fusion protein; and the chTCR when co-expressed with a CD3(^_4-1BB BRM costimulatory domain fusion protein (wherein a native basic rich motif (SEQ ID NO.: 156) is absent from the 4-1BB costimulatory domain; “4-1BB BRM” is also designated as “4-1BBABRM” herein), with knockout of endogenous TCRa, TCRP, and CD3(^. In the TCRa / TCRp / CD3^ knockout setting, chTCR expression was higher when co-expressed with the CD3^_4-1BB_BRM costimulatory domain fusion protein as compared to when co-expressed the CD3^_4-1BB costimulatory domain fusion protein. (C) Expression of the anti-CD19 chTCR, reported as the (left) geometric mean of binding to recombinant CD19, (center) percentage of chTCR-positive cells, and (right) geometric mean of TCR. In each chart, the furthest left bar ("Split") shows anti-CD19 chTCR expression in T cells with knockout of endogenous TCRa and TCRP only, and the remaining bars (from second-left to right-most: “Split”; “+CD3z”;

[0024] “+CD3z_4-lBB”; and “+CD3z_4-lBBABRM” (z.e., +CD3z_4-lBBABRM) show anti-CD19 chTCR expression in T cells with knockout of endogenous TCRa, TCRP, and CD3(^, the chTCR being co-expressed with exogenous CD3(^ or with a CD3(7costimulatory domain fusion protein of the present disclosure as indicated. Figures 42A and 42B relate to expression of an anti-CD19 split-scFv chTCR in host T cells with knockout of endogenous TCRa and TCRP, and with or without co-expression of exogenous CD3(^ or a CD3(7costimulatory domain fusion protein. (A) Expression of chTCR when expressed alone or co-expressed with exogenous CD3(^ or with a CD3(7co-stimulatory domain fusion protein of the present disclosure (CD3^_4-1BB_BRM, CD3(^ fused to a CD28 costimulatory domain (CD3(^_CD28), CD3(^ fused to a CD226 costimulatory domain containing mutations as described herein (CD3(^_CD226AA), CD3(^ fused to a truncated CD2 costimulatory domain (CD3(^_CD2t), and CD3(^ fused to an 0X40 costimulatory domain (CD3^_OX40)), in T cells with knockout of endogenous TCRa and TCRp. (B) Shows (from left to right) expression of the anti-CD19 chTCR under the conditions as in (A), reported as shown on the y-axis.

[0025] Figures 43A-43D show function of T cells expressing an anti-CD19 chTCR (split-scFv format) alone or with exogenous CD3(^ or with a CD3(7costimulatory domain fusion protein of the present disclosure, in response to antigen. (A) Histograms showing CD19 expression in various Nalm-6 cell lines. Nalm-6 cells naturally express CD19. Nalm-6 CD19-knockout cells were generated, as well as Nalm-6 cells expressing low, mid, or high levels of CD19. The low, mid and high nomenclature refers to: <2000 CD19 molecules = Low; 2000 to 5000 CD19 molecules = Mid; >5000 CD19 molecules = high. In this experiment, the high measurement was 23999 CD19 molecules, the mid measurement was 2619 CD19 molecules, and the low measurement was 230 CD19 molecules. (B) Concentration of IL-2 produced by T cells transduced with vector encoding: anti-CD19 chTCR alone; anti-CD19 chTCR and CD3(^; or anti-CD19 chTCR and CD3^_4-1BBABRM, and co-cultured with (left) CD19hlghNalm-6 cells or (right) CD19lowNalm-6 cells. T cells transduced with anti-CD19 chTCR and CD3(^_4-1BBABRM show increased cytokine production as compared to T cells transduced with anti-CD19 chTCR alone and as compared to T cells transduced with anti-CD19 chTCR and CD3(^. (C) T cell proliferation following co-culture of CD19hlghNalm-6 cells (see (A)) with T cells transduced with: anti-CD19 chTCR; anti-CD19 chTCR and CD3(^; or anti-CD19 chTCR and CD3^_4-1BBABRM. Proliferation was measured using Cell Trace Violet (CTV) and is shown in histograms (left) and by percentage of CTV-low cells (right). (D) As described in (C), except that in this experiment, transduced T cells were co-cultured with CD19lowNalm-6 cells (see (A).

[0026] After co-culture with CD19lowNalm-6 cells, T cells transduced with anti-CD19 chTCR and CD3^_4-1BBABRM show increased proliferation as compared to T cells transduced with anti-CD19 chTCR alone and as compared to T cells transduced with anti-CD19 chTCR and CD3(^.

[0027] Figure 44 summarizes costimulatory domains used to construct certain CD3(7costimulatory domain fusion proteins. For some costimulatory domains, a native basic rich motif (BRM) is deleted. Fusion proteins comprising full-length CD3s (instead of CD3Q and a costimulatory domain are also prepared and tested.

[0028] Additional studies are conducted that: assess association of CD3 / costimulatory domain fusion proteins with chCTR by biochemical analysis; assess the effect of deleting a native BRM on expression on other CD3 / costimulatory domain fusion proteins; assess, in in vitro co-culture studies, effects of various co-stimulatory domain-containing fusion proteins on chTCR functions (including maintenance of function / protection from exhaustion); assess, in in vitro co-culture assays with antigen-low target cells the effects of various co-stimulation domains on antigen sensitivity of chTCRs; validate findings in in vivo studies; assess effects of CD3 / costimulatory domain fusion proteins on additional chTCR formats.

[0029] Figure 45 shows amino acid sequences related to certain CD3 / costimulatory domain fusion proteins of the present disclosure. The CD3 / costimulatory domain fusion proteins can be encoded by a polynucleotide and / or vector of the present disclosure and / or can be encoded and / or expressed by a host (e.g., T) cell of the present disclosure. The polypeptide sequences shown include a N-terminal signal peptide. It will be understood that a CD3 / costimulatory domain fusion protein can comprise all, part, or none of a signal peptide (e.g., a signal peptide present in the encoded amino acid sequence may be partly or fully cleaved during protein maturation). Moreover, if a signal peptide is present, it may be or comprise a signal peptide that is native to the protein or fusion protein (or to a portion, such as the extracellular portion, of a fusion protein), may comprise a signal peptide that is native to a different protein, and / or may comprise an engineered signal peptide. Accordingly, disclosed signal peptides and amino acid sequences comprising the same are non-limiting, and disclosed embodiments include amino acid sequences without a signal peptide, with a portion of a signal peptide, with a native signal peptide, with a non-native signal peptide, and / or with an engineered signal peptide. In some embodiments, an encoded CD3 / costimulatory domain fusion protein comprises a GM-CSF signal peptide.

[0030] Figure 46 shows histograms showing CD19 expression in various Nalm-6 cell lines. Nalm-6 cells naturally express CD19. Nalm-6 CD19-knockout cells were generated, as well as Nalm-6 cells expressing low, mid, or high levels of CD19. CD19 expression was quantified by number of CD19 molecules on the cell surface, as measured by Quantibrite™ beads.

[0031] Figure 47 shows amino acid sequences related to certain polypeptides of the present disclosure. The sequences in Figure 47 labeled “CD3z-CD226(BRMdeletion)” and “CD2z-CD2 (BRM deletion)” are CD3 / costimulatory domain fusion protein sequences. The other sequences in Figure 47 are engineered CD3 polypeptide sequences. The engineered CD3 polypeptides can be encoded by a polynucleotide and / or vector of the present disclosure and / or can be encoded and / or expressed by a host (e.g., T) cell of the present disclosure. The polypeptide sequences shown include a N-terminal signal peptide. It will be understood that an engineered CD3 polypeptide can comprise all, part, or none of a signal peptide (e.g., a signal peptide present in the encoded amino acid sequence may be partly or fully cleaved during protein maturation). Moreover, if a signal peptide is present, it may be or comprise a signal peptide that is native to polypeptide (or to a portion, such as an extracellular component, of a polypeptide), may comprise a signal peptide that is native to a different protein, and / or may comprise an engineered signal peptide. Accordingly, disclosed signal peptides and amino acid sequences comprising the same are non-limiting, and disclosed embodiments include amino acid sequences without a signal peptide, with a portion of a signal peptide, with a native signal peptide, with a non-native signal peptide, and / or with an engineered signal peptide. In some embodiments, an encoded engineered CD3 polypeptide comprises a GM-CSF signal peptide.

[0032] Figure 48 shows certain sgRNA sequences for use in gene editing, such as using a base editing system (e.g., a cytosine base editing system) and / or a CRISPR / Cas system. To the left of each sgRNA nucleotide sequence is the name of the sgRNA, which name indicates the target gene. “CD3z” sgRNAs target CD3 “CD3e” and “CD3E” sgRNAs target CD3s; “CD3d” and “CD3D” sgRNAs target CD3s; and “CD247” sgRNAs target CD247 (CD3Q.

[0033] Figures 49A-49D relate to certain embodiments of chTCR constructs of the present disclosure. (A) Left: schematic of a chTCR in “Split ChTCR” format and in “Full ChTCR” format, as indicated. Right: result of base editor knockout of endogenous TCRa and TCRP in T cells, measured by loss of CD3 and TCR expression. Right panel shows >90% knockout of endogenous TCRa and TCRP in the “TRAC+TRBC KO” condition. “Control sgRNA” included as a negative control. (B) Cell surface expression of: a CD19-specific CD19-CD28-CD3(^ (“CD19-28z”) single-chain chimeric antigen receptor (CAR); a CD19-specific CD19-4-1BB-CD3< (“CD19-BBz”) CAR; a CD19-specific “split chTCR”; and a CD19-specific “Full chTCR”, measured by staining with recombinant CD19 and anti-TCRaP antibody. (C) Left: representative images from western blots for LAT pTyr200, LAT, and Actin in T cells expressing the indicated receptor after co-culture with Nalm-6 CD19lowcells. Right: heat map showing level of phosphorylated LAT at amino acid Y220 (pLAT Y220) in T cells expressing the indicated anti-CD19 receptor and activated with CD19 antigen. The scale at right shows heat (shading) reflecting fold-change versus T. LAT (linker for activation of T cells). (D) Representative TIRF microscopy images of T cells expressing the indicated anti-CD19 receptor interacting with a soluble lipid bilayer functionalized with soluble CD19, ICAM-1, and CD45. Scale bars = 10pm.

[0034] Figure 50 Far left: histogram showing CD19 expression levels on Nalm-6CD19kocells, Nalm-6Lowcells, Nalm-6Mldcells, and Nalm-6Hlghcells. Center-left: histogram showing proliferation of T cells expressing the indicated receptor, using Cell Trace Violet (CTV), in response to CD19-expressing Nalm-6lowcells. Center-right: percent of divided cells for T cells expressing the receptor indicated in the center-left panel, in response to CD19-expressing Nalm-6lowcells. Far-right: IL-2 production by T cells expressing a receptor as indicated in the leftcenter panel, in reponse to CD19-expressing Nalm-6lowcells. *p<.05; **p<.01; ***p< 001.

[0035] Figure 51A shows: (left) cartoon showing a CD19 / CD22 bispecific chTCR (Bi-ChTCR) in complex with CD3 proteins at a cell membrane. The Bi-ChTCR comprises an anti-CD19 scFv (VL-linker-VH orientation; comprising VL and VH amino acid sequences from anti-CD19 antibody FMC63) fused to TCR Ca, and an anti-CD22 scFv (VL-linker-VH orientation; comprising VL and VH amino acid sequences from anti-CD22 antibody 9A8) fused to TCR CP; (center) Representative bioluminescence images of NSG mice engrafted with a mixture of Nalm-6 GFP-FfLuc tumor cells (33% CD19+CD22+; 33% CD19KOCD22+; 33% CD19+CD22KO) at DO and treated at D4 with T cells expressing the indicated receptor. Figure 51B shows survival analysis of the mice. Figure 51C shows: (left) IL-2 production by Bi-ChTCR-T co-cultured for 24h with Nalm-6high(CD19highCD22high) or Nalm-6Low(CD19lowCD22low) cells; (right) bioluminescence analysis of NSG mice engrafted with a mixture (50% Nalm-6hlghand 50% Nalm-6Low) of Nalm-6 cells and treated with (1) a mixture of CD19 CAR-T cells and CD22 CAR-T cells or (2) Bi-ChTCR-T cells. Figure 51D shows survival analysis of the mice.

[0036] Figure 52A shows: (upper left) expression construct encoding “full scFv” anti-CD19 chTCR and CD3^_4-1BBABRM fusion protein; the expression construct can, for example, be comprised in a lentivirus; (lower left) histograms showing expression of endogenous CD3(^ in primary T cells in which base-editing was performed on endogenous CD3(^ (the bottom histogram), or in which cells received an irrelevant sgRNA (“Scramble”) or isotype control; (lower middle) IL-2 production by T cells expressing an anti-CD19 chTCR in “split” format, or the chTCR with exogenous CD3(^, or the chTCR with a CD3^_4-1BBABRM fusion protein, after co-culture with Nalm-6lowcells; (right) cartoons showing a chTCR in full format at a T cell membrane, co-expressed with a CD3^_4-1BBABRM fusion protein and under conditions of: endogenous TRAC and TRBC knockout but endogenous CD3(^ left intact, or with knockout of endogenous TRAC, TRBC, and CD3(^, as indicated. In the cartoon at left (endogenous TRAC and TRBC knockout but endogenous CD3(^ left intact), a CD3^_4-1BBABRM fusion protein is shown along with an endogenous CD3(^. It will be understood that where endogenous CD3(^ is left intact and a chTCR is co-expressed with a CD3^_4-1BBABRM fusion protein, the chTCR may associate with a CD3^_4-1BBABRM fusion protein and an endogenous CD3(^, or with two CD3^_4-1BBABRM fusion proteins (as shown at right), or with two endogenous CD3(^ proteins.

[0037] Figure 52B provides flow cytometry plots showing: (top) expression of anti-CD19 chTCR (“full” scFv format) alone, with an unmodified exogenous CD3(^, or with a CD3^_4-1BBABRM fusion protein, in primary T cells, binding recombinant CD19; and (bottom) expression of anti-CD19 chTCR (“full” scFv format) with a CD3^_28ABRM fusion protein or with a CD3(^_4- 1BBABRM M1 fusion protein, in primary T cells, binding recombinant CD19.

[0038] Figure 53 provides a summary of certain engineered CD3 complex subunit polypeptides (engineered human CD3(^, CD3s, CD3y, CD36) of the present disclosure with engineering in one or more immunoreceptor tyrosine-based activation motif (ITAMs). The column labeled “Wildtype format” summarizes native tyrosines in the ITAM(s) present in the wild-type version of the indicated human CD3 protein, and the column labeled “Mutants” shows summarizes variants of the indicated CD3 protein in which one or more tyrosine in one (or more, in the case of CD3 tyrosine was substituted with a phenylalanine. It will be understood that a host (e.g., T cell) can be modified to express a express an engineered CD3 polypeptide, optionally with a targetbinding protein such as, for example, a chTCR, a HIT receptor (see, e.g., Mansilla-Soto, J., et al., HLA-independent T cell receptors for targeting tumors with low antigen density. Nature Medicine, 2022), a STAR receptor or a mutSTAR receptor (see, e.g., Liu et al., Sci. Transl. Med.

[0039] 13, eabb5191 (2021)), a CAR, a TCR, or a scTCR. As described herein, an expression construct (e.g., comprised in a lentiviral vector) encoding an IT AM-engineered CD3 polypeptide of the present disclosure can further encode a target-binding protein. In some embodiments, a vector, such as a lentiviral vector, can encode a chTCR, such as a bispecific chTCR, and an engineered CD3 polypeptide.

[0040] Figures 54A and 54B relate to studies using cytometry Time of Flight (CyTOF) to analyze signaling and phenotype of anti-CD19 (monospecific) chTCR-T cells longitudinally (0, 2, 5, 10 and 45 minutes, 6, 24, 48 and 72 hours) following co-culture with Nalm-6Hlghcells. Figure 54A: (Upper left) The antibody panel for CyTOF included markers to measure activation of major signaling pathways and to profile T cell metabolism, phenotype and proliferation. (Upper right) Schematic of the CyTOF methodology. Purified chTCR-T cells were stained with CFSE proliferation dye and 0.5xl0A6 chTCR-T cells were co-cultured with 0.5xl0A6 Nalm-6Hlghcells in triplicates. After each incubation period, cells were immediately fixed and cryopreserved to be stained simultaneously at the end of the 72hr experiment. Cells were then barcoded and samples were combined before staining with surface markers, permeabilization, and staining with intracellular markers. Samples were assayed and data acquired on a Helios mass cytometer. Data from all timepoints were integrated and projected after dimensionality-reduction to visualize subsets of interest. Pseudo-time diffusion of the different markers was applied using cell division as an internal reference. (Lower right) DREMI analysis showing dependency on pCD3(^ activation for LAT activation in CD19 ChTCR stimulated with Nalm-6Hlghcells at indicated timepoints. Figure 54B: Signaling network analysis using Earth Mover’s Distance (EMD) values in anti-CD19 ChTCR and anti-CD19 CD28z CAR stimulated with Nalm-6Hlghcells for 10 minutes. EMD was used to quantify absolute signaling strength for each analyte accounting for baseline marker expression. Network analysis of signaling pathways was applied using EMD values. This method allows for integration of early signaling events with consequences on T cell phenotype, metabolism and proliferation at the single-cell level providing for a unique understanding of how receptor design translates into T cell functions. Experiments comparing monospecific CD19 chTCR-T cells and monospecific CD19_CD28z CAR-T cells showed higher activation of LAT and Zap70 for the ChTCR-T cells.

[0041] Figure 55 relates to a screen assessing activity of CD3 / costimulatory domain fusion proteins in T cells expressing a bispecific chTCR (Bi-ChTCR). A library of multi ci stronic lentiviral vectors is synthesized encoding the anti-CD19 / anti-CD22 Bi-ChTCR together with CD3 / costimulatory domain fusion proteins, each containing a unique barcode. The library contains constructs with costimulatory signaling domains fused to CD3(^ or CD3s, as both CD3(^ and CD3s are present in two copies in a Bi-ChTCR / CD3 complex. Fusion proteins comprising an intracellular domain from CD2 (full-length or truncated, as provided herein), CD27, CD28 (comprising native dileucine motif or diglycine motif in place of dileuceine motif), 4-1BB, 0X40, ICOS, or CD226 (wild-type or mutant as provided herein), optionally without BRM domains, are tested. Also included are 4-1BBABRM M1 and 4-1BBABRM M1 M1 domains. Constructs using the Bi-ChTCR alone, with unmodified CD3(^ or CD3s or containing only a barcode, serve as controls. Lentivirus is produced for each of the constructs, concentrated and snap frozen. CD8+ T cells are transduced with previously titrated individual lentiviral vectors before TRAC and TRBC knockout by cytidine base editing. In addition, endogenous CD3(^ or CD3s are knocked-out depending on the CD3 subunit (CD3^ or CD3s) used in the vector to maximize the number of Bi-ChTCR complexes that assemble with the CD3 / costimulatory domain fusion protein. Each construct is transduced into Bi-ChTCR-T cells and cells expressing the constructs are purified by flow cytometry, rested for 3 days, harvested, counted and pooled in equal numbers for in vitro and in vivo assays using Nalm-6Lowtumors to discern improvement in function.

[0042] In vitro repetitive cytotoxic assays are performed. Pooled Bi-ChTCR-T cells are repetitively stimulated with NLR Nalm-6Lowtumor and cell killing is imaged using an Incucyte assay. T cells from the same donor and expressing the Bi-ChTCR without any CD3 / costimulatory domain fusion protein are included in the assay to provide a baseline for target cell killing. Before every restimulation, an aliquot of Bi-ChTCR-T is centrifuged, supernatant is removed and the cell pellet is frozen at -80°C. The repetitive stimulation assay is terminated when the Bi-ChTCR-T cells without CD3 / costimulatory domain fusion protein lose the ability to kill Nalm-6Lowcells.

[0043] Pooled Bi-ChTCR-T cells are then harvested, a fraction is pelleted and cryopreserved and a second fraction is restimulated with Nalm-6Lowcells at 1: 1 ratio in presence of brefeldin A for 6h. Restimulated Bi-ChTCR-T is stained intracellularly for IL-2 and the cells expressing IL-2 are flow sorted, pelleted and cyropreserved.

[0044] Genomic DNA is extracted from frozen cell pellets and the barcodes are amplified by PCR. Illumina barcodes and adapters are added by another PCR amplification before sequencing. For each type of CD3 / costimulatory domain fusion protein, the frequency of the fusion protein within the T cell population is then determined relative to the library composition before the assay at each restimulation timepoint, at endpoint, and within the IL-2 producing Bi-ChTCR-T cells.

[0045] An in vivo Nalm-6 stress test model is used to determine if the same CD3 / costimulatory domain fusion proteins that sustain Bi-ChTCR-T function in vitro also improve persistence and anti-tumor activity in vivo. This model is aggressive and ChTCR anti-tumor activity can be titrated with T cell dose. A pool of CD8+ Bi-ChTCR-T cells collectively expressing each of the CD3 / costimulatory domain fusion proteins as described above is generated. A putatively subcurative T cell dose (IxlO6Bi-ChTCR-T cells total) is transferred to NSG mice engrafted with 0.5xl0A6 GFP-FfLuc Nalm-6lowcells 3 days prior. An aliquot of Bi-ChTCR-T cells before injection is harvested, pelleted and frozen. Bone marrow is harvested from 3 mice at D7, 14 and 21. Bi-ChTCR-T cells are isolated from bone marrow, flow sorted, pelleted and frozen at each time point. Genomic DNA is extracted from all samples before PCR amplification and sequencing. Bi-ChTCR-T cells with CD3 / costimulatory domain fusion proteins that are enriched in tumors are determined related to baseline. Together, these in vitro and in vivo studies identify, using an unbiased approach, CD3 / costimulatory domain fusion protein(s) effective at augmenting Bi-ChTCR-T cell functions. Variants are selected that meet at least two, and preferably three, of the following criteria: enriched after repetitive stimulation; maintained IL-2 production; preferentially persist in tumors in vivo.

[0046] CD3 / costimulatory domain fusion proteins are selected for individual validation. In vitro experiments are performed in 3 independent donors in parallel to ensure reproducibility of the findings. After DNA libray sequencing, FASTQ files are adapter-trimmed, deduplicated and aligned using a python script. CD3 / costimulatory domain fusion proteins are identified and counted using DESeq2 and a R scripts. Relative frequency for each CD3 / costimulatory domain fusion protein over time and within the IL-2 producing population is determined based on the average fold change in library abundance from baseline before stimulation. CD3 / costimulatory domain fusion proteins’ enrichment is compared using False Discovery Rate approach adjusted for multiple P values derived from a linear mixed effects model. Bi-ChTCR-T from 2 independent donors are used for in vivo experiments. CD3 / costimulatory domain fusion proteins enriching Bi-ChTCR-T cells in bone marrow at D7, 14 and 21 are identified and counted as described for in vitro experiments.

[0047] The pooled screen identifies candidate CD3 / costimulatory domain fusion proteins that provide co-stimulation in Bi-ChTCR-T cells. To avoid potential confounding effects of a mixture of cells expressing different CD3 / costimulatory domain fusion proteins, each construct enriched in the experiments described above is independently validated for function. To do this, Bi-ChTCR-T cells expressing candidate CD3 / costimulatory domain fusion proteins are generated. Bi-ChTCR-T cells with unmodified CD3(^ or CD3s are used for comparison. The expression level of the Bi-ChTCR (staining for TCRaP and binding to recombinant CD19 protein) are compared for each construct by flow cytometry on day 7. Bi-ChTCR-T cells are purified by flow cytometry for analysis.

[0048] Adding costimulation to CARs can result in tonic signaling that drives toxicities and T cell dysfunction. To evaluate whether the candidate CD3 / costimulatory domain fusion proteins increase tonic signaling in Bi-ChTCR-T cells, vectors encoding the fusion proteisn are expressed in a Jurkat TCRaP KO cell line with 3 reporter genes for T cell activation (NFAT-eGFP, NFkB-CFP and AP-l-mCherry) and reporter induction is measured as a proxy for tonic signaling.

[0049] Vector designs for which Bi-ChTCR expression level is high and with no or minimal evidence for tonic signaling are selected for functional evaluation.

[0050] Repetitive in vitro stimulation: A repetitive cytotoxic assay is used to serially restimulate Bi-ChTCR-T cells expressing individual CD3 / costimulatory domain fusion proteins with NLR Nalm-6Lowcells and validate whether these candidates extend the duration that Bi-ChTCR-T cells maintain cytotoxic function compared to Bi-ChTCR-T cells expressing unmodified CD3(^ or CD3s. Natural co-stimulation molecules differ in the signaling pathways they alter in T cells and can augment different T cell function(s). Similarly, CD3 / costimulatory domain fusion proteins may engage different signaling pathways at activation. To delineate how each CD3 / costimulatory domain fusion protein contributes to augmenting anti-tumor functions, Bi-ChTCR-T cell proliferation, phenotype (differentiation and activation / exhaustion) and cytokine production are also analyzed during the restimulation assay. Bi-ChTCR-T cells are harvested between each restimulation and a fraction of the harvested cells are analyzed by spectral flow cytometry to define T cell differentiation (CD45RA, CCR7, CD62L), activation status and expression of inhibitory receptors (CD25, PD-1, TIM-3, TIGIT, LAG-3 and CD39). Counting beads are added to determine absolute Bi-ChTCR-T cell counts and fold expansion for each CD3 / costimulatory domain fusion protein is determined. Culture supernatants are collected 24h after starting each restimulation and cytokines production (IL-2 and IFN-y) analyzed by ELISA.

[0051] In vivo anti-tumor activity: Selected combinations of Bi-ChTCR: CD3 / costimulatory domain fusion protein that show improved function compared to Bi-ChTCR T cells with unmodified CD3 subunit in the repetitive stimulation assay are tested for in vivo antitumor efficacy against Nalm-6Lowcells. NSG mice are engrafted with 0.5xl06GFP-FfLuc Nalm-6Lowcells and treated 3 days later with IxlO6Bi-ChTCR-expressing or Bi-ChTCR: CD3 / costimulatory domain fusion protein-expressing T cells. Anti-tumor activity is monitored by bioluminescence and mice are followed for survival. Bone marrow is harvested at Day 14 (D14) from 3 mice per group to determine absolute numbers of Bi-ChTCR-T cells and T cell phenotype by flow cytometry focusing on differentiation, activation and exhaustion markers as described above. If mice remain tumor-free by D45, they are rechallenged with tumor cells to determine if T cells persisted and were able to reject tumors.

[0052] Susceptibility to exhaustion: Excessive co-stimulation could be detrimental to cell persistence and promote exhaustion. To evaluate this, anti -tumor activity of Bi-ChTCR: CD3 / costimulatory domain fusion protein T cells is tested at high antigen levels. NSG mice are engrafted with 0.5xl06GFP-FfLuc Nalm-6hlghcells and receive IxlO6Bi-ChTCR-T cells. Anti-tumor activity of Bi-ChTCR: CD3 / costimulatory domain fusion proteinexpressing T cells is monitored by bioluminescence and mice survival is tracked. Bone marrow is harvested on D14 from 3 mice per group to determine Bi-ChTCR-T cell number and phenotype. This experiment determines if expressing a CD3 / costimulatory domain fusion protein in a Bi-ChTCR-T cell has any detrimental effects.

[0053] Data Analysis: Bi-ChTCR-T cells generated from 3 independent donors are used for in vitro and in vivo experiments. For repetitive stimulation assay, tumor cell killing is determined and groups compared as in Project 1, part 1 in Example 4. Bi-ChTCR-T cell cytokine production, proliferation and phenotype are compared between groups using a one-way ANOVA with Dunnett’s test. For in vivo experiments, T cell frequency, phenotype, and tumor growth is compared between treated groups using a t-test and a linear mixed model for comparing the data over all time points. Survival is compared using a log-rank test. 8 mice per group are used, with 3 sacrificed at D14 to interrogate Bi-ChTCR-T cell absolute numbers and phenotype. If rechallenge experiments are performed, 3 treatment-naive NSG mice are injected with tumor cells to serve as control group.

[0054] These studies identify CD3 / costimulatory domain fusion proteins that provide costimulation and improve anti -tumor activity of Bi-ChTCR-T cells in vitro and in vivo against AgLowtumor cells. If CD3 / costimulatory domain fusion proteins result in excessive costimulation in response to AgHlghtumor cells, experiments are performed to test whether eliminating the knockout of endogenous CD3(^ or CD3s when generating Bi-ChTCR-T cells to allow assembly of complexes that lack, or that contain heterodimers comprising one copy of, the CD3 / costimulatory domain fusion protein. This can provide a dose-dependent effect and modulate the amount of co-stimulation that T cell receives. Western blot data from the Bi-ChTCR pull down provide insight into how much of the CD3 / costimulatory domain fusion protein is incorporated into the Bi-ChTCR complex and the assays described above are repeated.

[0055] Figures 56A-56D relate to testing of IT AM-engineered CD3 proteins in anti-CD19 / anti-CD22 Bi-ChTCR T cells with knockout of the corresponding endogenous CD3(^. Figure 56A, top: schematic of expression vector encoding Bi-ChTCR and an IT AM-inactivated CD3 protein. CD3(^ IT AMs can be inactivated by, for example, introducing point mutations converting two tyrosines to phenylalanines within IT AM sequences. Eight (8) tricistronic lentiviral vectors were constructed, encoding the Bi-ChTCR and IT AM-engineered CD3(^ encompassing all IT AM activation / inactivation combinations (Figure 53). These constructs were expressed in primary CD8+ T cells by lentiviral delivery along with endogenous TCRaP and CD3(^ KO using CBE. T cells expressing Bi-ChTCR alone and KO for endogenous TCR a and P chains only were used as control for Bi-ChTCR expression determined by flow cytometry on D7. Wildtype CD3(^ (“CD3^_WT”) and all IT AM-engineered CD3(^ proteins tested restored Bi-ChTCR expression (Figures 56A and 56B) and Bi-ChTCR expression level was comparable to or slightly higher than in the control condition without CD3(^ KO, suggesting that CD3(^ is rate-limiting for Bi-ChTCR expression and that providing exogenous CD3(^ or an IT AM-engineered CD3(^ can improve Bi-ChTCR expression even when endogenous CD3(^ is knocked out (Figure 56C, left). sgRNAs were designed and generated that target additional CD3 subunits (CD3y, CD36, or CD3s) and it was demonstrated using flow cytometry and DNA sequencing that cytosine base editing (CBE) can very efficiently (>90% KO) KO these CD3 subunits (Figure 56C, right), demonstrating the feasibility of knocking-out a selected CD3 protein and re-expressing the CD3 protein with engineering in one or more IT AM.

[0056] Bi-ChTCR-T cells that express the “1XX” IT AM-engineered CD3(^ or the “6F” IT AM-engineered CD3(^ maintain Ag sensitivity in vitro: Efforts were made evaluate how ITAM-engineered CD3(^ polypeptides affect Bi-ChTCR-T cell functions. Bi-ChTCR-T cells generated with unmodified, “1XX”, or “6F” CD3(^ as described above (see also Figure 47) were sort purified for functional comparison. These ITAM-engineered CD3(^ polypeptides were selected for initial analysis. When co-cultured with Nalm-6hlghand Nalm-6Lowcells, 1XX Bi-ChTCR-T cells and 6F Bi-ChTCR-T cells demonstrated similar levels of IL-2 production and proliferation as compared to Bi-ChTCR-T cells with an unmodified CD3(^ (Figure 56D). Thus, reducing the number of functional IT AMs in CD3(^ does not impact the antigen-sensitivity of Bi-ChTCR-T cells in this assay.

[0057] Figure 57 shows (upper left) a cartoon representation of a double-stranded DNA expression vector. An EFla promoter (e.g., SEQ ID NO:7) is disposed on a first DNA strand and is operably linked to a polynucleotide encoding a chTCR. A MNDU3 (shown as “MND” in these Figures) promoter (e.g., SEQ ID NO:8) is disposed on an opposite DNA strand and is operably linked to a polynucleotide encoding a CD3 polypeptide or CD3 fusion polypeptide. At bottom right is a cartoon showing the encoded polypeptide expression products and promoters in further detail.

[0058] Figure 58 shows (upper left) a cartoon representation of a double-stranded DNA expression vector similar to that shown in Figure 57, except that the MNDU3 promoter is operably linked to a polynucleotide encoding an EGFR tag (e.g., a truncated EGFR tag as provided herein), and (bottom right) a cartoon showing the encoded polypeptide expression products and promoters in further detail.

[0059] Figure 59 shows a cartoon representation of a double-stranded DNA expression vector. An EFla promoter is operably linked to a polynucleotide encoding a chTCR in “full scFv” format and a second promoter (selected from hPGK, CMV, MNDU3, and RPBSA), disposed on an opposite strand to the first promoter, is operably linked to a polynucleotide encoding a truncated EGFR tag (“tEGFR”).

[0060] Figure 60 shows, at left, expression of an anti-CD19 chTCR (“full” scFv format, comprising a scFv comprising the VL and VH of FMC63 disposed on TRAC) in primary CD8+ human T cells when encoded by various lentiviral expression vectors. Each point represents data from cells isolated from a different healthy T cell donor. The expression vectors are:

[0061] “Full ChTCR WT (no tag)” encodes the full chTCR under control of the EFla promoter but does not encode the tEGFR. The full chTCR expression product comprises the TRAC-containing polypeptide separate from the TRBC-containing polypeptide by a RAKR sequence and a GSG-P2A self-cleaving peptide (i.e., a P2A sequence preceded immediately by a short Gly-Ser-Gly linker).

[0062] “Full ChTCR P2A Middle tEGFR” encodes the following under control of the EFla promoter: TRBC_RAKR GSG_P2A tEGFR _RAKR _GSG_P2A FMC63 scFv_TRAC “Full ChTCR P2A End tEGFR” encodes the following under control of the EFla promoter: TRBC RAKR _GSG_P2A FMC63 scFv_TRAC_RAKR GSG_P2A tEGFR “Full ChTCR hPGK tEGFR”, “Full ChTCR CMV tEGFR”, “Full ChTCR MND tEGFR”, and “Full ChTCR RPBSA tEGFR”, vectors encode (TRBC_RAKR GSG_P2A FMC63 scFv_TRAC) under control of the the EFla promoter, and the tEGFR under conrol of a hPGK, CMV, MNDU3, or RPBSA promoter, respectively. The bar graph at the upper right of Figure 60 shows transduction efficiency (percentage of T cells positive for binding recombinant CD19) of the indicated expression constructs in primary CD8+ human T cells.

[0063] Figures 61A-61C provide additional data for primary CD8+ human T cells transduced with the expression vectors shown in Figure 60: (61 A) amount of CD19 binding, a measure of how many anti-CD19 chTCR molecules are being expressed; (61B) expression of TRAC / TRBC; (61C) expression of tEGFR.

[0064] Figure 62 shows a cartoon representation of a double-standed DNA expression vector. An EFla promoter is disposed on a first DNA strand and is operably linked to a polynucleotide encoding a chTCR in full-scFv format (anti -CD19 scFv comprising VL and VH of FMC63 disposed on TRAC). A MNDU3 promoter or EFla promoter is disposed on an opposite DNA strand and is operably linked to a polynucleotide encoding a wild-type human CD3(^ polypeptide.

[0065] Figure 63 shows (left) flow cytometry data for binding recombinant CD19 and expression of TRAC / TRBC and (right) % CD19-binding, of primary CD8+ human T cells transduced with the indicated expression vector. Certain expression vectors used two promoters: EFla and EF la, or EF 1 a and MNDU3.

[0066] “WT Full ChTCR” encodes the following under control of an EFla promoter:

[0067] TRBC RAKR _GSG_P2A FMC63 scFv TRAC RAKR GSG P2A ( 1)32

[0068] “Full ChTCR P2A Middle WT CD3z” encodes the following under control of an EFla promoter: TRBC RAKR _GSG_P2A ( 1)32 RAKR _GSG_P2A FMC63 scFv_TRAC “Full ChTCR P2A EFla WT CD3z” encodes: under control of a first EFla promoter, TRBC_RAKR GSG_P2A FMC63 scFv_TRAC, and under control of a second EFla promoter on the opposite strand, CD3.

[0069] “Full ChTCR P2A MND WT CD3z” encodes: under control of an EFla promoter, TRBC_RAKR_GSG_P2A_FMC63 scFv_TRAC, and under control of a MNDU3 promoter on the opposite strand, CD3.

[0070] Two batches of transduced primary CD8+ human T cells were used to generate the bar graphs at the right of the figure (one batch per graph).

[0071] Figure 64 shows median fluorescence intensity (MFI) readouts indicating CD19-binding, TRAC / TRBC expression, and CD3(^ expression in primary CD8+ human T cells transduced with the indicated construct (as in Figure 63).

[0072] Figure 65 shows flow cytometry data showing (left) expression of tEGFR in primary CD8+ human T cells transduced with a DNA expression construct encoding: a bispecific chTCR (anti-BCMA x anti-CD19, with scFv comprising VH and VL from Cl 1D5.3 fused to TRBC and scFv comprising VL and VH from FMC63 fused to TRAC) under control of an EFla promoter; and tEGFR under control of a MNDU3 promoter, and (right) BCMA-binding and TRAC / TRBC expression in tEGFR+ T cells.

[0073] Figures 66A-70 show data related to IT AM inactivation in human CD3(^ by sequence engineering as disclosed herein. Primary CD8+ human T cells were transduced with lentiviral vector encoding anti-CD19 chTCR (FMC63 VL-linker-VH scFv disposed on TRAC) alone or with the indicated CD3(^ polypeptide (wild-type or IT AM-engineered, see Figure 47).

[0074] Additionally, CRISPR / Cas editing or cytosine base editing was used to knock out endogenous TCRaP only (in chTCR-alone condition) or endogenous TCRaP and CD3(^ (all other conditions). In these constructs, a single promoter (EFla) was used to drive expression.

[0075] Figures 66A-66C show results from flow cytometry studies assessing expression of CD3(^ and TRBC, and binding to recombinant CD19 APC, by T cells.

[0076] Figure 67A shows (left) percentage of primary CD8+ human T cells transduced with the indicated construct and (right) geometric mean of CD3s expression in the cells.

[0077] Figure 67B shows (left) geometric mean of TRAC / TRBC expression in the transduced cells and (right) geometric mean of transduced T cells binding recombinant CD19.

[0078] Figures 67C-67D show that chTCR T cell function and sensitivity to antigen are maintained with ITAM-engineered CD3(^ constructs. Graphs show cell proliferation (67C) and IL-2 production (67D) by transduced T cells in the presence of CD19-knockoutNalm-6 cells, Nalm-6 cells expressing low levels of CD19, and Nalm-6 cells expressing high levels of CD19. The left-to-right order of the bars in each experimental condition corresponds to the top-to-bottom order of the constructs as listed in the key. For example, the left-most bar in each condition corresponds to “CD19_Full”.

[0079] Figure 68 shows data for a reduced set of constructs. (Left) IL-2 expression in co-culture with the indicated Nalm-6 cell lines by transduced T cells (anti-CD19 chTCR alone, or with CD3z_6F or CD3z_lXX variant), with PMA-Ionomycin as positive control and a negative control (“CTL”), and (right) percentage of CTV-low T cells in the presence of Nalm6 cells expressing low levels of CD19.

[0080] Figures 69 and 70 show results from Western blot experiments confirming that, in T cells transduced with the anti-CD19 chTCR + CD3z_XXX IT AM, CD3(^ phosphorylation is absent but, unexpectedly, phosphorylation for other key T cell signaling molecules is similar to that observed in the chTCR-only and chTCR + wild-type CD3z settings.

[0081] Figures 71A-72D show data related to IT AM engineering in human CD3(^. Primary CD8+ human T cells were transduced with lentiviral vector encoding anti-CD19 chTCR (FMC63 VL-linker-VH scFv disposed on TRAC) alone or with the indicated CD3(^ polypeptide (wildtype or ITAM-engineered, see Figure 47). Additionally, CRISPR / Cas editing or cytosine base editing was used to knock out endogenous TCRaP only (in chTCR-alone condition) or endogenous TCRaP and CD3(^ (all other conditions). “CD3 111” refers to a CD3(^ wherein ITAM 1 is wild-type and IT AMs 2 and 3 are replaced by IT AM 1. “CD3 222” refers to a CD3(^ wherein ITAM 2 is wild-type and ITAMs 1 and 3 are replaced by the sequence from ITAM 2. “CD3 333” refers to a CD3(^ wherein ITAM 3 is wild-type and ITAMs 1 and 2 are replaced by ITAM 3. “CD3 elelel” refers to a CD3(^ wherein all 3 ITAMs are replaced with the ITAM from CD3s. Figures 71A-71B show results from flow cytometry studies assessing expression of CD3 and TRBC, and binding to recombinant CD19 APC, by transduced T cells.

[0082] Figure 72A shows (left-to-right) percentage of primary CD8+ human T cells positive for expression of the chTCR, geometric mean of TRAC / TRBC expression in the T cells, and geometric mean of CD19-binding.

[0083] Figure 72B shows proliferation of primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19-knockoutNalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing endogenous (“wild-type”) levels of CD19.

[0084] Figure 72C shows IL-2 production by primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19 -knockout Nalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing endogenous (“wild-type”) levels of CD19.

[0085] Figure 72D shows IFN-y production by primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19-knockoutNalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19.

[0086] Figures 73-74E show data related to ITAM engineering in human CD36. Primary CD8+ human T cells were transduced with lentiviral vector encoding anti-CD19 chTCR (FMC63 VL-linker-VH scFv disposed on TRAC) alone or with the indicated CD36 polypeptide (wild-type or ITAM-engineered, see Figure 47). Additionally, CRISPR / Cas editing or cytosine base editing was used to knock out endogenous TCRaP only (in chTCR-alone condition) or endogenous TCRaP and CD36 (all other conditions). “CD3d WT” refers to wild-type CD36. “CD3d FF” refers to a CD36 wherein each of the two tyrosines in the single ITAM is mutated to phenylalanine (see also “CD3d_2F (X) in Figure 47). “CD3d FY” refers to a CD36 wherein only the first tyrosine of the ITAM is mutated to phenylalanine (see also “CD3d_lF (FY) in Figure 47).

[0087] Figure 73 shows results from flow cytometry studies assessing expression of CD3 and binding to recombinant CD19 APC by transduced T cells.

[0088] Figures 74A-74B show: percentage of primary CD8+ human T cells positive for expression of the chTCR, geometric mean of CD3 expression in the T cells, geometric mean of TRAC / TRBC expression in the T cells, and geometric mean of CD19-binding by the T cells.

[0089] Figure 74C shows proliferation of primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19 -knockout Nalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19.

[0090] Figure 74D shows IL-2 production by primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19 -knockout Nalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19.

[0091] Figure 74E shows IFN-y production by primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19-knockoutNalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19. Figures 75-76D show data related to IT AM engineering in human CD3s. Primary CD8+ human T cells were transduced with lentiviral vector encoding anti-CD19 chTCR (FMC63 VL-linker-VH scFv disposed on TRAC) alone or with the indicated CD3s polypeptide (wild-type or ITAM-engineered, see Figure 47). Additionally, CRISPR / Cas editing or cytosine base editing was used to knock out endogenous TCRaP only (in chTCR-alone condition) or endogenous TCRaP and CD3s (all other conditions). “CD3s WT” refers to wild-type CD36. “CD3e FF” refers to a CD3s wherein each of the two tyrosines in the single ITAM is mutated to phenylalanine (see also “CD3e_2F (X)” in Figure 47).

[0092] Figure 75 shows results from flow cytometry studies assessing expression of CD3 and and binding to recombinant CD19 APC by transduced T cells.

[0093] Figure 76A shows (left) percentage of primary CD8+ human T cells positive for expression of the chTCR and (right) geometric mean of CD3 expression in the T cells. The text “SS776_” in the figure key, preceding “CD19_Full_CD3e_2F”, is irrelevant.

[0094] Figure 76B shows (left) geometric mean of binding to CD19 by primary CD8+ human T cells transduced with the indicated construct and (right) geometric mean of TRAC / TRBC expression in the cells. The text “SS776_” in the figure key, preceding “CD19_Full_CD3e_2F”, is irrelevant.

[0095] Figure 76C shows IL-2 production by primary CD8+ human T cells transduced with the indicated construct, in the presence of: negative control (CTL), CD19-knockout Nalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19. The text “SS776_” in the figure key, preceding “CD19_Full_CD3e_2F”, is irrelevant.

[0096] Figure 76D shows proliferation of primary CD8+ human T cells transduced with the indicated construct, in the presence of: negative control (CTL), CD19-knockout Nalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19. The text “SS776_” in the figure key, preceding “CD19_Full_CD3e_2F”, is irrelevant.

[0097] Figures 77-78E show data related to ITAM engineering in human CD3y. Primary CD8+ human T cells were transduced with lentiviral vector encoding anti-CD19 chTCR (FMC63 VL-linker-VH scFv disposed on TRAC) alone or with the indicated CD3s polypeptide (wild-type or ITAM-engineered, see Figure 47). Additionally, CRISPR / Cas editing or cytosine base editing was used to knock out endogenous TCRaP only (in chTCR-alone condition) or endogenous TCRaP and CD3y (all other conditions). “CD3g WT” refers to wild-type CD36. “CD3g FF” refers to a CD3y wherein each of the two tyrosines in the single ITAM is mutated to phenylalanine (see also “CD3g_2F (X)” in Figure 47).

[0098] Figure 77 shows results from flow cytometry studies assessing expression of CD3 and and binding to recombinant CD19 APC by transduced T cells.

[0099] Figure 78A shows (left) percentage of primary CD8+ human T cells positive for expression of the chTCR and (right) geometric mean of CD3s expression in the T cells.

[0100] Figure 78B shows (left) geometric mean of TRAC / TRBC expression in the T cells and (right) geometric mean of CD3s expression in the T cells. Figure 78C shows proliferation of primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19 -knockout Nalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19.

[0101] Figure 78D shows production of IL-2 by primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19-knockoutNalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19.

[0102] Figure 78E shows production of IFN-y by primary CD8+ human T cells transduced with the indicated construct, in the presence of: CD19-knockoutNalm6 cells, Nalm6 cells expressing low levels of CD19, and Nalm6 cells expressing high levels of CD19.

[0103] Figures 79 and 80 show non-limiting examples of plasmid maps comprising dualpromoter (EFla, MNDu3) expression constructs of the present disclosure.

[0104] DETAILED DESCRIPTION

[0105] The present disclosure relates generally to compositions and methods for cellular (e.g., T cell) immunotherapy, such as for treating cancers, infections, autoimmune diseases, or neurodegenerative diseases.

[0106] Disclosed embodiments include means for initiating, providing, boosting, and / or improving signaling in a host (e.g., T) cell that further expresses a target (e.g., antigen)-binding protein when the target-binding protein encounters the target. In some embodiments, the targetbinding protein comprises a chimeric T cell receptor (chTCR, also called a TCR / CAR) as described herein, a TCR, a CAR, a single-chain TCR (scTCR), a mutSTAR receptor, a Co-STAR receptor, a STAR receptor, or a HIT receptor. Certain embodiments provide means (e.g., fusion proteins, polypeptides, and expression constructs) for improving expression, activity, target-sensitivity, or any combination thereof, of the target-binding protein. In some embodiments, a host cell has endogenous expression of a TCRa, a TCRP, a CD3(^, a CD3s, or any combination thereof. In some embodiments, a host cell has diminished, attenuated, or knocked-out expression of an endogenous TCRa, TCRP, CD3(^, CD3s, or any combination thereof.

[0107] It will be understood that certain polypeptides may be depicted using a Greek alphabet symbol (e.g., CD3y, CD36, CD3s, CD3(^, TCRa, TCRP) or by the corresponding English alphabet character (CD3g, CD3d, CD3e, CD3z, TCRa, TCRb). These can be used interchangeably unless the context clearly provides otherwise.

[0108] Some embodiments provide fusion proteins that comprise a CD3(^ or a fragment or variant thereof that is functional to produce a CD3(^ signal (and / or to initiate an effector signal), and a costimulatory portion (CDSi^costimulatory domain fusion proteins). Some embodiments provide fusion proteins that comprise a CD3s or a fragment or variant thereof that is functional to produce a CD3s signal (and / or to initiate an effector signal), and a costimulatory portion (CD3s / costimulatory domain fusion proteins). In some embodiments, a fusion protein, when expressed by a host (e.g., T) cell that further expresses a target (e.g., antigen)-binding protein, can provide a costimulatory signal, an effector (CD3(^ or CD3s) signal, or both to the host cell when the target-binding protein encounters the target.

[0109] Some embodiments provide a polynucleotide or vector that encodes, or a host cell that encodes or expresses, (i) a chTCR according to the present disclosure and (ii) a CD3(^ or a functional fragment or variant thereof. Some embodiments provide polynucleotide or vector that encodes, or a host cell that encodes or expresses, (i) a chTCR according to the present disclosure and (ii) a CD3s or a functional fragment or variant thereof. Some embodiments provide a polynucleotide or vector that encodes, or a host cell that encodes or expresses, (i) a chTCR according to the present disclosure and (ii) a CD3 / costimulatory domain fusion protein comprising CD3(^ and a costimulatory domain from a 4-1BB or a functional fragment or variant thereof.

[0110] A host cell can comprise, for example, a hematopoietic progenitor cell, an immune system cell, an induced pluripotent stem cell, an embryonic stem cell, or a peripheral blood mononuclear cell. In some embodiments, a host cell comprises a human cell. In some embodiments, a host cell comprises a T cell (such as a human T cell). The target-binding protein can be exogenous to the host cell and / or the host cell can comprise a chromosomal gene knockout of, for example, an endogenous CD3(^, an endogenous CD3s, an endogenous TCRa, and endogenous TCRP, or any combination thereof.

[0111] Certain embodiments provide CD3 / costimulatory domain fusion proteins that comprise an extracellular component of a CD3(^ or CDs protein (or a functional fragment or variant thereof), a transmembrane component that optionally comprises, consists essentially of, or consists of a transmembrane domain of the CD3(^ or CDs protein (or a functional fragment or variant thereof), and an intracellular component that comprises (i) an intracellular domain of the CD3(^ or CDs protein (or a functional fragment or variant thereof) and (ii) a costimulatory portion that comprises, consists essentially of, or consists of, a costimulatory domain or a functional fragment or variant thereof. In some embodiments: the extracellular component of a CD3 / costimulatory domain fusion protein comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO.: 152; the transmembrane component of the CD3 / costimulatory domain fusion protein comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO.:240; and the intracellular component of the CD3 / costimulatory domain fusion protein comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO.:241. In other embodiments: the extracellular component of a CD3 / costimulatory domain fusion protein comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO.:242; the transmembrane component of the CD3 / costimulatory domain fusion protein comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO.:243; and the intracellular component of the CD3 / costimulatory domain fusion protein comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising, consisting essentially of, or consisting of, the amino acid sequence set forth in SEQ ID NO.:244.

[0112] In certain embodiments, the costimulatory domain or a functional fragment or variant thereof is from or is derived from 4-1BB, CD226, CD2, CD28, 0X40, CD27, CD3s, CD3< CD36, CD3y, CD79A, CD79B, SLAMF1, ICOS, DAP10, CD25, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, LIGHT, CD30, Lek, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, R0R2, Ryk, Slp76, pTa, TCRa, TCRp, TRIM, Zap70, or PTCH2, or any combination thereof. In some embodiments, a functional fragment or variant of a costimulatory domain does not comprise a native basic rich motif (BRM). In some embodiments, a functional fragment or variant of a costimulatory domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an intracellular domain of a 4- IBB, a CD226, a

[0113]

[0114] or a R0R2, or a Ryk, or a Slp76, or a pTa, or a TCRa, or a TCRP, or a TRIM, or a Zap70, or a PTCH2, or to a fragment thereof that does not comprise a native BRM, or any combination thereof.

[0115] In certain embodiments, a CD3 / costimulatory domain fusion protein is provided that comprises any of the amino acid sequences, or any combination of two or more of the amino acid sequences, shown in Figure 45. In some embodiments, a CD3 / costimulatory domain fusion protein is provided that comprises, consists essentially of, or consists of one or more of the following sequences shown in Figure 45: CD3z; CD3e; CD3z_4-lBB; CD3z_4-lBB-BRM; CD3z_4-lBB-BRM_ 7; CD3z_4-lBB-BRM_ 7 7; CD3z_CD226mut; CD3z_CD226mut-BRM; CD3z_CD28; CD3z_CD28-BRM; CD3z CD2; and CD3z_CD2tr. In certain embodiments, a variant or fragment of an amino acid sequence shown in Figure 45 is provided, wherein the variant or fragment does not comprise a signal peptide or comprises an alternative signal peptide.

[0116] It will be understood that a functional fragment or variant of a referenced protein or protein portion or domain substantially retains one or more function of the referenced protein or protein portion or domain. For example, a CD3 / costimulatory domain fusion protein comprising a functional fragment or variant of a CD3(^ extracellular domain, a functional fragment or variant of a CD3(^ transmembrane domain, and / or a functional fragment or variant of a CD3(^ intracellular domain substantially retains the ability to initiate a CD3(^ signal when a host T cell expressing the CD3 / costimulatory domain fusion protein and a target (e.g., antigen)-binding protein encounters the target, and preferably substantially retains a dimer-forming capability of a native CD3(^. In some embodiments, a CD3 / costimulatory domain fusion protein molecule comprising a functional fragment or variant of a CD3(^ extracellular domain, a functional fragment or variant of a CD3(^ transmembrane domain, and / or a functional fragment or variant of a CD3(^ intracellular domain can form a dimer with a CD3(^ protein and / or with another such CD3 / costimulatory domain fusion protein molecule. CD3(^ dimers are discussed in, for example, Call ME, Schnell JR, Xu C, Lutz RA, Chou JJ, Wucherpfennig KW. The structure of the zetazeta transmembrane dimer reveals features essential for its assembly with the T cell receptor. Cell. 2006 Oct 20;127(2):355-68. doi: 10.1016 / j.cell.2006.08.044. PMID: 17055436; PMCID:

[0117] PMC3466601, which teachings are incorporated by reference herein. For example, formation of CD3(^ dimers can involve formation of a disulfide bond through a native cysteine amino acid (e.g, the bold, underlined cysteine in the following amino acid sequence:

[0118] LCYLLDGILFIYGVILTALFL (SEQ ID NO.:240). Additionally or alternatively, for example, a cysteine may be introduced in a non-native position (e.g, by a substitution mutation to cysteine or by insertion of a cysteine) of a CD3(^ or a CD3(^ portion of a CD3 / costimulatory domain fusion protein, such as within the transmembrane component of the CD3 / costimulatory domain fusion protein or or within about 5 amino acids or within about 10 amino acids or within about 15 amino acids or within about 20 amino acids thereof.

[0119] As another example, a CD3 / costimulatory domain fusion protein comprising a functional fragment or variant of a CD3s extracellular domain, a functional fragment or variant of a CD3s transmembrane domain, and / or a functional fragment or variant of a CD3s intracellular domain substantially retains the ability to initiate a CD3s signal when a host T cell expressing the CD3s / costimulatory domain fusion protein and a target (e.g., antigen)-binding protein encounters the target, and preferably substantially retains the ability to form a heterodimer with a an endogenous CD36 and / or with an endogenous CD3y.

[0120] As another example, a CD3 / costimulatory domain fusion protein comprising a functional fragment or variant of a costimulatory domain at least partially retains, and preferably substantially retains, the ability of a reference costimulatory protein to initiate, contribute to, produce, or provide a costimulatory signal when a host T cell expressing the CD3 / costimulatory domain fusion protein and a target (e.g., antigen)-binding protein encounters the target. Functional fragments and variants of certain proteins and protein domains are discussed further herein.

[0121] Certain embodiments provide a CD3 / costimulatory domain fusion protein comprising, consisting essentially of, or consisting of: an extracellular component comprising, consisting essentially of, or consisting of the extracellular domain of a CD3(^ (e.g., QSFGLLDPK (SEQ ID NO.: 152)), or a functional fragment or variant thereof, wherein the variant optionally comprises one, two, three, or four amino acid substitutions, insertions, and / or deletions relative to SEQ ID NO.: 152; an intracellular component comprising, consisting essentially of, or consisting of (i) an intracellular signaling domain of the CD3(^ or a functional fragment or variant thereof and (ii) a costimulatory portion comprising, consisting essentially of, or consisting of (1) a costimulatory domain of a 4- IBB, or a functional fragment or variant thereof, linked or fused to an amino acid sequence that is not the intracellular signaling domain of the CD3(^ or a functional fragment or variant thereof; (2) a costimulatory domain of a CD226, or a functional fragment or variant thereof, wherein, optionally, the variant comprises one or more (e.g. substitution, e.g. nonconservative) mutation that (a) provides increased expression of the CD3 / costimulatory domain fusion protein in a T cell exposed to PVR, as compared to expression of endogenous CD226 by the T cell exposed to PVR, and / or (b) disrupts a Src kinase phosphorylation site on the CD3 / costimulatory domain fusion protein, and / or (c) reduces ubiquitination of the CD3 / costimulatory domain fusion protein by CBL-B, wherein, further optionally, the mutated CD226 costimulatory domain or a functional fragment or variant thereof comprises a substitution mutation at a position corresponding to one or more of positions K295, Y319, and K333, optionally comprising K295A, Y319F, and / or K333 A mutations; (3) a costimulatory domain of a CD28, or a functional fragment or variant thereof; (4) a costimulatory domain of a CD2, or a functional fragment or variant thereof; and / or (5) a costimulatory domain of an 0X40, or a functional fragment or variant thereof; and and a transmembrane component disposed between and connecting the extracellular component and the intracellular component, wherein, optionally, the transmembrane component comprises, consists essentially of, or consists of the transmembrane domain of the CD3(^.

[0122] Certain embodiments provide a CD3 / costimulatory domain fusion protein comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of: the amino acid sequence set forth in SEQ ID NO.: 182; the amino acid sequence set forth in SEQ ID NO.: 183; the amino acid sequence set forth in SEQ ID NO.: 184; the amino acid sequence set forth in SEQ ID NO.: 185; the amino acid sequence set forth in SEQ ID NO.: 186; the amino acid sequence set forth in SEQ ID NO.: 187; the amino acid sequence set forth in SEQ ID NO.: 188; the amino acid sequence set forth in SEQ ID NO.: 189; the amino acid sequence set forth in SEQ ID NO.:253; the amino acid sequence set forth in SEQ ID NO.:252; the amino acid sequence set forth in SEQ ID NO.: 190; the amino acid sequence set forth in SEQ ID NO.: 191; the amino acid sequence set forth in SEQ ID NO.: 192; the amino acid sequence set forth in SEQ ID NO.: 193; the amino acid sequence set forth in SEQ ID NO.: 194; the amino acid sequence set forth in SEQ ID NO.: 195; the amino acid sequence set forth in SEQ ID NO.: 196; the amino acid sequence set forth in SEQ ID NO.:197; the amino acid sequence set forth in SEQ ID NO.:198; the amino acid sequence set forth in SEQ ID NO.: 199; the amino acid sequence set forth in SEQ ID NO.:200; the amino acid sequence set forth in SEQ ID NO.:201; the amino acid sequence set forth in SEQ ID NO.:255; the amino acid sequence set forth in SEQ ID NO.:254; the amino acid sequence set forth in SEQ ID NO.:202; or the amino acid sequence set forth in SEQ ID NO.:203.

[0123] Certain embodiments provide a CD3 / costimulatory domain fusion protein comprising, consisting essentially of, or consisting of: an extracellular component comprising, consisting essentially of, or consisting of the extracellular domain of a CD3s or a functional fragment or variant thereof; an intracellular component comprising, consisting essentially of, or consisting of (i) an intracellular signaling domain of the CD3s or a functional fragment or variant thereof and (ii) a costimulatory portion, wherein the costimulatory portion optionally comprises a costimulatory domain, or a functional fragment or variant thereof, from CD2, CD226, CD28, 4-1BB, 0X40, CD27, CD36, CD3y, CD3i CD79A, CD79B, SLAMF1, ICOS, DAP10, CD25, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, LIGHT, CD30, Lek, LAG3, LAT, LRP, NKG2D, N0TCH1, N0TCH2, N0TCH3, N0TCH4, R0R2, Ryk, Slp76, pTa, TCRa, TCRp, TRIM, Zap70, PTCH2, or any combination thereof; and a transmembrane component disposed between and connecting the extracellular component and the intracellular component, wherein, optionally, the transmembrane component comprises, consists essentially of, or consists of the transmembrane domain of the CD3s.

[0124] Certain embodiments provide a CD3 / costimulatory domain fusion protein comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of: the amino acid sequence set forth in SEQ ID NO.:207; the amino acid sequence set forth in SEQ ID NO.:208; the amino acid sequence set forth in SEQ ID NO.:209; the amino acid sequence set forth in SEQ ID NO.:210; the amino acid sequence set forth in SEQ ID NO.:211; the amino acid sequence set forth in SEQ ID NO.:212; the amino acid sequence set forth in SEQ ID NO.:213; the amino acid sequence set forth in SEQ ID NO.:214; the amino acid sequence set forth in SEQ ID NO.:215; the amino acid sequence set forth in SEQ ID NO.:216; the amino acid sequence set forth in SEQ ID NO.:217; the amino acid sequence set forth in SEQ ID NO.:218; the amino acid sequence set forth in SEQ ID NO.:257; the amino acid sequence set forth in SEQ ID NO.:256; the amino acid sequence set forth in SEQ ID NO.:219; the amino acid sequence set forth in SEQ ID NO.:220; the amino acid sequence set forth in SEQ ID NO.:221; the amino acid sequence set forth in SEQ ID NO.:222; the amino acid sequence set forth in SEQ ID NO.:223; the amino acid sequence set forth in SEQ ID NO.:224; the amino acid sequence set forth in SEQ ID NO.:225; the amino acid sequence set forth in SEQ ID NO.:226; the amino acid sequence set forth in SEQ ID NO.:227; the amino acid sequence set forth in SEQ ID NO.:228; the amino acid sequence set forth in SEQ ID NO.:229; the amino acid sequence set forth in SEQ ID NO.:230; the amino acid sequence set forth in SEQ ID NO.:259; the amino acid sequence set forth in SEQ ID NO.:258; the amino acid sequence set forth in SEQ ID NO.:231; or the amino acid sequence set forth in SEQ ID NO.:232.

[0125] In some embodiments, when a CD3 / costimulatory domain fusion protein of the present disclosure is expressed by a human T cell that further expresses a target-binding protein (e.g., a TCR, a CAR, or a chTCR (also referred to as a chimeric TCR or a TCR / CAR)), the T cell has increased production of one or more cytokine (e.g., IL-2, IFN-^, or both) when in the presence of the target, and / or has increased proliferation when in the presence of the target, as compared to a reference human T cell (i.e., a human T cell that is substantially identical to the human T cell expressing the CD3 / costimulatory domain fusion protein and target-binding protein) that does not express the CD3 / costimulatory domain fusion protein.

[0126] Also provided is a CD3 / costimulatory domain fusion protein comprising an extracellular component, an intracellular component, and a transmembrane component disposed between and connecting the extracellular and intracellular components, wherein the intracellular component comprises, consists essentially of, or consists of: the amino acid sequence set forth in SEQ ID NO.:233; the amino acid sequence set forth in SEQ ID NO.:234; the amino acid sequence set forth in SEQ ID NO.:235; or the amino acid sequence set forth in SEQ ID NO.:236. In some embodiments, the intracellular component of the fusin protein further comprises an intracellular signaling domain of a CD3(^ or an intracellular signaling domain of a CD3s.

[0127] Also provided are polynucleotides that encode a disclosed CD3 / costimulatory domain fusion protein (and optionally further encode a target (e.g., antigen)-binding protein, such as a chTCR (also called chimeric TCR or TCR / CAR), a TCR, or a CAR), vectors that comprise a polynucleotide, host cells that comprise a polynucleotide and / or vector, host cells that express a disclosed CD3 / costimulatory domain fusion protein (and optionally further encode a target (e.g., antigen)-binding protein, such as a chTCR (also called chimeric TCR or TCR / CAR), a TCR, or a CAR), and methods of using and making the same.

[0128] Also provided is a polynucleotide encoding (i) a chTCR and (ii) a CD3(^ or a functional portion or variant thereof, wherein, optionally, the CD3(^ comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:155 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 153.

[0129] Also provided is a polynucleotide encoding (i) a chTCR and (ii) a CD3s, wherein, optionally, the CD3s comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:206 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:204.

[0130] Also provided is a polynucleotide encoding (i) a chTCR and (ii) a CD3 / costimulatory domain fusion protein comprising: an extracellular component comprising the extracellular domain of a CD3(^, or a functional portion or variant thereof; an intracellular component comprising an intracellular signaling domain of the CD3(^ and costimulatory domain; and a transmembrane component, wherein the transmembrane component is optionally the transmembrane domain of the CD3(^.

[0131] Also provided is a polynucleotide encoding (i) a chTCR and (ii) a CD3 / costimulatory domain fusion protein comprising: an extracellular component comprising the extracellular domain of a CD3s, or a functional portion or variant thereof; an intracellular component comprising an intracellular signaling domain of the CD3s and costimulatory domain; and a transmembrane component, wherein the transmembrane component is optionally the transmembrane domain of the CD3s.

[0132] Also provided is a host cell expressing (i) a chTCR and (ii) a non-endogenous CD3(^ or a functional portion or variant thereof, wherein, optionally, the CD3(^ comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 155 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 153.

[0133] Also provided is a host cell expressing (i) a chTCR and (ii) a non-endogenous CD3s, wherein, optionally, the CD3s comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:206 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:204.

[0134] Pharmaceutical compositions comprising a disclosed CD3 / costimulatory domain fusion protein, polynucleotide, vector, and / or host cell are also provided, as are methods of using the CD3 / costimulatory domain fusion proteins, polynucleotides, vectors, host cell, and pharmaceutical compositions (e.g., to treat a disease such as for example a cancer). Also provided are methods of making a host cell. Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in one or more CD3(^ ITAM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues are each substituted with a different amino acid residue, as described herein, in any one, any two, or all three of the CD3(^ ITAM motifs.

[0135] Also provided is a polypeptide that comprises a CD3s polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in the CD3s ITAM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD3s ITAM motif are each substituted with a different amino acid residue, as described herein.

[0136] Also provided is a polypeptide that comprises a CD3y polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in the CD3y ITAM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD3y ITAM motif are each substituted with a different amino acid residue, as described herein.

[0137] Also provided is a polypeptide that comprises a CD36 polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in the CD36 ITAM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD3y ITAM motif are each substituted with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD36 ITAM motif are each substituted with a different amino acid residue, as described herein.

[0138] Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising three copies of CD3(^ IT AMI: CD3(^ IT AMI, a second copy of CD3(^ IT AMI in place of CD3(^ ITAM2, and a third copy of CD3(^ IT AMI in place of CD3(^ ITAM3.

[0139] Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising three copies of CD3(^ ITAM3: CD3(^ ITAM3 in place of CD3(^ IT AMI, CD3(^ ITAM3 in place of CD3(^ ITAM2, and CD3(^ ITAM3.

[0140] Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising CD3s ITAM1 in place of CD3(^ IT AMI, a second copy of CD3s ITAM1 in place of CD3< ITAM2, and a third copy of CD3s IT AMI in place of CD3(^ ITAM3. Also provided is a polypeptide that comprises a CD3s polypeptide or variant thereof comprising CD3(^ ITAM1 in place of CD3s IT AM.

[0141] Also provided is a polypeptide that comprises a CD3s polypeptide or variant thereof comprising: substitution of one or more native lysine amino acid residues, and / or a native arginine residue, in a CD3s basic rich sequence (BRS) motif with a different amino acid, as described herein; replacement of one or more proline amino acid residues in a native CD3s proline rich sequence (PRS) motif with a different amino acid, as described herein; or substitution of a native “RK” in CD3s ITAM motif with two amino acids, as described herein.

[0142] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: (i) in place of the amino acid sequence of SEQ ID NO.:262, an amino acid sequence according to SEQ ID NO.:263; (ii) in place of the amino acid sequence of SEQ ID NO.:264, an amino acid sequence according to SEQ ID NO.:265; and / or (iii) in place of the amino acid sequence of SEQ ID NO.:266, an amino acid sequence according to SEQ ID NO.:267. In some embodiments, (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152), or a variant thereof having one, two, or three amino acid substitutions; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID No.:240) or an amino acid sequence comprising one, two, three, or four substitutions relative to SEQ ID NO.:240. In some embodiments, (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152); and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID NO.:240). In some embodiments, the intracellular component comprises amino acid sequences according to SEQ ID NOs.: (A) 263, 264, and 266; (B) 263, 264, and 267; (C) 263, 265, and 266; (D) 263, 265, and 267; (E) 262, 264, and 267; (F) 262, 265, and 266; or (G) 262, 265, and 267. In some embodiments, the intracellular component comprises amino acid sequences according to SEQ ID NOs.: (A) 450, 264, and 266; (B) 450, 264, and 452; (C) 450, 451, and 266; (D) 450, 451, and 452; (E) 262, 451, and 266; (F) 262, 264, and 452; or (G) 450, 451, and 452. In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:284, an amino acid sequence according to SEQ ID NO.:285, preferably the amino acid sequence of SEQ ID NO.:453. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID No.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID No.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

[0143] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD36 extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD36 transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD36 intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:296, an amino acid sequence according to any one of SEQ ID NOs.:297-298a, preferably the amino acid sequence of SEQ ID NO.:455, the amino acid sequence of SEQ ID NO.:456, or the amino acid sequence of SEQ ID NO.:457. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:458, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:459 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:458, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:459 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459.

[0144] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3y extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3y transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3y intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:290, an amino acid sequence according to SEQ ID NO.:291, preferably the amino acid sequence of SEQ ID NO.:454. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:460, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:461 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:461.

[0145] In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:460, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:461 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459. In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: SEQ ID NO.:262; in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:262; and in place of the amino acid sequence of SEQ ID NO.:266, the amino acid sequence according to SEQ ID NO.:262.

[0146] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: (i) in place of the amino acid sequence of SEQ ID NO.:262, the amino acid sequence of SEQ ID NO.:266; (ii) in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:266; and (iii) the amino acid sequence of SEQ ID NO.:266.

[0147] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: (i) in place of the amino acid sequence of SEQ ID NO.:262, the amino acid sequence of SEQ ID NO.:284; (ii) in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:284; and (iii) place of the amino acid sequence of SEQ ID NO.:266, the amino acid sequence of SEQ ID NO.:284.

[0148] In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152), or a variant thereof having one, two, or three amino acid substitutions; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID No.:240) or an amino acid sequence comprising one, two, three, or four substitutions relative to SEQ ID NO.:240. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152); and (2)the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID NO.:240).

[0149] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:284, the amino acid sequence of SEQ ID NO.:266.

[0150] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising: (i) in place of the amino acid sequence KNRKAKAK, a variant thereof wherein one or more lysine amino acid residue is independently substituted with a neutral amino acid residue and / or wherein the arginine amino acid is substituted with a neutral amino acid residue, optionally a serine, further optionally, the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence KNRKAKAK, the amino acid sequence SNSSASAS; (ii) in place of the amino acid sequence PPPVPNPDY, a variant thereof wherein one or more proline amino acid residue is independently substituted with an alanine amino acid residue, a serine amino acid residue, or a valine amino acid residue, preferably an alanine amino acid residue, optionally wherein the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence PPPVPNPDY, the amino acid sequence PAPVANPDY; or (iii) in place of the amino acid sequence of SEQ ID NO.:284, a variant thereof wherein the first arginine amino acid residue and the lysine residue are each independently substituted with a neutral amino acid residue, preferably an alanine, optionally wherein the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence of SEQ ID NO.:284, the amino acid sequence YEPIAAGQRDLYSGL.

[0151] In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

[0152] In some embodiments, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:268-281, 286, 287, 292, 293, and 299-316.

[0153] In some embodiments, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of any one of the following constructs shown in Figure 47: CD3z_12X; CD3z_lX3; CD3z X23; CD3z 1XX; CD3z X2X; CD3z XX3; CD3z XXX or 6F; CD3e_2F; CD3g_2F; CD3g_WT; CD3d_WT; CD3z_WT; CD3e_WT; CD3d_2F; CD3d_lF; CD3z_lll; CD3z_333; CD3z_CD3elelel; CD3e_CD3zl; CD3e_BRS; CD3e_PRS; CD3 KR, CD3z-CD226 (BRM deletion); CD3z-CD2 (BRM deletion), optionally with the respective CD3(^ signal peptide or CD3s or CD3d or CD3g signal peptide removed or comprising a GM-CSF signal peptide in place of the respective CD3(^ signal peptide or CD3s signal peptide or CD3d signal peptide or CD3g signal peptide.

[0154] In some embodiments, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of construct CD3z_222. In some embodiments, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:478 or SEQ ID NO:479.

[0155] Also provided are polynucleotides that encode a polypeptide (and, optionally, a targetbinding protein such as for example a chTCR), vectors that comprise a polypeptide-encoding polynucleotide (and, optionally, a target-binding protein such as for example a chTCR), host cells that comprise a polypeptide-encoding vector, host cells that express a polypeptide (and, optionally, a target-binding protein such as for example a chTCR), and related compositions and uses.

[0156] In some embodiments, any presently disclosed CD3 / costimulatory domain fusion protein does not comprise, in an extracellular portion or extracellular component thereof, (1) a targetbinding domain (e.g., a scFv, a VH, a VL, a Fab, a Fd, a scFab, a dsFv, a VHH, a centyrin, a TCR variable domain, or the like) and / or (2) a hinge (e.g., an immunoglobulin hinge or a variant thereof, a CD8 hinge or a variant thereof, or the like) and / or (3) a linker amino acid sequence.

[0157] In some embodiments, any presently disclosed engineered CD3 polypeptide does not comprise, in an extracellular portion or extracellular component thereof, (1) a target-binding domain (e.g., a scFv, a VH, a VL, a Fab, a Fd, a scFab, a dsFv, a VHH, a centyrin, a TCR variable domain, or the like) and / or (2) a hinge (e.g., an immunoglobulin hinge or a variant thereof, a CD8 hinge or a variant thereof, or the like) and / or (3) a linker amino acid sequence.

[0158] In some embodiments, any presently disclosed CD3 / costimulatory domain fusion protein comprises an extracellular portion or extracellular component that consists essentially of or consists of the extracellular domain of a human CD3(^ or the extracellular domain of a human CD3s.

[0159] In some embodiments, any presently disclosed engineered CD3 polypeptide comprises an extracellular portion or extracellular component that consists essentially of or consists of the extracellular domain of a human CD3(^ or the extracellular domain of a human CD3s.

[0160] In some embodiments, a target-binding protein comprises a polypeptide dimer (referred-to herein as a chimeric TCR or a chTCR or a TCR / CAR) that confers to a host cell (e.g. a T cell) target-specificity (including, in some contexts, high binding affinity and / or non-MHC -restricted binding) of a CAR while leveraging natural T cell signaling properties. TCR / CARs can be efficiently expressed at the surface of, for example, host T cells and confer to host T cells a diversified and highly sensitive signaling complex that acts with native T cell signaling properties. In some embodiments, a T cell expressing a TCR / CAR has one or more of the following properties as compared to a T cell expressing a CAR that binds the same target(s): increased sensitivity to antigen; increased production of one or more cytokine (e.g, IFN-y, IL-2); increased killing against cells expressing the target(s); increased proliferation when in the presence of the target(s); reduced basal or non-specific activation; increased survival of a model mammal comprising cells expressing the target(s) (e.g, target-expressing cancer cells); increased antitumor activity against tumor cells expressing the target(s); lower target EC50 for production of IL-2 and / or IFN-y; higher avidity (e.g., a greater amount of TCR / CAR-expressing cells bound to target); and association of the TCR / CAR with one or more CD3 proteins.

[0161] In some embodiments, a TCR / CAR comprises (1) a first polypeptide comprising a first TCR constant domain and (2) a second polypeptide comprising a second TCR constant domain, wherein the first TCR constant domain and the second TCR constant domain associate with one another. The association can comprise native interactions between cognate TCR constant domains (e.g. a native disulfide bond), engineered interactions between the TCR constant domains e.g. one or more disulfide bonds introduced by protein engineering, knob-into-hole-type interactions, and / or charge-pair interactions), or both. One or both of the first polypeptide and the second polypeptide further comprises a target-binding domain disposed N-terminal to the TCR constant domains, and the target-binding domain does not comprise TCR variable regions. In certain embodiments, one or both of the first polypeptide and the second polypeptide comprises a target-binding domain disposed N-terminal to the TCR constant domains, wherein the target binding domain is selected from the target binding domains described herein. In certain embodiments, one or both of the first polypeptide and the second polypeptide comprises a target-binding domain disposed N-terminal to the TCR constant domains, wherein the target binding domain is selected from the target binding domains described herein and one or both of the first polypeptide and the second polypeptide may further comprise a TCR variable domain, provided that one or both fo the first polypeptide and the second polypeptide comprise a targetbinding domain that is not, or does not comprise, a TCR variable domain.

[0162] TCR / CARs are preferably heterodimers ( / .<., the TCR constant domains of the two polypeptides are different to one another), though TCR / CARs wherein the TCR constant domains of the two polypeptides are the same or substantially the same (e.g. are homodimeric with respect to the constant domains; see e.g. Groettrup et al. EMBO J. 77(7):2735-2745 (1992)) are also contemplated. In some embodiments, the first TCR constant domain comprises a TCR alpha-chain constant domain (Ca) and the second TCR constant domain comprises a TCR betachain constant domain (CP). In other embodiments, the first TCR constant domain comprises a CP and the second TCR constant domain comprises a Ca.

[0163] In some embodiments, the first polypeptide and / or the second polypeptide of a TCR / CAR comprises an intracellular portion that consists essentially of or that consists of the intracellular portion of the respective TCR constant domain. In some embodiments, the first polypeptide and / or the second polypeptide (preferably, both) does not comprise an intracellular signaling component (e.g. effector domain) from a CD3 protein, such as CD3(^. In some embodiments, the first polypeptide and / or the second polypeptide (preferably, both) does not comprise an intracellular costimulatory domain from a costimulatory protein, such as CD28, 4- IBB, ICOS, CD27, 0X40, DAP 10, or any combination thereof. In some embodiments, the first polypeptide and / or the second polypeptide (preferably, both) does not comprise an immunoglobulin CHI, an immunoglobulin CH2, an immunoglobulin CH3, and / or an immunoglobulin CL (light chain constant domain). In some embodiments, the first polypeptide and / or the second polypeptide (preferably, both) does not comprise an immunoglobulin CHI, an immunoglobulin CH2, an immunoglobulin CH3, and / or an immunoglobulin CL (light chain constant domain) disposed C-terminal of the TCR constant domain(s).

[0164] The target-binding domain can comprise any naturally occurring or engineered binding domain suitable for binding a target of interest, such as, for example, an antibody heavy chain variable domain (VH), an antibody light chain variable domain (VL), a VH and a VL, a single- chain variable fragment (scFv) comprising VH-linker-VL or VL-linker-VH, a fragment antigenbinding region (Fab), a single-chain Fab, an antigen-binding fragment of a heavy chain-only antibody (VHH, also referred-to as a nanobody), a killer immunoreceptor from a NK cell, a designed ankyrin repeat protein (DARPin (Binz etal., J. Mol. Biol. 332 489, 2003 and Binz et al., Nat. Biotechnol. 22:515, 2004)), a10FNIII domain such as an Adnectin™ or monobody ((Richards et al., J. Mol. Biol. 326: 1475, 2003; Parker et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381: 1238-1252)), a lectin binding domain, a receptor ectodomain or functional portion or fragment thereof, provided that the receptor ectodomain does not comprise a TCR variable domain, a ligand such as e.g. a cytokine, a fully synthetic polypeptide e.g. designed in silico, such as using the AlphaFold modeling program), a fibrinogen domain (see, e.g., Weisel etal., Science 230:1388, 1985), Kunitz domains (see, e.g., US Patent No. 6,423,498), a cysteine-knot miniprotein (Vita et al. (1995) Proc. Nat'L Acad. Sci. (USA) 92:6404-6408; Martin et al. (2002) Nat. Biotechnol. 21:11, 2002 and Huang et al. (2005) Structure 13:155, 2005; Lui etal. Nature Communications 77:295 (2020)), a tetratricopeptide repeat domain (Main et al., Structure 77:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008), a leucine-rich repeat domain (Stumpp et al., J. Mol. Biol. 332:411, 2003), a lipocalin domain (see, e.g., WO 2006 / 095164, Beste et al., Proc. Nat'l. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et al., Proc. Nat'l. Acad. Sci. (USA) 106:8198, 2009), an armadillo repeat protein (see, e.g., Madhurantakam etal., Protein Sci. 21: 1015, 2012; PCT Patent Application Publication No. WO 2009 / 040338), an affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), an affibody, an avimer, a knottin, a fynomer, an atrimer, cytotoxic T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen. Proteo. 10:155, 2013) or the like (Nord et al, Protein Eng. 5:601, 1995; Nord etal., Nat. Biotechnol. 15:112, 1997; Nord et al., Euro. J.

[0165] Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1251, 2005; Boersma and Pliickthun, Curr. Opin. Biotechnol. 2:849, 2011), a centyrin, or the like, or any combination thereof.

[0166] In some embodiments, a target-binding domain is “split” across the first polypeptide and the second polypeptide, meaning that the component parts of a target-binding domain are dispersed between the first polypeptide and the second polypeptide; for example, where a VH and a VL together function to bind to a target, the VH may be comprised in the first polypeptide and the VL is comprised in the second polypeptide, or vice versa. In some embodiments, a target-binding domain is fully comprised in one TCR constant domain-containing polypeptide. For example, a scFv (VH-linker-VL or VL-linker-VH) may be comprised in the first polypeptide or the second polypeptide of a TCR / CAR; such an arrangement can be described as “full-scFv”.

[0167] In some embodiments, both of the first polypeptide and the second polypeptide fully comprise a target-binding domain. For example, the first polypeptide can comprise a Ca linked or fused to a first scFv and the second polypeptide can comprise a CP linked or fused to a second scFv. A target binding domain can be fused directly to a TCR constant domain or can be linked thereto by a linker, such as, for example, a hinge sequence. In some embodiments, a VH or a VL of an antibody is sufficient to confer specific binding to a target (e.g., binding interactions between the antibody and its target occur, or can occur, through only VH and the target or only VL and the target); accordingly, in certain embodiments, a TCR / CAR, or a first polypeptide and / or a second polypeptide of a TCR / CAR, comprises only a VH or a VL as a binding domain.

[0168] Certain embodiments provide multispecific (e.g. bispecific) TCR / CARs. In some contexts, a multispecific TCR / CAR binds to two or more antigens that are expressed by a cancer; for example, to target multiple myeloma, a multispecific TCR / CAR may target any two or more of: BCMA, GPRC5D, SLAMF7, and CD229. In some embodiments, a multispecific TCR / CAR binds to CD19 and CD22, or binds to CD19 and BCMA, or binds to BCMA and SLAMF7, or binds to BCMA and CD229, or binds to SLAMF7 and CD229. In certain embodiments, a target comprises a protein ligand and a binding domain is from a receptor for the ligand. For example, a binding domain can comprise a receptor ectodomain from Bcl2 and a target comprises BIM.

[0169] Also provided are fusion polypeptides that comprise a binding-domain-containing TCR / CAR polypeptide of the present disclosure. Any of the presently disclosed first or second TCR / CAR polypeptides may be provided as an isolated polypeptide, provided that the polypeptide comprises a binding domain, and not accompanied by a cognate TCR / CAR polypeptide. Polynucleotides and vectors that encode the fusion polypeptides are also provided.

[0170] In some embodiments, a presently disclosed protein is expressed as membrane-bound molecule at a cell surface.

[0171] Certain embodiments provide a host cell that encodes or expresses a CD3 / costimulatory domain fusion protein of the present disclosure, and optionally further encodes or expresses a target-binding protein (e.g., a chTCR or TCR / CAR).

[0172] Certain other embodiments provide a host cell that encodes or expresses an engineered CD3 polypeptide of the present disclosure, and optionally further encodes or expresses a targetbinding protein (e.g., a chTCR or TCR / CAR).

[0173] A TCR / CAR (polypeptide dimer) can be expressed as a membrane-bound protein or protein dimer at a cell surface of a host cell. Also provided are host cells that comprise a polynucleotide or vector encoding a presently disclosed TCR / CAR. In certain embodiments, a host cell comprises a hematopoietic progenitor cell, a hematopoeitic stem cell, or an immune system cell, such as a human immune system cell. In certain embodiments, an immune system cell comprises a T cell, aNK-T cell, or a macrophage. In certain embodiments, a T cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, an ap+ T cell, a 76+ T cell, or any combination thereof. In certain embodiments, a T cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

[0174] In some embodiments, a host cell comprises a chromosomal knockout of a CD3C locus, of a CD3e locus, of a TIGIT locus, of a TCR locus (e.g. TRAC, TRBC), of a CD8 locus, of a CD4 locus, of a PD-1 locus, of a LAG-3 locus, of a TIM3 locus, of an HLA locus e.g. a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a pi microglobulin, or a P2 microglobulin), of a TGFfiRl locus, of a TGFfiR locus, of a LAT locus, of an A2AR locus, of a Fas locus, of a FasL locus, of a B7-H3 locus, of a B7-H4 locus, of an IDO locus, of a VISTA locus, of a SIGLEC7 locus, of a SIGLEC9 locus, of a CBLB locus, of a RASA2 locus, of a UBASH3A locus, of a GISH locus, or of any combination thereof.

[0175] In some embodiments, a host cell expresses a TCR / CAR and comprises a chromosomal knockout of one or more genes expressing a protein that is recognized by the TCR / CAR. For example, in some embodiments, a host cell expresses a TCR / CAR that binds SLAMF7 and comprises a chromosomal knockout of a SLAMF7 locus. In some embodiments, a host cell expresses a TCR / CAR that binds CD229 and comprises a chromosomal knockout of a CD229 locus. Adminstering a plurality of such host cells to a subject may reduce fratricidal lysis as between the host cells. In certain further embodiments, the host cell is a T cell. The T cell can further comprise a chromosomal gene knockout of a CD4 gene locus and / or of a CD8 gene locus.

[0176] In further aspects, expression constructs are provided, wherein the expression constructs comprise a polynucleotide of the present disclosure operably linked to an expression control sequence (e.g., a promoter). An exemplary promoter sequence includes an EFla promoter or a MNDu3 promoter. In certain embodiments, the expression construct is comprised in a vector. An exemplary vector may comprise a polynucleotide capable of transporting another polynucleotide to which it has been linked, or which is capable of replication in a host organism. Some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g., lentiviral vector, retroviral vector). Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors"). According to related embodiments, it is further understood that, if one or more agents (e.g., polynucleotides encoding fusion proteins as described herein) are co-administered to a subject, that each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.

[0177] As shown herein, the combination of an EFla promoter and a MNDU3 promoter provides advantages for transducing into and expressing multiple expression products of interest. An EFla core promoter and / or an MND minimal promoter can be used, alternatively.

[0178] In certain embodiments, an expression construct encodes a chTCR of the present disclosure and at least one additional expression product (e.g., polypeptide) of interest. The expression construct comprises an EFla promoter (SEQ ID NO:7) and a MNDu3 promoter (SEQ ID NO:8), optionally on opposing polynucleotide strands and oriented in opposite directions. One of the EFla promoter and the MNDu3 promoter is operably linked to a polynucleotide encoding the chTCR, and the other of the EFla promoter and the MNDu3 promoter is operably linked to a polynucleotide encoding the at least one additional expression product of interest. In some embodiments, the EFla promoter is operably linked to the polynucleotide encoding the 30 chTCR and the MNDu3 promoter is operably linked to the polynucleotide encoding the at least one expression product of interest. Non-limiting examples of expression construct architectures comprising an EFla promoter and a MNDu3 promoter are shown in certain of the Figures. In Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide additional definitions of certain terms to be used herein. Still more definitions are set forth throughout this disclosure.

[0179] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, is to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. "About" includes ±15%, ±10%, and ±5%. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the terms "include," "have," and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0180] "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.

[0181] The term "consisting essentially of' is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a protein domain, linker, signal peptide) or a protein (which may have one or more domains, regions, or modules) "consists essentially of' a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy -terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

[0182] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, / .<., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0183] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).

[0184] A "conservative substitution" refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W. H. Freeman and Company. Variant proteins, peptides, polypeptides, and amino acid sequences of the present disclosure can, in certain embodiments, comprise one or more conservative substitutions relative to a reference amino acid sequence.

[0185] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers. As used herein, "fusion protein" or "fusion polypeptide" refers to a protein that, in a single chain, has at least two distinct domains and / or motifs, wherein the domains or motifs are not naturally found together (e.g., in the given arrangement, order, or number, or at all) in a protein. In certain embodiments, a fusion protein comprises at least two distinct domains and / or motifs that are not found together in a single naturally occurring peptide or polypeptide. In certain embodiments, a fusion protein comprises amino acid sequences from two or more distinct polypeptides. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized. A fusion protein may further contain other components, such as a tag, a linker, or a transduction marker. In certain embodiments, a fusion protein expressed or produced by a host cell (e.g., a T cell) locates to the cell surface, where the fusion protein can be anchored to the cell membrane.

[0186] "Nucleic acid molecule" or "polynucleotide" refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.

[0187] Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C.

[0188] Nucleic acid molecule variants retain the capacity to encode a protein or a binding domain thereof having a functionality described herein, such as specifically binding a target molecule.

[0189] "Percent sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters which originally load with the software when first initialized.

[0190] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and / or such nucleic acid or polypeptide could be part of a composition e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. In some embodiments, a composition of the present disclosure can be "isolated" in the sense that it is physically separated from and not comprised within a subject to whom the composition can be, was, or is to be administered.

[0191] The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).

[0192] A "functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs, in some contexts slightly, in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding," "similar affinity" or "similar activity" when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant) or avidity; or an assay measuring TCR signaling or an activity stimulated thereby (e.g. as exemplified herein, such as measuring IFN-g production, IL-2 production, intracellular calcium flux, cellular avidity as determined by the percentage of cells in a sample that bind to antigen, proliferation, specific cytotoxicity against a target cell, NF AT expression, NFkB expression, AP-1 expression, Nur77 expression) optionally in the presence of PVR or PVR-expressing cells.)) As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., T cell signaling and / or activity following binding to antigen). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of this disclosure has "similar binding" or "similar activity" when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity), such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release).

[0193] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules (e.g., receptors, ligands, etc.) may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector). It will be appreciated that in the case of a host cell that comprises a heterologous polynucleotide, the polynucleotide is "heterologous" to progeny of the host cell, whether or not the progeny were themselves manipulated to, for example, introduce the polynucleotide.

[0194] The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof.

[0195] As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject. The term "expression", as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0196] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

[0197] As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid," "expression plasmid," "virus" and "vector" are often used interchangeably.

[0198] The term "introduced" in the context of inserting a nucleic acid molecule into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). As used herein, the term "engineered," "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.

[0199] The term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther. 5:108, 2003; Mates et al., Nat. Genet. 41:753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

[0200] As used herein, the term "host" refers to a cell (e.g., T cell) or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a protein of interest. In certain embodiments, a host cell may optionally possess or be modified to include other genetic modifications that confer desired properties related or unrelated to, e.g., biosynthesis of the heterologous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous host cell protein; expression of an antigen-binding protein).

[0201] As used herein, "enriched" or "depleted" with respect to amounts of cell types in a mixture refers to an increase in the number of the "enriched" type, a decrease in the number of the "depleted" cells, or both, in a mixture of cells resulting from one or more enriching or depleting processes or steps. Thus, depending upon the source of an original population of cells subjected to an enriching process, a mixture or composition may contain 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more (in number or count) of the "enriched" cells. Cells subjected to a depleting process can result in a mixture or composition containing 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% percent or less (in number or count) of the "depleted" cells. In certain embodiments, amounts of a certain cell type in a mixture will be enriched and amounts of a different cell type will be depleted, such as enriching for CD4+cells while depleting CD8+cells, or enriching for CD62L+cells while depleting CD62L cells, or combinations thereof.

[0202] "T cell receptor" (TCR) refers to a multi-protein complex (each component protein having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rdEd., Current Biology Publications, p. 4:33, 1997) capable of binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and P chains (also known as TCRa and TCRP, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively). The extracellular portion of TCR chains (e.g., a-chain, P-chain) contain two immunoglobulin domains, a variable domain (e.g., a-chain variable domain or Va, P-chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5thed.) at the N-terminus, and one constant domain (e.g., a- chain constant domain or Ca, typically amino acids 117 to 259 based on Kabat, 0-chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. Non-limiting examples of TCR constant domain amino acid sequences are provided herein. The variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U. S. A. 57:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The source of a TCR or TCR binding domain as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit, non-human primate, or other mammal.

[0203] TCR / CARs of the present disclosure (also referred to as chTCRs) are structurally distinct from TCRs.

[0204] "CD3" is a multi-protein complex of six chains (see, Abbas and Lichtman, 2003;

[0205] Janeway etal., p. 172 and 178, 1999). In mammals, the complex generally comprises a CD3y chain, a CD36 chain, two CD3s chains, and a homodimer of CD3(^ chains. The CD3y, CD36, and CD3s chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD36, and CD3s chains are negatively charged, which is thought to allow these chains to associate with positively charged regions of T cell receptor chains. The intracellular tails of the CD3 complex proteins contain immunoreceptor tyrosine-based activation motifs or IT AMs, which are thought to be important for T cell signaling in response to antigen binding. CD3y, CD36, CD3s, and CD3(^ may be referred to as CD3 “subunits”.

[0206] CD3, as well as the protein subunits, domains, and sequences therefrom, may be from various animal species, including human, mouse, rat, or other mammals. In certain embodiments, CD3 proteins, subunits, domains, and / or sequences are human. In certain embodiments, CD3 proteins, subunits, domains, and / or sequences are engineered from human CD3 proteins, subunits, domains, and / or sequences.

[0207] In certain embodiments, a TCR or a chTCR is found on the surface of T cells (also referred to as T lymphocytes) and associates with the CD3 complex.

[0208] In some embodiments, a chTCR can associate with an endogenous CD3 complex.

[0209] "Major histocompatibility complex molecules" (MHC molecules) refer to glycoproteins that deliver peptide antigens to a cell surface. MHC class I molecules are heterodimers consisting of a membrane spanning a chain (with three a domains) and a non-covalently associated P2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and P, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide: MHC complex is recognized by CD8+T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4+T cells. An MHC molecule may be from various animal species, including human, mouse, rat, cat, dog, goat, horse, or other mammals. "CD4" refers to an immunoglobulin co-receptor glycoprotein that can assist the TCR in binding to antigen: MHC and communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKB P01730). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and includes four immunoglobulin domains (DI to D4) that are expressed at the cell surface. During antigen recognition, CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII P2, while the TCR complex binds antigen: MHCII al / pi).

[0210] As used herein, the term "CD8 co-receptor" or "CD8" means the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer. The CD8 co-receptor can assist in the function of cytotoxic T cells (CD8+) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 27:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 7:81-88, 2004). In humans, there are five (5) different CD8 beta chains (see UniProtKB identifier Pl 0966) and a single CD8 alpha chain (see UniProtKB identifier P01732).

[0211] "Chimeric antigen receptor" (CAR) refers to a fusion protein engineered to contain two or more amino acid sequences (which may be naturally occurring amino acid sequences) linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as an antigen-specific receptor when present on a surface of a cell. CARs of the present disclosure are single-chain fusion proteins that include an extracellular portion comprising an antigen-binding domain (e.g., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a scFv or scTCR derived from an antibody or TCR (respectively) specific for a cancer antigen, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell, a designed ankyrin repeat protein (DARPin), an engineered fibronectin type three domain (also referred-to as a monobody) such as an Adnectin™, a ligand (e.g., a cytokine, if the target is a cytokine receptor), a receptor ectodomain (e.g., a cytokine receptor, if the target is a cytokine) or the like) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain etal., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3): 220 (2016); Stone etal., Cancer Immunol. Immunother., 63(11):1163 (2014)). In certain embodiments, a CAR comprises an antigenspecific TCR binding domain (see, e.g., Walseng et al., Scientific Reports 7: 10713, 2017; it will be understood that TCR / CARs of the present disclosure possess a distinct structure to CARs).

[0212] The term "variable region" or "variable domain" refers to the domain an antibody heavy or light chain (or, for TCRs, of a TCR of a TCR a-chain or P-chain (or y-chain and 8-chain for y6 TCRs)), that is involved in binding to antigen (i.e., contains amino acids and / or other structures that contact antigen and result in binding). The variable domains of cognate chains generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs. In both TCRs and antibodies, framework regions separate CDRs and CDRs are situated between framework regions (i.e., in primary structure).

[0213] The terms "complementarity determining region," and "CDR," are synonymous with "hypervariable region" or "HVR," and refer to sequences of amino acids within TCR or antibody variable regions, which, in general, confer antigen specificity and / or binding affinity and are separated from one another in primary structure by framework sequence. In some cases, framework amino acids can also contribute to binding, e.g., may also contact the antigen or antigen-containing molecule. In general, there are three CDRs in each variable region (e.g., three CDRs in each of the antibody heavy chain and light chain variable regions). Variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).

[0214] "Antigen" or " Ag" as used herein refers to an immunogenic molecule that can provoke an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells (e.g., T cells), secretion of cytokines, or any combination thereof. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen. In any of the presently disclosed embodiments, a target can be, or can comprise, an antigen.

[0215] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0216] "Treat" or "treatment" or "ameliorate" refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). In general, an appropriate dose or treatment regimen comprising a host cell of the present disclosure, and optionally an adjuvant, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof. In some embodiments, a benefit of a cellular immunotherapy of this disclosure can further include a reduction (e.g., in number or severity) or absence of a cytokine-related toxicity, such as a cytokine release syndrome.

[0217] A "therapeutically effective amount" or "effective amount" of a composition of this disclosure, refers to an amount of the composition sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner. In the case of cancers, benefits can include, for example, a reduction in the size, area, volume, and / or density of a tumor, and / or a reduction or reversal in the rate of tumor growth or spread of cancer.

[0218] When referring to an individual active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.

[0219] The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refer to biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject and generally recognized as safe or not causing a serious adverse event.

[0220] As used herein, "statistically significant" refers to a p-value of 0.050 or less when calculated using the Student’s t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.

[0221] As used herein, the term "adoptive immune therapy" or "adoptive immunotherapy" refers to administration of naturally occurring or genetically engineered, disease-antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient). or CD3s Costimulatory Domain Fusion Proteins

[0222] Certain embodiments provide a fusion protein comprising, consisting essentially of, or consisting of: an extracellular component comprising, consisting essentially of, or consisting of the extracellular domain of a CD3(^ (e.g., QSFGLLDPK (SEQ ID NO.: 152)), or a functional fragment or variant thereof, wherein the variant optionally comprises one, two, three, or four amino acid substitutions, insertions, and / or deletions relative to SEQ ID NO.: 152;

[0223] an intracellular component comprising, consisting essentially of, or consisting of (i) an intracellular signaling domain of the CD3(^, or a functional fragment or variant thereof, and (ii) a costimulatory portion comprising, consisting essentially of, or consisting of (1) a costimulatory domain of a 4- IBB, or a functional fragment or variant thereof, linked or fused to an amino acid sequence that is not the intracellular signaling domain of the CD3(^ or a functional fragment or variant thereof; (2) a costimulatory domain of a CD226, or a functional fragment or variant thereof, wherein, optionally, the variant comprises one or more (e.g. substitution, e.g. nonconservative) mutation that (a) provides increased expression of the fusion protein in a T cell exposed to PVR, as compared to expression of endogenous CD226 by the T cell exposed to PVR, and / or (b) disrupts a Src kinase phosphorylation site on the fusion protein, and / or (c) reduces ubiquitination of the fusion protein by CBL-B, wherein, further optionally, the mutated CD226 costimulatory domain or a functional fragment or variant thereof comprises a substitution mutation at a position corresponding to one or more of positions K295, Y319, and K333, optionally comprising K295A, Y319F, and / or K333 A mutations; (3) a costimulatory domain of a CD28, or a functional fragment or variant thereof; (4) a costimulatory domain of a CD2, or a functional fragment or variant thereof; and / or (5) a costimulatory domain of an 0X40, or a functional fragment or variant thereof; and a transmembrane component disposed between and connecting the extracellular component and the intracellular component, wherein, optionally, the transmembrane component comprises, consists essentially of, or consists of the transmembrane domain of the CD3(^ or a functional fragment or variant thereof.

[0224] In some embodiments, the extracellular domain of a CD3(^ or a functional fragment or variant thereof, the intracellular domain of the CD3(^ or a fragment thereof, and, optionally, the transmembrane domain of the CD3(^ or a fragment thereof, are human.

[0225] In some embodiments, the costimulatory portion of the intracellular domain of the fusion protein comprises a costimulatory domain of CD27, CD3e, CD36, CD3y, CD79A, CD79B, SLAMF1, ICOS, DAP10, CD25, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, LIGHT, CD30, Lek, LAG3, LAT, LRP, NKG2D, N0TCH1, N0TCH2, N0TCH3, N0TCH4, R0R2, Ryk, Slp76, pTa, TCRa, TCRP, TRIM, Zap70, PTCH2, or a functional fragment or variant thereof, or any combination thereof. In some embodiments, the extracellular component, transmembrane component, and intracellular domain of a CD3(^ together comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO.: 153, wherein SEQ ID NO.: 153 is optionally preceded immediately by a signal peptide, wherein, further optionally, the signal peptide comprises, consists essentially of, or consists of the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO.: 154) wherein, still further optionally, the fusion protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO.:155.

[0226] In some embodiments, the intracellular component, the intracellular domain of the CD3(^ (or a functional fragment or variant thereof) is disposed N-terminal to the costimulatory portion. In certain other embodiments, the intracellular domain of the CD3(^ (or a functional fragment or variant thereof) is disposed C-terminal to the costimulatory portion.

[0227] In some embodiments: (a) the functional variant or fragment of a costimulatory domain of a 4- IBB does not comprise a(n, e.g., native) basic rich motif (e.g., does not comprise the amino acid sequence KRGRKKLLYIFKQPF (SEQ ID NO.: 156)) and / or the amino acid sequence that is not the intracellular signaling domain of the CD3(^ comprises, consists essentially of, or consists of DYHNPGYLVVLPDSTP (Ml-GS; SEQ ID NO: 157), EELDENYVPMNPNSPP (M2; SEQ ID NO.:158), EEGAPDYENLQELNHP (M3; SEQ ID NO.:159), LGSNQEEAYVTMSSFYQNQ (M4; SEQ ID NO:160), LPMDTEVYESPFADPEEIR (M5; SEQ ID NO: 161), KPMAESITYAAVARHSAG (M6; SEQ ID NO.: 162), LPTWSTPVQPMALIVLG (M7; SEQ ID NO.: 163), PAPSIDRSTKPPLDRSL (M8; SEQ ID NO: 164), GSNTAAPVQETLHGCQ (M9; SEQ ID NO: 165), DDSLPHPQQATDDSGHES (MIO; SEQ ID NO.:166), KAPHAKQEPQEINFPDDLP (Mil; SEQ ID NO.:167), GSGPGSRPTAVEGLALGSS (M12; SEQ ID NO.:168),

[0228] SAGS AGS AGS AGS AGS AG (Ml 3; SEQ ID NO: 169); (GSDYHNPGYLVVLPDSTP)X, wherein X is any integer >1 and is preferably 1 or 2 (Ml; SEQ ID NO.: 170); or any combination of two or more of the foregoing; (b) the costimulatory domain of a CD226 or a functional fragment or variant thereof comprises a K295A mutation, a Y319F mutation, a K333 A mutation, K295A and Y319F mutations, K295A and K333A mutations, K333A and Y319F mutations, or K295A, Y319F, and K333A mutations, and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence NRRRRRERRD (SEQ ID NO.: 171)); (c) the costimulatory domain of a CD28, or a functional fragment or variant thereof, comprises a native dileucine (LL) or comprises a diglycine (GG) in place of the native LL, and / or comprises an amino acid substitution at any one or more of amino acids Y191, Y206, Y209, Y218, P196, P199, P208, and P211, as described in International Application No. PCT / US2019 / 019014 (e.g., comprises Y191F, Y206F, Y209F, Y218F, P196A, P199A, P208A, P211A, or any combination thereof)), and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence RSKRSR (SEQ ID NO.: 172)); or (d) the costimulatory domain of a CD2, or a functional fragment or variant thereof does not comprise the amino acid sequence KRKKQRSRR (SEQ ID NO.: 173) and / or comprises a truncation of from 1 to 44 N-terminal amino acids of the CD2 intracellular domain.

[0229] In certain embodiments, the extracellular component, the transmembrane component, and the intracellular domain of a CD3(^ together comprise, consist essentially of, or consist of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO.: 153, or to the amino acid sequence set forth in SEQ ID NO.: 155. In certain embodiments, the costimulatory portion of the intracellular component comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to:

[0230] the amino acid sequence set forth in SEQ ID NO.: 174; the amino acid sequence set forth in SEQ ID NO.: 175; the amino acid sequence set forth in SEQ ID NO.:79; the amino acid sequence set forth in SEQ ID NO.: 176; the amino acid sequence set forth in SEQ ID NO.:348;

[0231] an amino acid sequence according to the consensus sequence RSKRSRX1X2HSDX3MNMTX4RRX5GPTRKHX6QX7X8AX9PRDFAAX10RS wherein Xi is L or G, X2 is L or G, Xi and X2 preferably being L-L or G-G, X3 is Y or F, X4 is P or A, X5 is P or A, X6 is Y or F, X7 is P or A, X8 is Y or F, X9 is P or A, and X10 is Y or F (SEQ ID NO.: 177), optionally to the amino acid sequence set forth in SEQ ID NO.: 178 or to the amino acid sequence set forth in SEQ ID NO.: 179; the amino acid sequence set forth in SEQ ID NO.: 180; the amino acid sequence set forth in SEQ ID NO.: 181; the amino acid sequence set forth in SEQ ID NO.:246; the amino acid sequence set forth in SEQ ID NO.:247; or the amino acid sequence set forth in SEQ ID NO.: 142.

[0232] Certain embodiments provide a fusion protein comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of: the amino acid sequence set forth in SEQ ID NO.: 182; the amino acid sequence set forth in SEQ ID NO.: 183; the amino acid sequence set forth in SEQ ID NO.: 184; the amino acid sequence set forth in SEQ ID NO.: 185; the amino acid sequence set forth in SEQ ID NO.: 186; the amino acid sequence set forth in SEQ ID NO.: 187; the amino acid sequence set forth in SEQ ID NO.: 188; the amino acid sequence set forth in SEQ ID NO.: 189; the amino acid sequence set forth in SEQ ID NO.:253; the amino acid sequence set forth in SEQ ID NO.:252; the amino acid sequence set forth in SEQ ID NO.: 190; the amino acid sequence set forth in SEQ ID NO.: 191; the amino acid sequence set forth in SEQ ID NO.: 192; the amino acid sequence set forth in SEQ ID NO.: 193; the amino acid sequence set forth in SEQ ID NO.: 194; the amino acid sequence set forth in SEQ ID NO.: 195; the amino acid sequence set forth in SEQ ID NO.:196; the amino acid sequence set forth in SEQ ID NO.:197; the amino acid sequence set forth in SEQ ID NO.: 198; the amino acid sequence set forth in SEQ ID NO.: 199; the amino acid sequence set forth in SEQ ID NO.:200; the amino acid sequence set forth in SEQ ID NO.:201; the amino acid sequence set forth in SEQ ID NO.:255; the amino acid sequence set forth in SEQ ID NO.:254; the amino acid sequence set forth in SEQ ID NO.:202; or the amino acid sequence set forth in SEQ ID NO.:203. Certain embodiments provide a fusion protein comprising, consisting essentially of, or consisting of:

[0233] an extracellular component comprising, consisting essentially of, or consisting of the extracellular domain of a CD3s, or a functional fragment or variant thereof;

[0234] an intracellular component comprising, consisting essentially of, or consisting of (i) an intracellular signaling domain of the CD3s, or a functional fragment or variant thereof and (ii) a costimulatory portion, wherein the costimulatory portion optionally comprises a costimulatory domain, or a functional fragment or variant thereof, from CD2, CD226, CD28, 4- IBB, 0X40, CD27, CD36, CD3y, CD3i CD79A, CD79B, SLAMF1, ICOS, DAP10, CD25, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, LIGHT, CD30, Lek, LAG3, LAT, LRP, NKG2D, N0TCH1, N0TCH2, N0TCH3, N0TCH4, R0R2, Ryk, Slp76, pTa, TCRa, TCRp, TRIM, Zap70, PTCH2, or any combination thereof; and

[0235] a transmembrane component disposed between and connecting the extracellular component and the intracellular component, wherein, optionally, the transmembrane component comprises, consists essentially of, or consists of the transmembrane domain of the CD3s, or a functional fragment or variant thereof.

[0236] In some embodiments, the costimulatory portion comprises, consists essentially of, or consists of:

[0237] (1) a costimulatory domain of a 4-1BB, or a functional fragment or variant thereof, optionally being linked or fused to an amino acid sequence that is not the intracellular signaling domain of the CD3s or a functional fragment or variant thereof; (2) a costimulatory domain of a CD226, or a functional fragment or variant thereof, wherein, optionally, the variant comprises one or more (e.g. substitution, e.g. non-conservative) mutation that (a) provides increased expression of the fusion protein in a T cell exposed to PVR, as compared to expression of endogenous CD226 by the T cell exposed to PVR, and / or (b) disrupts a Src kinase phosphorylation site on the fusion protein, and / or (c) reduces ubiquitination of the fusion protein by CBL-B, wherein, further optionally, the mutated CD226 costimulatory domain or a functional fragment or variant thereof comprises a substitution mutation at a position corresponding to one or more of positions K295, Y319, and K333, optionally comprising K295A, Y319F, and / or K333 A mutations; (3) a costimulatory domain of a CD28, or a functional fragment or variant thereof; (4) a costimulatory domain of a CD2, or a functional fragment or variant thereof; and / or (5) a costimulatory domain of an 0X40, or a functional fragment or variant thereof. In some embodiments, the extracellular component, the transmembrane component, and the intracellular domain of a CD3s together comprise, consist essentially of, or consist of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO.:204, optionally preceded immediately by a signal peptide, wherein, further optionally, the signal peptide comprises, consists essentially of, or consists of the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) wherein, still further optionally, the fusion protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO.:206.

[0238] In some embodiments, in the intracellular component, the intracellular domain of the CD3s (or a functional fragment or variant thereof) is disposed N-terminal to the costimulatory portion. In other embodiments, in the intracellular component, the intracellular domain of the CD3s (or a functional fragment or variant thereof) is disposed C-terminal to the costimulatory portion.

[0239] In some embodiments:

[0240] (a) the functional variant or fragment of a costimulatory domain of a 4- IBB does not comprise a(n, e.g., native) basic rich motif (e.g., does not comprise the amino acid sequence KRGRKKLLYIFKQPF (SEQ ID NO.: 156)) and / or the amino acid sequence that is not the intracellular signaling domain of the CD3(^ comprises, consists essentially of, or consists of DYHNPGYLVVLPDSTP (Ml-GS; SEQ ID NO: 157), EELDENYVPMNPNSPP (M2; SEQ ID NO: 158), EEGAPDYENLQELNHP (M3; SEQ ID NO: 159), LGSNQEEAYVTMSSFYQNQ (M4; SEQ ID NO: 160), LPMDTEVYESPFADPEEIR (M5; SEQ ID NO: 161), KPMAESITYAAVARHSAG (M6; SEQ ID NO: 162), LPTWSTPVQPMALIVLG (M7; SEQ ID NO: 163), PAPSIDRSTKPPLDRSL (M8; SEQ ID NO: 164), GSNTAAPVQETLHGCQ (M9; SEQ ID NO.:165), DDSLPHPQQATDDSGHES (MIO; SEQ ID NO.:166), KAPHAKQEPQEINFPDDLP (Mil; SEQ ID NO: 167), GSGPGSRPTAVEGLALGSS (M12; SEQ ID NO: 168), SAGS AGS AGS AGS AGS AG (Ml 3; SEQ ID NO: 169);

[0241] (GSDYHNPGYLVVLPDSTP)x, wherein x is any integer >1 and is preferably 1 or 2 (Ml; SEQ ID NO.: 170); or any combination of two or more of the foregoing; (b) the costimulatory domain of a CD226 or a functional fragment or variant thereof comprises a K295A mutation, a Y319F mutation, a K333 A mutation, K295A and Y319F mutations, K295A and K333A mutations, K333A and Y319F mutations, or K295A, Y319F, and K333A mutations, and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence NRRRRRERRD (SEQ ID NO: 171));

[0242] (c) the costimulatory domain of a CD28, or a functional fragment or variant thereof, comprises a native dileucine (LL) or comprises a diglycine (GG) in place of the native LL, and / or comprises an amino acid substitution at any one or more of amino acids Y191, Y206, Y209, Y218, Pl 96, Pl 99, P208, and P211, as described in International Application No.

[0243] PCT / US2019 / 019014 (e.g., comprises Y191F, Y206F, Y209F, Y218F, P196A, P199A, P208A, P211 A, or any combination thereof, and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence RSKRSR (SEQ ID NO.: 172)); or

[0244] (d) the costimulatory domain of a CD2, or a functional fragment or variant thereof does not comprise the amino acid sequence KRKKQRSRR (SEQ ID NO.: 173) and / or comprises a truncation of from 1 to 44 N-terminal amino acids of the CD2 intracellular domain.

[0245] 16. The fusion protein of any one of claims 10-15, wherein the costimulatory portion of the intracellular component comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to:

[0246] the amino acid sequence MRPVQTTQEEDGCSCRFPEEEEGGCELGSDYHNPGYLVVLPDSTP (SEQ ID NO: 174); the amino acid sequence MRPVQTTQEEDGCSCRFPEEEEGGCELGSDYHNPGYLVVLPDSTPGSDYHNPGYLVVLP DSTP (SEQ ID NO: 175);

[0247] the amino acid sequence NRRRRRERRDLFTESWDTQAAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPATR

[0248] V (SEQ ID NO: 79);

[0249] the amino acid sequence LFTESWDTQAAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPATRV (SEQ ID NO.: 176);

[0250] the amino acid sequence LFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO.:348); an amino acid sequence according to the consensus sequence RSKRSRX1X2HSDX3MNMTX4RRX5GPTRKHX6QX7X8AX9PRDFAAX10RS wherein Xi is L or G, X2 is L or G, Xi and X2 preferably being L-L or G-G, X3 is Y or F, X4 is P or A, X5 is P or A, X6 is Y or F, X7 is P or A, X8 is Y or F, X9 is P or A, and X10 is Y or F (SEQ ID NO.: 177), optionally to the amino acid sequence RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO.: 178) or to the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 179);

[0251] the amino acid sequence GGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 180);

[0252] the amino acid sequence LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 181);

[0253] the amino acid sequence KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ IDNO.:246);

[0254] the amino acid sequence QHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPH GAAENSLSPSSN (SEQ ID NO.:247);

[0255] or

[0256] the amino acid sequence ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 142).

[0257] Also provided is a fusion protein comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of: the amino acid sequence set forth in SEQ ID NO.:207; the amino acid sequence set forth in SEQ ID NO.:208; the amino acid sequence set forth in SEQ ID NO.:209; the amino acid sequence set forth in SEQ ID NO.:210; the amino acid sequence DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICIT GGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL YSGLNQRRI (SEQ ID NO.:211); the amino acid sequence set forth in SEQ ID NO.:212; the amino acid sequence set forth in SEQ ID NO.:213; the amino acid sequence set forth in SEQ ID NO.:214; the amino acid sequence set forth in SEQ ID NO.:215; the amino acid sequence set forth in SEQ ID NO.:216; the amino acid sequence set forth in SEQ ID NO.:217; the amino acid sequence set forth in SEQ ID NO.:218;

[0258] the amino acid sequence DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICIT GGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL YSGLNQRRILFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV

[0259] (SEQ ID NO.:257);

[0260] the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPG SEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLY LRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGR QRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRILFTESWDTQKAPNNYRSPISTSQP TNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO.:256); the amino acid sequence set forth in SEQ ID NO.:219; the amino acid sequence set forth in SEQ ID NO.:220; the amino acid sequence set forth in SEQ ID NO.:221; the amino acid sequence set forth in SEQ ID NO.:222; the amino acid sequence set forth in SEQ ID NO.:223; the amino acid sequence set forth in SEQ ID NO.:224; the amino acid sequence set forth in SEQ ID NO.:225; the amino acid sequence set forth in SEQ ID NO.:226; the amino acid sequence set forth in SEQ ID NO.:227; the amino acid sequence set forth in SEQ ID NO.:228; the amino acid sequence set forth in SEQ ID NO.:229; the amino acid sequence set forth in SEQ ID NO.:230;

[0261] the amino acid sequence DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICIT GGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL YSGLNQRRIGSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPL PRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO.:259);

[0262] the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARV CENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQ NKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGSQHPPPPPGHRSQAPSHRPPPPGHRVQ HQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO.:258); the amino acid sequence set forth in_SEQ ID NO.:231; or the amino acid sequence set forth in SEQ ID NO.:232.

[0263] In some embodiments, when the fusion protein is expressed by a human T cell that further expresses a target (e.g., antigen)-binding protein (e.g., a TCR, a CAR, or a chTCR (also referred to as a chimeric TCR or a TCR / CAR)), the T cell has increased production of one or more cytokine (e.g., IL-2, IFN-, or both) when in the presence of the antigen, and / or has increased proliferation when in the presence of the target, as compared to a reference human T cell (i.e., a T cell that is substantially identical to the human T cell) that does not express the fusion protein.

[0264] In some embodiments, the intracellular component comprises a linker disposed between and connecting: (i) the intracellular domain of the CD3(^ or a functional fragment or variant thereof and the costimulatory portion; and / or (ii) the costimulatory domain or a functional fragment or variant thereof and the amino acid sequence that is not the CD3(^ intracellular signaling domain or a functional fragment or portion thereof, wherein, optionally, the linker comprises: a (GlyxSery)n sequence, wherein x, y, and n are each independently 1 or more), a Whitlow linker, a Townsend linker, an alanine linker (e.g., AAA), a proline-glycine linker (e.g., PG or GP or GPP or PGP), or any combination thereof.

[0265] In some embodiments, the intracellular component comprises a linker disposed between and connecting: (i) the intracellular domain of the CD3s or a functional fragment or variant thereof and the costimulatory portion; and / or (ii) the costimulatory domain or a functional fragment or variant thereof and the amino acid sequence that is not the CD3s intracellular signaling domain or a functional fragment or portion thereof, wherein, optionally, the linker comprises: a (GlyxSery)n sequence, wherein x, y, and n are each independently 1 or more), a Whitlow linker, a Townsend linker, an alanine linker (e.g., AAA), a proline-glycine linker (e.g., PG or GP or GPP or PGP), or any combination thereof.

[0266] In some embodiments, when expressed by a host cell (e.g., a human T cell), two molecules of a CD3(7costimulatory domain fusion protein are capable of together forming a protein homodimer; a single molecule of a CD3(7costimulatory domain fusion protein is capable of forming a protein heterodimer with a single molecule of an endogenous CD3(^ protein; or both.

[0267] In some embodiments, when expressed by a host cell (e.g., a human T cell), a single molecule of a CD3s / costimulatory domain fusion protein is capable of forming a protein heterodimer with a single molecule of an endogenous CD36 or CD3y protein. Also provided is a fusion protein comprising an extracellular component, an intracellular component, and a transmembrane component disposed between and connecting the extracellular and intracellular components, wherein the intracellular component comprises, consists essentially of, or consists of:

[0268] the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSDYHNPGYLVVLPDST

[0269] P (SEQ ID NO.:233);

[0270] the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSDYHNPGYLVVLPDST PGSDYHNPGYLVVLPDSTP (SEQ ID NO.:234);

[0271] the amino acid sequence MRPVQTTQEEDGCSCRFPEEEEGGCELGSDYHNPGYLVVLPDSTP (SEQ IDNO.:235); or the amino acid sequence MRPVQTTQEEDGCSCRFPEEEEGGCELGSDYHNPGYLVVLPDSTPGSDYHNPGYLVVLP DSTP (SEQ ID NO.:236).

[0272] In some embodiments, the intracellular component further comprises an intracellular signaling domain of a CD3(^ or an intracellular signaling domain of a CD3s.

[0273] As described further herein, in certain embodiments, a CD3 / costimulatory domain fusion protein is encoded by a polypeptide that further encodes a target-binding protein, such as a chTCR, a TCR, a scTCR, a mutSTAR, a STAR, a Co-STAR, a HIT receptor, or a CAR. In certain embodiments, a fusion protein is expressed by a host cell that further expresses a targetbinding protein, such as a chTCR, a TCR, a scTCR, a mutSTAR, a STAR, a Co-STAR, a HIT receptor, or a CAR.

[0274] Engineered CD3 Polypeptides

[0275] Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in one or more CD3(^ IT AM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues are each substituted with a different amino acid residue, as described herein, in any one, any two, or all three of the CD3(^ IT AM motifs.

[0276] Also provided is a polypeptide that comprises a CD3s polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in the CD3s IT AM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD3s ITAM motif are each substituted with a different amino acid residue, as described herein.

[0277] Also provided is a polypeptide that comprises a CD3y polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in the CD3y ITAM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD3y ITAM motif are each substituted with a different amino acid residue, as described herein.

[0278] Also provided is a polypeptide that comprises a CD36 polypeptide or variant thereof comprising one or more amino acid substitution of a native tyrosine amino acid residue in the CD36 ITAM motif with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD3y ITAM motif are each substituted with a different amino acid residue, as described herein. In some embodiments, two native tyrosine amino acid residues in the CD36 ITAM motif are each substituted with a different amino acid residue, as described herein.

[0279] Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising three copies of CD3(^ IT AMI: CD3(^ IT AMI, a second copy of CD3(^ IT AMI in place of CD3(^ IT M2, and a third copy of CD3(^ IT AMI in place of CD3(^ IT M3.

[0280] Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising three copies of CD3(^ IT M3: CD3(^ IT M3 in place of CD3(^ IT AMI, CD3(^ ITAM3 in place of CD3(^ ITAM2, and CD3(^ ITAM3.

[0281] Also provided is a polypeptide that comprises a CD3(^ polypeptide or variant thereof comprising CD3s ITAM1 in place of CD3(^ IT AMI, a second copy of CD3s ITAM1 in place of CD3< ITAM2, and a third copy of CD3s IT AMI in place of CD3(^ ITAM3.

[0282] Also provided is a polypeptide that comprises a CD3s polypeptide or variant thereof comprising CD3(^ IT AMI in place of CD3s ITAM.

[0283] Also provided is a polypeptide that comprises a CD3s polypeptide or variant thereof comprising: substitution of one or more native lysine amino acid residues, and / or a native arginine residue, in a CD3s basic rich sequence (BRS) motif with a different amino acid, as described herein; replacement of one or more proline amino acid residues in a native CD3s proline rich sequence (PRS) motif with a different amino acid, as described herein; or substitution of a native “RK” in CD3s ITAM motif with two amino acids, as described herein.

[0284] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: (i) in place of the amino acid sequence of SEQ ID NO.:262, an amino acid sequence according to SEQ ID NO.:263; (ii) in place of the amino acid sequence of SEQ ID NO.:264, an amino acid sequence according to SEQ ID NO.:265; and / or (iii) in place of the amino acid sequence of SEQ ID NO.:266, an amino acid sequence according to SEQ ID NO.:267. In some embodiments, (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152), or a variant thereof having one, two, or three amino acid substitutions; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID No.:240) or an amino acid sequence comprising one, two, three, or four substitutions relative to SEQ ID NO.:240. In some embodiments, (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152); and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID NO.:240). In some embodiments, the intracellular component comprises amino acid sequences according to SEQ ID NOs.: (A) 263, 264, and 266; (B) 263, 264, and 267; (C) 263, 265, and 266; (D) 263, 265, and 267; (E) 262, 264, and 267; (F) 262, 265, and 266; or (G) 262, 265, and 267. In some embodiments, the intracellular component comprises amino acid sequences according to SEQ ID NOs.: (A) 450, 264, and 266; (B) 450, 264, and 452; (C) 450, 451, and 266; (D) 450, 451, and 452; (E) 262, 451, and 266; (F) 262, 264, and 452; or (G) 450, 451, and 452.

[0285] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:284, an amino acid sequence according to SEQ ID NO.:285, preferably the amino acid sequence of SEQ ID NO.:453. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID No.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID No.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

[0286] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD36 extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD36 transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD36 intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:296, an amino acid sequence according to any one of SEQ ID NOs.:297-298a, preferably the amino acid sequence of SEQ ID NO.:455, the amino acid sequence of SEQ ID NO.:456, or the amino acid sequence of SEQ ID NO.:457. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:458, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:459 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:458, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:459 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459.

[0287] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3y extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3y transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3y intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:290, an amino acid sequence according to SEQ ID NO.:291, preferably the amino acid sequence of SEQ ID NO.:454. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:460, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:461 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:461.

[0288] In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:460, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:461 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459. In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: SEQ ID NO.:262; in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:262; and in place of the amino acid sequence of SEQ ID NO.:266, the amino acid sequence according to SEQ ID NO.:262.

[0289] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: (i) in place of the amino acid sequence of SEQ ID NO.:262, the amino acid sequence of SEQ ID NO.:266; (ii) in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:266; and (iii) the amino acid sequence of SEQ ID NO.:266.

[0290] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising: (i) in place of the amino acid sequence of SEQ ID NO.:262, the amino acid sequence of SEQ ID NO.:284; (ii) in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:284; and (iii) place of the amino acid sequence of SEQ ID NO.:266, the amino acid sequence of SEQ ID NO.:284.

[0291] In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152), or a variant thereof having one, two, or three amino acid substitutions; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID No.:240) or an amino acid sequence comprising one, two, three, or four substitutions relative to SEQ ID NO.:240. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152); and (2)the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID NO.:240).

[0292] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:284, the amino acid sequence of SEQ ID NO.:266.

[0293] In some embodiments, the present disclosure provides a polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising: (i) in place of the amino acid sequence KNRKAKAK, a variant thereof wherein one or more lysine amino acid residue is independently substituted with a neutral amino acid residue and / or wherein the arginine amino acid is substituted with a neutral amino acid residue, optionally a serine, further optionally, the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence KNRKAKAK, the amino acid sequence SNSSASAS; (ii) in place of the amino acid sequence PPPVPNPDY, a variant thereof wherein one or more proline amino acid residue is independently substituted with an alanine amino acid residue, a serine amino acid residue, or a valine amino acid residue, preferably an alanine amino acid residue, optionally wherein the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence PPPVPNPDY, the amino acid sequence PAPVANPDY; or (iii) in place of the amino acid sequence of SEQ ID NO.:284, a variant thereof wherein the first arginine amino acid residue and the lysine residue are each independently substituted with a neutral amino acid residue, preferably an alanine, optionally wherein the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence of SEQ ID NO.:284, the amino acid sequence YEPIAAGQRDLYSGL.

[0294] In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID No.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243. In some embodiments: (1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242), or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID No.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

[0295] In some embodiments, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:268-281, 286, 287, 292, 293, and 299-316.

[0296] In some embodiments, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of any one of the following constructs shown in Figure 47: CD3z_12X; CD3z_lX3; CD3z X23; CD3z 1XX; CD3z X2X; CD3z XX3; CD3z XXX or 6F; CD3e_2F; CD3g_2F; CD3d_2F; CD3d_lF; CD3z_lll; CD3z_333; CD3z_CD3elelel;

[0297] CD3e_CD3zl; CD3e_BRS; CD3e_PRS; CD3 KR, optionally with the respective CD3(^ signal peptide or CD3s signal peptide removed or comprising a GM-CSF signal peptide in place of the respective CD3(^ signal peptide or CD3s signal peptide.

[0298] ChTCRs (also called TCR / CARs or chimeric TCRs)

[0299] Certain disclosed embodiments include hybrid receptor polypeptides (also referred-to herein as chimeric TCRs, chTCRs, or TCR / CARs) that confer to a host cell e.g. a T cell) targetspecificity (including, in some contexts, high binding affinity and / or non-MHC-restricted binding) of a CAR while leveraging natural T cell signaling properties. TCR / CARs can be efficiently expressed at the surface of host cells and confer to host cells a diversified and highly sensitive signaling complex that acts with native T cell signaling properties. In some embodiments, a TCR / CAR comprises (1) a first polypeptide comprising a first TCR constant domain and (2) a second polypeptide comprising a second TCR constant domain, wherein the first TCR constant domain and the second TCR constant domain associate to form a dimer. The association can comprise one or more native interaction between cognate TCR constant domains e.g. a native disulfide bond), one or more engineered interaction between the TCR constant domains e.g. one or more disulfide bonds introduced by protein engineering), or both. One or both of the first polypeptide and the second polypeptide further comprises a target-binding domain N-terminal to the TCR constant domain(s).

[0300] In some embodiments, the first TCR constant domain comprises a TCR alpha-chain constant domain (Ca) and the second TCR constant domain comprises a TCR beta-chain constant domain (CP). In other embodiments, the first TCR constant domain comprises a CP and the second TCR constant domain comprises a Ca.

[0301] In other embodiments, the first TCR constant domain comprises a TCR gamma-chain constant domain (Cy) and the second TCR constant domain comprises a TCR delta-chain constant domain (C8). In other embodiments, the first TCR constant domain comprises a C6 and the second TCR constant domain comprises a Cy. It will be understood that the terms “TRAC” and “TRBC”, when referring to a TCR / CAR, may be used interchangeably with the terms TCR Ca and TCR CP, respectively, and include embodiments comprising the variant sequences and modifications described herein. A TRAC or TRBC may, but need not necessarily, comprise the native amino acid sequence encoded by a(n e.g. human) TRAC or TRBC, respectively. When describing an endogenous gene locus encoding a TCR alpha chain constant domain or beta chain constant domain, the term TRAC or TRBC, respectively, may be used.

[0302] TCR constant domain sequences may be from, for example, human, mouse, marsupial (e.g. opossum, bandicoot, wallaby), shark, or non-human primate. In certain preferred embodiments, TCR constant domain sequences are human or comprise engineered variants of human sequences. TCR constant domains may be engineered to improve pairing, expression, stability, or any combination of these. See, e.g., Cohen etal., Cancer Res, 2007; Kuball etal., Blood 2007; and Haga-Friedman et al., Journal of Immunology 2009. Examples of engineering in TCR Ca and CP are illustrated in Figure 7; these can include mutation of a native amino acid to a cysteine so that a disulfide bond forms between the introduced cysteine of one TCR constant domain and a native cysteine of the other TCR constant domain. Such mutations can include T48C in Ca, T57C in CP, or both. Mutations to improve stability can include a mutation in the Ca transmembrane domain from the sequence LSVIGF (SEQ ID NO.:62) to the sequence LLVIVL (SEQ ID NO.:63) (“L-V-L” mutation; see Haga-Friedman et al., J Immunol 188:5538-5546 (2012), the TCR mutations and mutant TCR constant domain sequences of which are incorporated herein by reference). Also contemplated are embodiments wherein cognate TCR constant domains comprise mutations so that, for example, one TCR constant domain (e.g., one of Ca and CP) comprises an introduced “cavity” (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having smaller side chains) and the other (e.g., the other of Ca and CP) comprises a compensatory “protuberance” (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having larger side chains), similar to a “knob-into-hole” configuration used to promote preferential pairing of antibody heavy chains. Also contemplated are embodiments wherein TCR constant domain amino acids are mutated to introduce charge properties that favor pairing of the mutated constant domains.

[0303] Examples of mutations that may be made in Ca and CP to promote specific pairing by a knobs-into-holes-type mechanism or by a charge-pairing mechanism are provided in Voss et al., J. Immunol 7S0(l):391-401 (2008) doi.org / 10.4049 / jimmunol.180.1.391; see also U Patent No.

[0304] 9,062,127. The TCR constant domain mutations, mutated TCR constant domains, and methods used to identify sites for mutation, described in these documents, are incorporated herein by reference.

[0305] An example of a TCR Ca amino acid sequence is provided in UniProt KB P01848 (human TRAC): IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS (SEQ ID NO.:56)

[0306] An example of a TCR Ca amino acid sequence engineered to include threonine-to-cysteine and LVL mutations as described herein is provided in SEQ ID NO.: 57:

[0307] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLR ILLLKVAGFNLLMTLRLWS S.

[0308] Two human TCR CP isoforms are TRBC1 and TRBC2. An example of a TRBC1 amino acid sequence is provided in UniProt KB P01850:

[0309] DLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VS ATFWQNPR NHFRCQVQF Y GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSVSYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDF (SEQ ID NO.:58).

[0310] An example of a TRBC1 amino acid sequence engineered to include a serine-to-cysteine mutation is provided in SEQ ID NO.: 59:

[0311] DLNKVFPPEV AVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDP QPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKD

[0312] F.

[0313] An example of a TRBC2 amino acid sequence is provided in UniProt KB A0A5B9: DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VS ATFWQNPR NHFRCQVQF Y GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG (SEQ ID NO.: 60).

[0314] An example of a TRBC2 amino acid sequence engineered to include a serine-to-cysteine mutation is provided in SEQ ID NO: 61: DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVCTDPQ PLKEQPALND SRYCLSSRLR VS ATFWQNPR NHFRCQVQF Y GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG.

[0315] In any of the presently disclosed embodiments, a TCR / CAR can comprise a TCR Ca having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs.:56-57, and a TCR CP having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs.:58-61. In certain embodiments, a TCR / CAR comprises a TCR Ca and a TCR CP having at least least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to, or comprising or consisting of, the amino acid sequences set forth in SEQ ID NOs.: (i) 56 and 58, respectively; (ii) 56 and 59, respectively; (iii) 56 and 60, respectively; (iv) 56 and 61, respectively; (v) 57 and 58, respectively; (vi) 57 and 59, respectively; (vii) 57 and 60, respectively; or (viii) 57 and 61, respectively.

[0316] In preferred embodiments, a variant TCR Ca or CP maintains the same or substantially the same length and / or number of amino acids as compared to a native TCR Ca or CP, respectively, such that, in certain embodiments, the variation does not comprise a truncation in the length thereof as compared to a native TCR Ca or CP, respectively. In certain embodiments, a variant of a TCR CP (TRBC1) maintains the intracellular sequence VKRKDF (SEQ ID NO.:64). In certain embodiments, a variant of a TCR CP (TRBC1) maintains the intracellular sequence MAMVKRKDSRG (SEQ ID NO.:65). Variant TCR constant domains of the present disclosure are capable of associating with a cognate TCR constant domain and with one or more CD3 proteins. In other words, in certain embodiments, TCR / CARs can be assimilated into a TCR complex on a host (e.g. T) cell surface that comprises the TCR / CAR and CD3 proteins. In particular, TCR / CARs of the present disclosure, including those that comprise variant TCR constant domains, are capable of producing a TCR-CD3 complex signal in a host (e.g. T) cell when the TCR / CAR expressed by the host cell binds to its target(s).

[0317] In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) of a TCR / CAR comprises an intracellular portion that consists essentially of or that consists of the intracellular portion of the respective TCR constant domain. In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) does not comprise an intracellular signaling component (e.g. effector domain) from a CD3 protein, such as CD3(^. In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) does not comprise an intracellular costimulatory domain from a costimulatory protein, such as CD28, 4-1BB, ICOS, CD27, 0X40, DAP10, or any combination thereof.

[0318] In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) of a TCR / CAR does not comprise an immunoglobulin CH2 domain and / or an immunoglobulin CH3 domain and / or an immunoglobulin light chain constant domain. In some embodiments, the first polypeptide and / or the second polypeptide (preferably both) of a TCR / CAR does not comprise an immunoglobulin CH2 domain and / or an immunoglobulin CH3 domain and / or an immunoglobulin light chain constant domain disposed C-terminal to the TCR constant domain. In some embodiments, the polypeptide dimer does not comprise an immunoglobulin CH2-CH3 or an immunoglobulin CH2-CH3: CH2-CH3 dimer.

[0319] In some embodiments, a polypeptide dimer comprises a target-binding domain comprising (i) a VH comprised in the first polypeptide or the second polypeptide and (ii) a cognate VL comprised in the other of the first and the second polypeptide, wherein the target is not 2,4,6-trinitrophenyl (TNP), digoxin, or phosphorylcholine.

[0320] A "binding domain" (also referred to as a "binding region" or "binding moiety"), as used herein, refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target (e.g. antigen). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e., complex comprising two or more biological molecules), or other target of interest.

[0321] Herein, a “binding domain” also includes a subunit of a complete binding domain; e.g., if VH and VL together are required for binding to a target, VH alone or VL alone may be referred-to as a “binding domain”. It will be understood that TCR / CARs are capable of binding to a target. Exemplary binding domains useful in TCR / CARs include single chain immunoglobulin variable regions (e.g., scFv, scFab), Fabs, Fv, sdAbs such as nanobodies / VHH, VNAR, receptor ectodomains, ligands (e.g., cytokines, chemokines), or (other) synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex or other target of interest (e.g., DARPins,10FNIII domains). In certain embodiments, the binding domain comprises a scFv, or ligand. In certain embodiments, the binding domain is chimeric, human, or humanized.

[0322] The target-binding domain can comprise any naturally occurring or engineered binding domain suitable for binding a target of interest, such as, for example, an antibody heavy chain variable domain (VH), an antibody light chain variable domain (VL), a VH and a VL, a singlechain variable fragment (scFv) comprising VH-linker-VL or VL-linker-VH, a fragment antigenbinding region (Fab), a single-chain Fab, an antigen-binding fragment of a heavy chain-only antibody (VHH, also referred-to as a nanobody), a killer immunoreceptor from a NK cell, a designed ankyrin repeat protein (DARPin (Binz etal., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:515, 2004)), a10FNIII domain such as an Adnectin™ or monobody ((Richards et al., J. Mol. Biol. 326: 1475, 2003; Parker et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381: 1238-1252)), a lectin binding domain, a receptor ectodomain or functional portion or fragment thereof, provided that the receptor ectodomain does not comprise a TCR variable domain, a ligand such as e.g. a cytokine, a fully synthetic polypeptide (e.g. designed in silico, such as using the AlphaFold modeling program), a fibrinogen domain (see, e.g., Weisel etal., Science 230:1388, 1985), Kunitz domains (see, e.g., US Patent No. 6,423,498), a cysteine-knot miniprotein (Vita et al. (1995) Proc. Nat'L Acad. Sci. (USA) 92:6404-6408; Martin et al. (2002) Nat. Biotechnol. 21:11, 2002 and Huang et al. (2005) Structure 13:155, 2005; Lui etal. Nature Communications 77:295 (2020)), a tetratricopeptide repeat domain (Main et al., Structure 77:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008), a leucine-rich repeat domains (Stumpp etal., J. Mol. Biol. 332:411, 2003), a lipocalin domain (see, e.g., WO 2006 / 095164, Beste et al., Proc. Nat'l. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et al., Proc. Nat'l. Acad. Sci. (USA) 106:8198, 2009), an armadillo repeat protein (see, e.g., Madhurantakam etal., Protein Sci. 21: 1015, 2012; PCT Patent Application Publication No. WO 2009 / 040338), an affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), an affibody, an avimer, a knottin, a fynomer, an atrimer, cytotoxic T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen. Proteo. 10:155, 2013) or the like (Nord et al, Protein Eng. 5:601, 1995; Nord etal., Nat. Biotechnol. 15:112, 1997; Nord et al., Euro. J. Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1257, 2005; Boersma and Pluckthun, Curr. Opin. Biotechnol. 2:849, 2011), a receptor ectodomain or functional portion or fragment thereof, provided that the receptor ectodomain does not comprise a TCR variable domain, a centyrin, or the like, or any combination thereof.

[0323] Binding domains of this disclosure can be generated as described herein or by a variety of methods known in the art (see, e.g., U. S. Patent Nos. 6,291,161 and 6,291,158). For example, binding domains of this disclosure may be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Additionally, traditional strategies for hybridoma development using a target of interest as an immunogen in convenient systems (e.g., mice, HuMAb mouse®, TC mouse™, KM-mouse®, llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains of this disclosure. Binding domains can be isolated from a human protein (see e.g. Traggiai et al., Nature Medicine 70(8):871-875 (2004)), designed in silico (e.g. using AlphaFold or a like program), isolated or derived a rat, a mouse, a hamster, or other rodent, can be from an avian source, can be from a bovine source, can be from a canine source, can be from a camelid (e.g. from camels, dromedaries, or llamas; Ghahroudi et al., FEBS Lett. 414: 521, 1997; Vincke etal., J. Biol. Chem. 284:3213, 2009; Hamers-Casterman et al., Nature 363:446, 1993 and Nguyen et al., J. Mol. Biol. 275:413, 1998), a shark such as a nurse shark (Roux etal., Proc. Nat'l. Acad. Sci. (USA) 95:11804, 1998), spotted ratfish (Nguyen etal., Immunogen. 54:39, 2002), or lamprey (Herrin et al., Proc. Nat'l. Acad. Sci. (USA) 105:2040, 2008 and Alder et al. Nat. Immunol.

[0324] 9:319, 2008). A binding domain can include sequences from a library that encodes random peptides or sequences from a library that encodes an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds.

[0325] In some contexts, a VH alone is sufficient to confer binding (i.e. a target-binding domain can comprise a VH and need not comprise a VL). In some contexts, a VL alone is sufficient to confer binding (i.e. a target-binding domain can comprise a VL and need not comprise a VH).

[0326] In some embodiments, a target-binding domain is “split” across the first polypeptide and the second polypeptide; for example, where a VH and a VL together function to bind to a target, the VH is comprised in the first polypeptide and the VL is comprised in the second polypeptide, or vice versa. The same may be the case, for example, with an antibody Fab; VH-CH1 may be comprised in the first polypeptide, and VL-CL may be comprised in the second polypeptide, or vice versa. Regarding Fab-type molecules that function as binding domains in the presently disclosed TCR / CARs, it will be understood that CHI and CL may be swapped (i.e. VH-CL, VL-CH1), and that other immunoglobulin constant domains can be utilized in place of CHI and CL (e.g., CH2-CH2 or CH3-CH3 may replace CHI -CL; see e.g. Wozniak-Knopp etal. PLoS One 73(4):e0195442 (2018)).

[0327] Accordingly, certain embodiments refer to a “split” format. It will be understood that in a “split-scFv” design, “scFv” refers to the VH and VL components that would form a scFv if linked by a linker; however, “split-scFv” designs typically do not include a peptide linker disposed between and connecting the VH and the VL. Thus, “split-scFv” refers to an arrangement wherein VH is comprised in a first polypeptide of a TCR / CAR and VL is comprised in a second polypeptide of a TCR / CAR, and VH and VL function to form a target-binding domain. In “split” formats, VH and VL can be engineered to introduce one or more disulfide bond therebetween; see e.g. Reiter et al. Biochemistry 33:5451-5459 (1994), Brinkmann Antibody Engineering (2012) doi.org / 10.1007 / 978-3-642-01147-4_14: Weatherill et al. PEDS 25(7):321-329 (2012), and Metz et al. PEDS 25(10):571-580 (2012)). Such disulfide bonds may be introduced in “full” scFv for full scFab formats, as well.

[0328] In some embodiments, a target-binding domain is fully comprised in one TCR constant domain-containing polypeptide. For example, a scFv (VH-linker-VL or VL-linker-VH) may be comprised in the first polypeptide or the second polypeptide of a TCR / CAR; such an arrangement can be described as “full-scFv”.

[0329] In some embodiments, both the first polypeptide and the second polypeptide fully comprise a target-binding domain. For example, the first polypeptide can comprise a Ca linked or fused to a first scFv and the second polypeptide can comprise a CP linked or fused to a second scFv. In other embodiments, the first polypeptide can comprise a Ca linked or fused to a first VHH and the second polypeptide can comprise a CP linked or fused to a second VHH. In other embodiments, the first polypeptide can comprise a Ca linked or fused to a first Fab or scFab and the second polypeptide can comprise a CP linked or fused to a second Fab or scFab, respectively. In other embodiments, the first polypeptide can comprise a Ca linked or fused to a first ligand and the second polypeptide can comprise a CP linked or fused to a second ligand. For example, the first polypeptide can comprise a Ca linked or fused to a first receptor ectodomain and the second polypeptide can comprise a CP linked or fused to a second receptor ectodomain.

[0330] When present, two target-binding domains (it will be understood that any arrangement and combination of binding domains is contemplated; for example, one polypeptide can comprise a scFv and the other polypeptide can comprise a VHH, or one polypeptide can comprise a Fab and the other polypeptide can comprise a scFv, or one polypeptide can comprise a receptor ectodomain and the other polypeptide can comprise a DARPin, or the like) may be the same e.g. two copies of a same VHH may be present, one comprised in each of the first and the second polypeptide) or different. If different, the two binding domains may target different epitopes on the same target e.g. two epitopes present within a tumor antigen), or may target different targets altogether (e.g., may target two different tumor antigens, or may target a tumor antigen and a cytokine). Accordingly, certain embodiments provide multispecific (e.g. bispecific) TCR / CARs. In some contexts, a multispecific TCR / CAR binds to two or more antigens that are expressed by a cancer; for example, to target multiple myeloma, a multispecific TCR / CAR may target any two or more of: BCMA, GPRC5D, SLAMF7, and CD229. In some contexts, a multispecific TCR / CAR binds to two or more antigens that are expressed by a cancer; for example, to target multiple myeloma, a multispecific TCR / CAR may target any two or more of: BCMA, GPRC5D, SLAMF7, CD229, CD19, and CD22. Contemplated embodiments include those wherein a polypeptide chain comprises two or more binding domains (e.g., scFv-linker-scFv-TCR constant domain; VHH-linker-VHH-TCR constant domain; or the like).

[0331] A target can be a synthetic molecule or a biological antigen or other biomolecule. In some embodiments, a target is expressed on or by a cancer cell, a cell infected with a pathogen e.g. virus, fungus, parasite, bacteria) or is otherwise associated with an an infection, or is associated with an autoimmune disease or a neurodegenerative disease (e.g., tau, amyloid-beta, alpha-synuclein), or is a cytokine (e.g. TNFa, IL-13, IL-10) or a chemokine. In some embodiments, a target is or comprises a cancer antigen selected from BCMA, GPRC5D, CD19, R0R1, SLAMF7, CD229, PNE, EGFR, EGFRvIII, EGP-2, EGP-40, GD2, GD3, HPV E6, HPV E7, Her2, LI -CAM, Lewis A, Lewis Y, MUC1, MUC16, PSCA, PSMA, CD20, CD22, CD56, CD23, CD24, CD30, CD33, CD37, CD44v7 / 8, CD38, CD56, CD123, CA125, c-MET, FcRH5, WT1, folate receptor a, VEGF-a, VEGFR1, VEGFR2, IL-13Ra2, IL-llRa, MAGE-A1, PSA, ephrin A2, ephrin B2, NKG2D, NY-ESO-1, TAG-72, mesothelin, NY-ESO, 5T4, BCMA, FAP, Carbonic anhydrase 9, BRAF, a-fetoprotein, MAGE- A3, MAGE-A4, SSX-2, PRAME, HA-1, P2M, ETA, tyrosinase, KRAS, NRAS, a peptideMHC complex, and CEA. In some embodiments, a TCR / CAR comprises a Bcl-2 ectodomain or portion or variant thereof and a target is a Bcl-2 ligand.

[0332] In certain embodiments, a TCR / CAR is bispecific and binds to: (i) BCMA and GPRC5D; (ii) BCMA and SLAMF7; (iii) BCMA and CD229; (iv) GPRC5D and SLAMF7; (v) GPRC5D and CD229; or (vi) SLAMF7 and CD229. In certain embodiments, a TCR / CAR is bispecific and binds to: CD19 and BCMA; or to CD19 and CD22. In some embodiments, the bispecific TCR / CAR comprises two scFvs. In some embodiments, a bispecific TCR / CAR comprises two VHH and each VHH binds to a different epitope on BCMA.

[0333] Non-limiting examples of binding domains include those that comprise the VH, the VL, the HCDRs, and / or the LCDRs of: trastuzumab; pertuzumab; rituximab; erbituxumab; ublituxumab; 1.5.3; a BMCA-specific antibody such as J22.0-xi, J22.9-xi, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, CA8, A7D12.2, Cll D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SGI, S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115GO1, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4. E2.1, Vicky-1, pSCHLI333, pSCHLI372, pSCHLI373, and those other BCMA-specific antibodies and antigen-binding fragments disclosed in PCT Publication Nos. WO 2002 / 066516, WO 2007 / 062090, WO 2010 / 104949, WO 2011 / 108008, WO 2012 / 163805, WO 2014 / 068079, WO 2015 / 166073, WO 2014 / 122143, WO 2014 / 089335, WO 2016 / 090327, and WO 2016 / 079177; Ryan etal., Mol. Cancer. Ther.

[0334] 6(11):3009, 2007; and Abbas et al., Blood 725:1688, 2016; a ROR1 -specific antibody such as Rll, R12, Y4, Y13, Y27, or Y31; a CD19-specific antibody such as FMC63; a CD33-specific antibody such as gemtuzumab; a GPRC5D-specific antibody; a ROR1 -specific VHH; a VHH such as MB14; 3F8; alemtuzumab; XMAB-5574; pembrolizumab; nivolumab; a PD-l-specific antibody; elotuzomab; a SLAMF-specific antibody; a CD229-specific antibody; a PD-L1-specific antibody; a SARS-CoV-2-specific antibody, or an (e.g. cancer antigen-specific, pathogen-specific, autoimmune disease antigen-specific, or neurodegenerative-disease-specific) antibody or antigen-binding fragment approved for therapeutic and / or diagnostic use in humans by the US Food and Drug Administration, the European Medicines Agency, or both. In some embodiments, a binding domain comprises the VH, the VL, the HCDRs, and / or the LCDRs of 3F8, 8H9, Abagovomab, Abciximab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afasevikumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atlizumab, Atorolimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab,

[0335] Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Bleselumab, Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Cergutuzumab amunaleukin, Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab, Concizumab, CR6261, Crenezumab, Crotedumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin, Derlotuximab biotin, Detumomab, Dinutuximab, Donanemab, Diridavumab, Domagrozumab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Emicizumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erenumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Inolimomab, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lanadelumab, Landogrozumab, Laprituximab emtansine, Lebrikizumab, Lemalesomab, Lendalizumab, Lenzilumab, Lerdelimumab, Lecanemab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, MABpl, Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab, Mogamulizumab, Monalizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Murom onab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Naratuximah emtansine, Narnatumab, Natalizumab, Navicixizumab, Navivumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Plozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140 aka leronlimab, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Risankizumab, Rituximab, Rivabazumab pegol, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovalpituzumab tesirine, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sapelizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Sotrovimab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tamtuvetmab, Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Ticilimumab, Tigatuzumab, Tildrakizumab, Timolumab, Tisotumab vedotin, TNX-650, Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab, Vadastuximab talirine, Vandortuzumab vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Xentuzumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, or Zolimomab aritox.

[0336] In certain embodiments, a TCR / CAR comprises VH, the VL, the HCDRs, and / or the LCDRs of an antibody or antigen-binding fragment that is one of those described in U. S. Pat. No. 7,947,809 and U. S. Patent Application Publication No. 20090041784 (glucagon receptor), U. S. Pat. Nos. 7,939,070, 7,833,527, 7,767,206, and 7,786,284 (IL-17 receptor A), U. S. Pat. Nos.

[0337] 7,872,106 and 7,592,429 (Sclerostin), U. S. Pat. Nos. 7,871,611, 7,815,907, 7,037,498, 7,700,742, and U. S. Patent Application Publication No. 20100255538 (IGF-1 receptor), U. S. Pat. No. 7,868,140 (B7RP1), U. S. Pat. No. 7,807,159 and U. S. Patent Application Publication No. 20110091455 (myostatin), U. S. Pat. Nos. 7,736,644, 7,628,986, 7,524,496, and U. S. Patent Application Publication No. 20100111979 (deletion mutants of epidermal growth factor receptor), U. S. Pat. No. 7,728,110 (SARS coronavirus), U. S. Pat. No. 7,718,776 and U. S. Patent Application Publication No. 20100209435 (OPGL), U. S. Pat. Nos. 7,658,924 and 7,521,053 (Angiopoietin-2), U. S. Pat. Nos. 7,601,818, 7,795,413, U. S. Patent Application Publication No.

[0338] 20090155274, U. S. Patent Application Publication No. 20110040076 (NGF), U. S. Pat. No.

[0339] 7,579,186 (TGF-P type II receptor), U. S. Pat. No. 7,541,438 (connective tissue growth factor), U. S. Pat. No. 7,438,910 (IL1-R1), U. S. Pat. No. 7,423,128 (properdin), U. S. Pat. Nos. 7,411,057, 7,824,679, 7,109,003, 6,682,736, 7,132,281, and 7,807,797 (CTLA-4), U. S. Pat. Nos. 7,084,257, 7,790,859, 7,335,743, 7,084,257, and U. S. Patent Application Publication No. 20110045537 (interferon-gamma), U. S. Pat. No. 7,932,372 (MAdCAM), U. S. Pat. No. 7,906,625, U. S. Patent Application Publication No. 20080292639, and U. S. Patent Application Publication No.

[0340] 20110044986 (amyloid), U. S. Pat. Nos. 7,815,907 and 7,700,742 (insulin-like growth factor I), U. S. Pat. Nos. 7,566,772 and 7,964,193 (interleukin- 1 ), U. S. Pat. Nos. 7,563,442, 7,288,251, 7,338,660, 7,626,012, 7,618,633, and U. S. Patent Application Publication No. 20100098694 (CD40), U. S. Pat. No. 7,498,420 (c-Met), U. S. Pat. Nos. 7,326,414, 7,592,430, and 7,728,113 (M-CSF), U. S. Pat. Nos. 6,924,360, 7,067,131, and 7,090,844 (MUC18), U. S. Pat. Nos.

[0341] 6,235,883, 7,807,798, and U. S. Patent Application Publication No. 20100305307 (epidermal growth factor receptor), U. S. Pat. Nos. 6,716,587, 7,872,113, 7,465,450, 7,186,809, 7,317,090, and 7,638,606 (interleukin-4 receptor), U. S. Patent Application Publication No. 20110135657 (BETA-KLOTHO), U. S. Pat. Nos. 7,887,799 and 7,879,323 (fibroblast growth factor-like polypeptides), U. S. Pat. No. 7,867,494 (IgE), U. S. Patent Application Publication No.

[0342] 20100254975 (ALPHA-4 BETA-7), U. S. Patent Application Publication No. 20100197005 and U. S. Pat. No. 7,537,762 (ACTIVIN RECEPTOR-LIKE KINASE- 1), U. S. Pat. No. 7,585,500 and U. S. Patent Application Publication No. 20100047253 (IL-13), U. S. Patent Application Publication No. 20090263383 and U. S. Pat. No. 7,449,555 (CD148), U. S. Patent Application Publication No. 20090234106 (ACTIVIN A), U. S. Patent Application Publication No.

[0343] 20090226447 (angiopoietin-1 and angiopoietin-2), U. S. Patent Application Publication No.

[0344] 20090191212 (Angiopoietin-2), U. S. Patent Application Publicaiton No. 20090155164 (C-FMS), U. S. Pat. No. 7,537,762 (activin receptor-like kinase-1), U. S. Pat. No. 7,371,381 (galanin), U. S. Patent Application Publication No. 20070196376 (INSULIN-LIKE GROWTH FACTORS), U. S. Pat. Nos. 7,267,960 and 7,741,115 (LDCAM), U. S. Pat. No. 7,265,212 (CD45RB), U. S. Pat. No.

[0345] 7,709,611, U. S. Patent Application Publication No. 20060127393 and U. S. Patent Application Publication No. 20100040619 (DKK1), U. S. Pat. No. 7,807,795, U. S. Patent Application Publication No. 20030103978 and U. S. Pat. No. 7,923,008 (osteoprotegerin), U. S. Patent Application Publication No. 20090208489 (0V064), U. S. Patent Application Publication No. 20080286284 (PSMA), U. S. Pat. No. 7,888,482, U. S. Patent Application Publication No.

[0346] 20110165171, and U. S. Patent Application Publication No. 20110059063 (PAR2), U. S. Patent Application Publication No. 20110150888 (HEPCIDIN), U. S. Pat. No. 7,939,640 (B7L-1), U. S. Pat. No. 7,915,391 (c-Kit), U. S. Pat. Nos. 7,807,796, 7,193,058, and U. S. Pat. No. 7,427,669 (ULBP), U. S. Pat. Nos. 7,786,271, 7,304,144, and U. S. Patent Application Publication No.

[0347] 20090238823 (TSLP), U. S. Pat. No. 7,767,793 (SIGIRR), U. S. Pat. No. 7,705,130 (HER-3), U. S. Pat. No. 7,704,501 (ataxin-l-like polypeptide), U. S. Pat. Nos. 7,695,948 and 7,199,224 (TNF-a converting enzyme), U. S. Patent Application Publication No. 20090234106 (ACTIVIN A), U. S. Patent Application Publication No. 20090214559 and U. S. Pat. No. 7,438,910 (IL1-R1), U. S. Pat. No. 7,579,186 (TGF-P type II receptor), U. S. Pat. No. 7,569,387 (TNF receptor-like molecules), U. S. Pat. No. 7,541,438, (connective tissue growth factor), U. S. Pat. No. 7,521,048 (TRAIL receptor-2), U. S. Pat. Nos. 6,319,499, 7,081,523, and U. S. Patent Application Publication No. 20080182976 (erythropoietin receptor), U. S. Patent Application Publication No.

[0348] 20080166352 and U. S. Pat. No. 7,435,796 (B7RP1), U. S. Pat. No. 7,423,128 (properdin), U. S. Pat. Nos. 7,422,742 and 7,141,653 (interleukin-5), U. S. Pat. Nos. 6,740,522 and 7,411,050 (RANKE), U. S. Pat. No. 7,378,091 (carbonic anhydrase IX (CA IX) tumor antigen), U. S. Pat. Nos. 7,318,925 and 7,288,253 (parathyroid hormone), U. S. Pat. No. 7,285,269 (TNF), U. S. Pat. Nos. 6,692,740 and 7,270,817 (ACPL), U. S. Pat. No. 7,202,343 (monocyte chemo-attractant protein-1), U. S. Pat. No. 7,144,731 (SCF), U. S. Pat. Nos. 6,355,779 and 7,138,500 (4-1BB), U. S. Pat. No. 7,135,174 (PDGFD), U. S. Pat. No. 6,630,143 and U. S. Pat. No. 7,045,128 (Flt-3 ligand), U. S. Pat. No. 6,849,450 (metalloproteinase inhibitor), U. S. Pat. No. 6,596,852 (LERK-5), U. S. Pat. No. 6,232,447 (LERK-6), U. S. Pat. No. 6,500,429 (brain-derived neurotrophic factor), U. S. Pat. No. 6,184,359 (epithelium-derived T-cell factor), U. S. Pat. No. 6,143,874 (neurotrophic factor NNT-1), U. S. Patent Application Publication No. 20110027287 (PROPROTEIN CONVERTASE SUBTILISIN KEXIN TYPE 9 (PCSK9)), U. S. Patent Application Publication No. 20110014201 (IL-18 RECEPTOR), and U. S. Patent Application Publication No. 20090155164 (C-FMS). The above patents and published patent applications are incorporated herein by reference in their entirety for purposes of their disclosure of variable domain and CDR polypeptides, variable domain and CDR-encoding nucleic acids, host cells, vectors, methods of making the antibodies or antigen-binding fragments polypeptides encoding said variable domains, pharmaceutical compositions, and methods of treating diseases associated with the respective target of the variable domain-containing antibody or antigen-binding fragment. In certain embodiments, a TCR / CAR comprises VH, the VL, the HCDRs, and / or the LCDRs of an antibody or antigen-binding fragment that is one of: Muromonab-CD3 (product marketed with the brand name Orthoclone Okt3®), Abciximab (product marketed with the brand name Reopro®.), Rituximab (product marketed with the brand name Mab Thera®, Rituxan®), Basiliximab (product marketed with the brand name Simulect®), Daclizumab (product marketed with the brand name Zenapax®), Palivizumab (product marketed with the brand name Synagis®), Infliximab (product marketed with the brand name Remicade®), Trastuzumab (product marketed with the brand name Herceptin®), Alemtuzumab (product marketed with the brand name MabCampath®, Campath- 1H®), Adalimumab (product marketed with the brand name Humira®), Tositumomab-1131 (product marketed with the brand name Bexxar®), Efalizumab (product marketed with the brand name Raptiva®), Cetuximab (product marketed with the brand name Erbitux®), ITbritumomab tiuxetan (product marketed with the brand name Zevalin®), 1'Omalizumab (product marketed with the brand name Xolair®), Bevacizumab (product marketed with the brand name Avastin®), Natalizumab (product marketed with the brand name Tysabri®), Ranibizumab (product marketed with the brand name Lucentis®), Panitumumab (product marketed with the brand name Vectibix®), 1'Eculizumab (product marketed with the brand name Soliris®), Certolizumab pegol (product marketed with the brand name Cimzia®), Golimumab (product marketed with the brand name Simponi®), Canakinumab (product marketed with the brand name Ilarise), Catumaxomab (product marketed with the brand name Removab®), Ustekinumab (product marketed with the brand name Stelara®), Tocilizumab (product marketed with the brand name RoActemra®, Actemra®), Ofatumumab (product marketed with the brand name Arzerra®), Denosumab (product marketed with the brand name Prolia®), Belimumab (product marketed with the brand name Benlysta®), Raxibacumab, Ipilimumab (product marketed with the brand name Yervoy®), and Pertuzumab (product marketed with the brand name Peijeta®). In exemplary embodiments, the antibody is one of anti-TNF alpha antibodies such as adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol; anti -IL 1. beta, antibodies such as canakinumab; anti-IL12 / 23 (p40) antibodies such as ustekinumab and briakinumab; and anti-IL2R antibodies, such as daclizumab. Examples of suitable anti-cancer antibodies include, but are not limited to, anti-BAFF antibodies such as belimumab; anti-CD20 antibodies such as rituximab; anti-CD22 antibodies such as epratuzumab; anti-CD25 antibodies such as daclizumab; anti-CD30 antibodies such as iratumumab, anti-CD33 antibodies such as gemtuzumab, anti-CD52 antibodies such as alemtuzumab; anti-CD152 antibodies such as ipilimumab; anti-EGFR antibodies such as cetuximab; anti-HER2 antibodies such as trastuzumab and pertuzumab; anti-IL6 antibodies such as siltuximab; and anti-VEGF antibodies such as bevacizumab; and anti-IL6 receptor antibodies such as tocilizumab.

[0349] In certain embodiments, a target comprises a protein ligand and a binding domain is from a receptor for the ligand. For example, a binding domain can comprise a receptor ectodomain from Bcl2 and a target comprises BIM. In some embodiments, a binding domain comprises a “TCR-mimic” antibody fragment (e.g. scFv or VH and VL from a TCR-mimic antibody) and a target comprises a peptide antigen in complex with a MHC e.g. HLA) molecule. In some embodiments, the MHC molecule is a Class I MHC molecule. In other embodiments, the MHC molecule is a Class II MHC molecule. In certain further embodiments, a peptide antigen: HLA complex comprises WTI126 (RMFPNAPYL; SEQ ID N0.:66) / HLA-A*0201 or NY-ESO-I157 (SLLMWITQC; SEQ ID NO.:67) / HLA-A*0201. TCR-mimic antibodies may be prepared by the hybridoma methodology described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U. S. Pat. No.

[0350] 4,816,567). TCR-mimic antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 552:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), and Noy et al. Expert Rev. Anticancer Ther. 5(3):523-536 (2005); these techniques are incorporated herein in their entireties), for example. TCR-mimic antibodies may also be obtained using methods disclosed in PCT Publication No. WO 2004 / 076677A2.

[0351] In some embodiments, the binding domain is capable of specifically binding to the target. As used herein, "specifically binds" or "specific for" refers to an association or union of an target-binding protein or a binding domain to a target molecule with an affinity or Ka(i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M'1(which equals the ratio of the on-rate [Kon] to the off rate [Koir] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Target-binding proteins or binding domains may be classified as "high-affinity" binding proteins or binding domains or as "low-affinity" binding proteins or binding domains. "High-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Kaof at least 107M-1, at least 108M’1, at least 109M at least IO10M’ at least 1011M’1, at least 1012M-1, or at least 1013M’1. "Low-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Kaof up to 107M’1, up to 106M’1, or up to 105M’1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10'5M to 10’13M).

[0352] In some embodiments, a binding domain is engineered have a preferred affinity for the target. For example, without wishing to be bound by theory, higher binding affinities (e.g., picomolar or femtomolar Kd) may in some contexts initiate or contribute to a more intense signal than is desired upon binding to antigen (e.g., binding by a TCR / CAR comprising such a binding domain may contribute to T cell signaling that is more intense, longer, or both, than may be desired, and may contribute to tonic signaling and / or cell exhaustion). Accordingly, certain embodiments provide binding domains which are selected for or are modified to have affinity in a preferred range, such as in the range of about InM to about lOOnM Kd, as determined by surface plasmon resonance, which may be comparable to physiological affinity of a native TCR for antigen: MHC. By way of illustration, if a binding domain from an antibody with picomolar or femtomolar Kd for its antigen is selected for use in a TCR / CAR, the binding domain may first be engineered to decrease affinity for antigen to a range of about InM to about lOOnM Kd, such as by rational mutagenesis in one or more CDRs (e.g., by mutation from an amino acid with a larger side-chain (e.g. tryptophan or phenylalanine) to an amino acid with a smaller side-chain (e.g. alanine or serine)).

[0353] A variety of assays are known for identifying binding domains that specifically bind a particular target, as well as determining binding domain or antigen-binding protein affinities, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy, isothermal titration calorimetry (ITC), and surface plasmon resonance (Biacore®) analysis (see, e.g, Scatchard et al., Ann. N. Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U. S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). Assays for apparent affinity or relative affinity are also known. In certain examples, apparent affinity for a target-binding protein is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers. In some examples, apparent KD of a binding protein or binding domain is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent KD being determined as the concentration of ligand that yielded half-maximal binding.

[0354] Non-limiting examples of binding domain amino acid sequences are provided in SEQ ID NOs.:97-109. In some embodiments, a TCR / CAR comprises a binding domain comprising: (i) SEQ ID NO.:97 and SEQ ID NO.:98, optionally comprised in a scFv, such as having the sequence of SEQ ID NO.: 99 or SEQ ID NO.: 100; (ii) SEQ ID NO.: 101 and 102, optionally in a scFv, such as having the sequence of SEQ ID NO.: 103; (iii) SEQ ID NO.: 104 and SEQ ID NO.: 105, optionally in a scFv, such as having the sequence of SEQ ID NO.: 106; (iv) SEQ ID NO.: 107, optionally comprised in SEQ ID NO.: 108; or (v) SEQ ID NO.: 109. In certain further embodiments, the binding domain comprising (i), (ii), (iii), (iv), and / or (v) above is fused or linked to a TCR Ca, to a TCR CP, or to both of a TCRa and a TCRP (e.g., where a VH and a VL are present, in a “split” format or in a “full format”). In some embodiments, the TCR Ca has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO.:56. In some embodiments, the TCR Ca has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO.:57. In some embodiments, the TCR CP has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO.:58. In some embodiments, the TCR CP has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO.:59. In some embodiments, the TCR CP has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO.:60. In some embodiments, the TCR CP has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO.:61.

[0355] The target-binding domain or a component thereof may be directly fused to a TCR constant domain ( / .<?., there is no intervening amino acid or amino acid sequence between the target-binding domain or component thereof and the TCR constant domain) or may be linked to a TCR constant domain by a linker, which may function as a hinge. Accordingly, in some embodiments, a hinge sequence is disposed between and connects the target-binding domain or portion thereof and a TCR constant domain. A hinge sequence may be from an immunoglobulin superfamily molecule (e.g. an antibody hinge or a CD8 hinge, or an engineered variant hinge based thereon, or a synthetic flexible linker such as a glycine-serine linker) and can confer desired structure and flexibility for binding to the target. A hinge sequence can be selected and / or engineered for preferred characteristics, such as, for example, a desired length, a desired flexibility, a desired reduced interaction or lack of interaction of interaction with a Fc receptor (e.g. aFcyR).

[0356] Non-limiting examples of hinge sequences are provided in SEQ ID NOs.:42-55. These include a (G4S)3 linker (SEQ ID NO.:54); it will be understood that this linker and other synthetic linkers may function as a hinge, or may be present elsewhere in a TCR / CAR or CCR of the present disclosure (e.g., between adjacent domains of a polypeptide, such as between VH and VL in a scFv). Linkers include an (A)nlinker, wherein n is 1 or more, a GS linker, a GSG linker, a GPP linker, a (GlyxSery)nlinker wherein X, Y, and N are not zero, and may each independently be from 1-10, a Townsend linker (GSGGSGGSGGTG; SEQ ID NO.:68), a Whitlow linker aka linker 218 (GSTSGSGKPGSGEGSTKG; SEQ ID NO.:69), or a linker comprising or consisting of any one of the following amino acid sequences: GSGKPGSGEG (SEQ ID NO.:70);

[0357] GKPGSGEG (SEQ ID NO.:71); SGKPGSGE (SEQ ID NO.:72); EGKSSGSGSESKVD (SEQ ID NO.:73), or BPXXXZ, wherein each X is independently a glycine (G) or serine (S), B is a positively charged amino acid and Z is glycine (G) or a negatively charged amino acid (SEQ ID NO.:74), or the like).

[0358] Non-limiting examples of TCR / CAR polypeptide amino acid sequences are provided in SEQ ID NOs.: 110-125. In certain embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise, consist essentially of, or consist of, amino acid sequences having at least least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to, or comprising or consisting of, the amino acid sequences set forth in SEQ ID NOs.: (i) 110 and 111, respectively; (ii) 112 and 113, respectively; (iii) 118 and 119; (iv) 116 and 117; (v) 120 and 121; or (vi) 122 or 123 and 124 or 125, respectively. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 114 and the second polypeptide consists essentially of or consists of a TCR Cp. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 115 and the second polypeptide consists essentially of or consists of a TCR Ca. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 115 and the second polypeptide consists essentially of or consists of a TCR Cp. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 116 or SEQ ID NO.: 117 and the second polypeptide consists essentially of or consists of a TCR Ca. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 118 and the second polypeptide consists essentially of or consists of a TCR Ca. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 119 and the second polypeptide consists essentially of or consists of a TCR Cp. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 120 and the second polypeptide consists essentially of or consists of a TCR Cp. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 121 and the second polypeptide consists essentially of or consists of a TCR Ca. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 122 or SEQ ID NO.: 123 and the second polypeptide consists essentially of or consists of a TCR Cp. In some embodiments, a TCR / CAR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 124 or SEQ ID NO.: 125 and the second polypeptide consists essentially of or consists of a TCR Ca.

[0359] In some embodiments, a first polypeptide, the second polypeptide, or both, of a TCR / CAR has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to, or comprising or consisting of, the amino acid sequence set forth in SEQ IDN0.:114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125.

[0360] Table 1 provides non-limiting examples of certain TCR / CAR designs. It will be understood that a scFv can be in VH-linker-VL or VL-linker-VH orientation. It will be understood that a scFab can be in VH-CH1 -linker- VL-CL orientation or VL-CL-linker-VH-CHl orientation (or, alternatively, that CHI and CL can be exchanged or CH3 can replace CHI and CL; e.g., VH-CL-linker-VL-CHl, VL-CH1 -linker- VH-CL, VH-CH3 -linker- VL-CH3, VL-CH3-linker-VH-VH3). It will be understood that any TCR / CAR can comprise a hinge disposed between a binding domain and a TCR constant domain. It will be understood that where two or more binding domains are present, they may be the same or different. If different, they may comprise a different amino acid sequence to one another but still bind a same (or overlapping) epitope or a same target, or they may bind different epitopes on a same target or may bind different targets. It will be understood that in Table 1, “none” refers to the absence of a target or antigen-binding domain; the absence of an antigen-binding domain does not exclude the presence of amino acid sequence N-terminal to the TCR constant domain. For example, in some embodiments of the present disclosure, one of the first polypeptide and the second polypeptide comprises, N-terminal to the TCR constant domain thereof, one or more tag and optionally one or more linker. A tag can include, for example, any tag or combinations of tags known in the art, including those tags and combinations of tags described herein. It will be understood that a polypeptide dimer (TCR / CAR) can comprise one or more additional binding domain further to the binding domain(s) specified in Table 1; for example, a polypeptide of a polypeptide dimer may comprise two, three, four, or more binding domains, e.g. linked in a series.

[0361] Table 1

[0362]

[0363]

[0364] Additionally or alternatively, any first and / or second polypeptide of a TCR / CAR as in Table 1 can comprise, as a binding domain: a cytokine; a chemokine; a synthetic polypeptide selected for its specific ability to bind to a biological molecule, a molecular complex or other target of interest (e.g., DARPins,10FNIII domains); a killer immunoreceptor from a NK cell; a designed ankyrin repeat protein (DARPin); a10FNIII domain such as an Adnectin™ or monobody; a lectin binding domain; a fibrinogen domain; a cysteine-knot miniprotein; a tetratricopeptide repeat domain; a lipocalin domain; an armadillo repeat protein; an affibody; an avimer; a knottin; a fynomer; an atrimer; cytotoxic T-lymphocyte associated protein-4; a centyrin; or any combination thereof.

[0365] In some embodiments, a first and a second polypeptide of a TCR / CAR each comprise a scFv.

[0366] Also provided are embodiments wherein a first polypeptide of a TCR / CAR and a second polypeptide of a TCR / CAR comprise, as a binding domain: (i) a VNAR and a VNAR, respectively (wherein the VNARs can be the same or different); (ii) a VNAR and no binding domain, respectively; (iii) no binding domain and a VNAR, respectively; (iv) a VNAR and scFv, respectively; (v) a scFv and a VNAR, respectively; (vi) a VNAR and a scFab, respectively; (vii) a scFab and a VNAR, respectively; (viii) a VNAR and a VHH or a VHH-linker-VHH, respectively; (ix) a VHH or a VHH-linker-VHHH and a VNAR, respectively; (x) a VNAR and a protein ligand-binding domain, respectively; or (xi) a protein ligand-binding domain and a VNAR, respectively.

[0367] Additionally or alternatively, a TCR / CAR can comprise a VNAR and one or more of: a cytokine; a chemokine; a synthetic polypeptide selected for its specific ability to bind to a biological molecule, a molecular complex or other target of interest (e.g., DARPins,10FNIII domains); a killer immunoreceptor from a NK cell; a designed ankyrin repeat protein (DARPin); a10FNIII domain such as an Adnectin™ or monobody; a lectin binding domain; a fibrinogen domain; a cysteine-knot miniprotein; a tetratricopeptide repeat domain; a lipocalin domain; an armadillo repeat protein; an affibody; an avimer; a knottin; a fynomer; an atrimer; cytotoxic T-lymphocyte associated protein-4; a centyrin; or any combination thereof.

[0368] Also provided are embodiments wherein a VH or a VL of an antibody is sufficient for binding and a first polypeptide of a TCR / CAR comprises the VH or VL and the second polypeptide of the TCR / CAR does not comprise a binding domain, or comprises the cognate VL or VH of the first polypeptide, or comprises a different binding domain. In some embodiments, a first polypeptide of a TCR / CAR and a second polypeptide of a TCR / CAR comprise, as a binding domain: (i) an antigen-binding VH or VL and a VNAR, respectively; (ii) an antigenbinding VH or VL and no binding domain, respectively; (iii) no binding domain and an antigenbinding VH or VL, respectively; (iv) a VNAR and an antigen-binding VH or VL, respectively; (v) an antigen-binding VH or VL and an antigen-binding VH or VL, respectively; (vi) an antigen-binding VH or VL and a scFab, respectively; (vii) a scFab and an antigen-binding VH or VL, respectively; (viii) an antigen-binding VH or VL and a VHH or a VHH-linker-VHH, respectively; (ix) a VHH or a VHH-linker-VHH and an antigen-binding VH or VL, respectively; (x) an antigen-binding VH or VL and a protein ligand-binding domain, respectively; or (xi) a protein ligand-binding domain and an antigen-binding VH or VL, respectively.

[0369] Additionally or alternatively, a TCR / CAR can comprise an antigen-binding VH or antigen-binding VL and one or more of: a cytokine; a chemokine; a synthetic polypeptide selected for its specific ability to bind to a biological molecule, a molecular complex or other target of interest (e.g., DARPins,10FNIII domains); a killer immunoreceptor from a NK cell; a designed ankyrin repeat protein (DARPin); a10FNIII domain such as an Adnectin™ or monobody; a lectin binding domain; a fibrinogen domain; a cysteine-knot miniprotein; a tetratricopeptide repeat domain; a lipocalin domain; an armadillo repeat protein; an affibody; an avimer; a knottin; a fynomer; an atrimer; cytotoxic T-lymphocyte associated protein-4; a centyrin; or any combination thereof.

[0370] It will be appreciated that either or both of the first polypeptide and the second polypeptide can comprise any binding domain, such as described herein. It will be appreciated that the first polypeptide can comprise a TCR Ca and the second polypeptide can comprise a TCR CP, or the first polypeptide can comprise a TCR CP and the second polypeptide can comprise a TCR Ca.

[0371] Any TCR constant domain-containing polypeptide of a TCR / CAR can also be provided as an isolated polypeptide. For example, a Ca-containing or CP-containing polypeptide can be provided, wherein the Ca-containing or CP-containing polypeptide comprises a binding domain as provided herein.

[0372] Additional TCR / CARs and components thereof are provided herein.

[0373] In some embodiments, a polynucleotide or expression construct of the present disclosure comprises DNA. In some embodiments, a polynucleotide or expression construct of the present disclosure comprises RNA. In some embodiments, a polynucleotide or expression construct of the present disclosure comprises mRNA. In some embodiments, a polynucleotide or expression construct of the present disclsoure comprises circRNA, saRNA, or both. A polynucleotide or expression construct can, for example, be comprised, contained, and / or delivered to a subject or a host cell in a carrier, wherein the carrier comprises a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), a lipopolyplex (LPP), a cationic polypeptide, a polymeric nanoparticle, or a nanoscale platform, such as a nanoemulsion.

[0374] A polynucleotide can be codon-optimized for expression in a host cell. A polynucleotide can be comprised in a vector, such as, for example, a viral vector, such as a lentiviral vector or a retroviral vector. A polynucleotide or vector can include one or more additional features to facilitate desired expression of the encoded polypeptide(s), such as one or more promoter, one or more sequence encoding a signal peptide (also known as a leader peptide or leader sequence or transit peptide), one or more sequence encoding a furin cleavage sequence, one or more sequence encoding a self-cleaving peptide, or any combination thereof. Non-limiting examples of promoters include an EFla promoter (SEQ ID NO.:7) and a MNDu3 promoter (SEQ ID NO.: 8).

[0375] Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell. A signal peptide may be removed, at least in part, from the polypeptide during or once localization or secretion is completed. Polypeptides that have a signal peptide are referred to herein as a "pre-protein" and polypeptides having their signal peptide removed are referred to herein as "mature" proteins or polypeptides. Signal peptides can be at the N-terminal or C-terminal end of an encoded polypeptide. Non-limiting examples of signal peptides include: the signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126; from GM-CSF); the signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 127; from CD8a); the signal peptide MRPRLWLLLAAQLTVLHGNSV (SEQ ID NO: 128; from CD8P); the signal peptide METDTLLLWVLLLWVPGSTG (SEQ ID NO.: 150, from murine IgG, kappa light chain); and the signal peptide MDMRVPAQLLGLLLLWLRGARC (from human Ig kappa). Signal peptides from CD3(^, CD36, CD3y,and CD3s are known and amino acid sequences of these are provided herein. It will be appreciated that any suitable naturally occurring or engineered signal peptide can be employed. In some contexts, a signal peptide that is native to the encoded polypeptide or ectodomain of a polypeptide is used; for example, a CD226 signal peptide (MDYPTLLLALLHVYRALC; SEQ ID NO.:76) may be used with a CD226-based OCR, a TIGIT signal peptide (MRWCLLLIWAQGLRQAPLASG; SEQ ID NO.:81) may be used with a TIGIT-based CCR, and a receptor ectodomain signal peptide may be used in a receptor ectodomain-containing TCR / CAR. Certain signal peptides and char...

Claims

1. CLAIMS2.What is claimed is:

1. A T cell comprising: (i) a polynucleotide encoding a chTCR and (ii) a heterologous polynucleotide encoding a wild-type CD3 polypeptide, a polynucleotide encoding an engineered CD3 polypeptide as provided herein, or a polynucleotide encoding a CD3: costimulatory domain fusion protein as provided herein.

2. An expression construct comprising: (i) a polynucleotide encoding a chTCR and (ii) a polynucleotide encoding: a wild-type CD3 polypeptide, an engineered CD3 polypeptide as provided herein, or a CD3: costimulatory domain fusion protein as provided herein, optionally wherein the expression construct comprises an EFla promoter operably linked to the polynucleotide of (i) and a MNDU3 promoter operably linked to the polynucleotide of (ii), or wherein the expression construct comprises a MNDU3 promoter operably linked to the polynucleotide of (i) and an EFla promoter operably linked to the polynucleotide of (ii), and / or wherein the EFla promoter and the MNDU3 promoter are disposed on opposite strands of the expression construct.

3. A fusion protein comprising, consisting essentially of, or consisting of:6.an extracellular component comprising, consisting essentially of, or consisting of the extracellular domain of a CD3(^ (e.g., QSFGLLDPK (SEQ ID NO.:152)), or a functional fragment or variant thereof, wherein the variant optionally comprises one, two, three, or four amino acid substitutions, insertions, and / or deletions relative to SEQ ID NO.: 152;7.an intracellular component comprising, consisting essentially of, or consisting of (i) an intracellular signaling domain of the CD3(^, or a functional fragment or variant thereof, and (ii) a costimulatory portion comprising, consisting essentially of, or consisting of (1) a costimulatory domain of a 4- IBB, or a functional fragment or variant thereof, linked or fused to an amino acid sequence that is not the intracellular signaling domain of the CD3(^ or a functional fragment or variant thereof; (2) a costimulatory domain of a CD226, or a functional fragment or variant thereof, wherein, optionally, the variant comprises one or more (e.g. substitution, e.g. nonconservative) mutation that (a) provides increased expression of the fusion protein in a T cell exposed to PVR, as compared to expression of endogenous CD226 by the T cell exposed to PVR, and / or (b) disrupts a Src kinase phosphorylation site on the fusion protein, and / or (c) reduces ubiquitination of the fusion protein by CBL-B, wherein, further optionally, the mutated CD226 costimulatory domain or a functional fragment or variant thereof comprises a substitution mutation at a position corresponding to one or more of positions K295, Y319, and K333, optionally comprising K295A, Y319F, and / or K333 A mutations; (3) a costimulatory domain of a CD28, or a functional fragment or variant thereof; (4) a costimulatory domain of a CD2, or a functional fragment or variant thereof; (5) a costimulatory domain of an 0X40, or a functional fragment or variant thereof; (6) a costimulatory domain or a functional fragment or variant thereof from any one of CD27, CD3e, CD36, CD3y, CD79A, CD79B, SLAMF1, ICOS, DAP10, GITR, CD25, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, LIGHT, CD30, Lek, LAG3, LAT, LRP, NKG2D, N0TCH1, N0TCH2, N0TCH3, N0TCH4, R0R2, Ryk, Slp76, pTa, TCRa, TCRP, TRIM, Zap70, and PTCH2; and / or (7) a combination of any two or more of (l)-(6); and a transmembrane component disposed between and connecting the extracellular component and the intracellular component, wherein, optionally, the transmembrane component comprises, consists essentially of, or consists of the transmembrane domain of the CD3(^, or a functional fragment or variant thereof.

4. The fusion protein of claim 3, wherein:9.the extracellular domain of a CD3(^ or a functional fragment or variant thereof, the intracellular domain of the CD3(^ or a functional fragment thereof, and, optionally, the transmembrane domain of the CD3(^ or a functional fragment thereof, are human; or10.the extracellular component, transmembrane component, and intracellular domain of a CD3(^ together comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO.: 153, wherein SEQ ID NO.: 153 is optionally preceded immediately by a signal peptide, wherein, further optionally, the signal peptide comprises, consists essentially of, or consists of the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) or the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 126), wherein, still further optionally, the fusion protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO.: 155, or a variant of SEQ ID NO.: 155 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126)).

5. The fusion protein of claim 3 or 4, wherein:12.in the intracellular component, the intracellular signaling domain of the CD3(^ (or a functional fragment or variant thereof) is disposed N-terminal to the costimulatory portion; or, in the intracellular component, the intracellular signaling domain of the CD3(^ (or a functional fragment or variant thereof) is disposed C-terminal to the costimulatory portion.

6. The fusion protein of any one of claims 3-5, wherein:14.(a) the functional variant or fragment of a costimulatory domain of a 4- IBB does not comprise a(n, e.g., native) basic rich motif (e.g., does not comprise the amino acid sequence KRGRKKLLYIFKQPF (SEQ ID NO.: 156)) and / or the amino acid sequence that is not the intracellular signaling domain of the CD3(^ comprises, consists essentially of, or consists of DYHNPGYLVVLPDSTP (Ml-GS; SEQ ID NO: 157), EELDENYVPMNPNSPP (M2; SEQ ID NO: 158), EEGAPDYENLQELNHP (M3; SEQ ID NO: 159), LGSNQEEAYVTMSSFYQNQ (M4; SEQ ID NO: 160), LPMDTEVYESPFADPEEIR (M5; SEQ ID NO: 161), KPMAESITYAAVARHSAG (M6; SEQ ID NO: 162), LPTWSTPVQPMALIVLG (M7; SEQ ID NO: 163), PAPSIDRSTKPPLDRSL (M8; SEQ ID NO.: 164), GSNTAAPVQETLHGCQ (M9; SEQ ID NO.:165), DDSLPHPQQATDDSGHES (MIO; SEQ ID NO.:166), KAPHAKQEPQEINFPDDLP (Mil; SEQ ID NO: 167), GSGPGSRPTAVEGLALGSS (M12; SEQ ID NO: 168), SAGSAGS AGS AGS AGS AG (Ml 3; SEQ ID NO: 169);15.(GSDYHNPGYLVVLPDSTP)x, wherein X is any integer >1 and is preferably 1 or 2 (Ml; SEQ ID NO.: 170); or any combination of two or more of the foregoing;16.(b) the costimulatory domain of a CD226 or a functional fragment or variant thereof comprises a K295A mutation, a Y319F mutation, a K333 A mutation, K295A and Y319F mutations, K295A and K333A mutations, K333A and Y319F mutations, or K295A, Y319F, and K333A mutations, and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence NRRRRRERRD (SEQ ID NO: 171));17.(c) the costimulatory domain of a CD28, or a functional fragment or variant thereof, comprises a native dileucine (LL) or comprises a diglycine (GG) in place of the native LL, and / or comprises an amino acid substitution at any one or more of amino acids Y191, Y206, Y209, Y218, Pl 96, Pl 99, P208, and P211, as described in International Application No.18.PCT / US2019 / 019014 (e.g., comprises Y191F, Y206F, Y209F, Y218F, P196A, P199A, P208A, P211 A, or any combination thereof)), and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence RSKRSR (SEQ ID NO.: 172)); or19.(d) the costimulatory domain of a CD2, or a functional fragment or variant thereof does not comprise the amino acid sequence KRKKQRSRR (SEQ ID NO.: 173) and / or comprises a truncation of from 1 to 44 N-terminal amino acids of the CD2 intracellular domain.

7. The fusion protein of any one of claims 3-6, wherein the extracellular component, the transmembrane component, and the intracellular signaling domain of a CD3(^ together comprise, consist essentially of, or consist of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO.: 153, or to the amino acid sequence set forth in SEQ ID NO.: 155, or to a variant of SEQ ID NO: 155 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126).

8. The fusion protein of any one of claims 3-7, wherein the costimulatory portion of the intracellular component comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to: the amino acid sequence set forth in SEQ ID NO.: 174; the amino acid sequence set forth in SEQ ID NO.: 175; the amino acid sequence set forth in SEQ ID NO.:79; the amino acid sequence setforth in SEQ ID NO.: 176; the amino acid sequence set forth in SEQ ID NO.:348; an amino acid sequence according to the consensus sequence RSKRSRX1X2HSDX3MNMTX4RRX5GPTRKHX6QX7X8AX9PRDFAAX10RS wherein Xi is L or G, X2 is L or G, Xi and X2 preferably being L-L or G-G, X3 is Y or F, X4 is P or A, X5 is P or A, X6 is Y or F, X7 is P or A, X8 is Y or F, X9 is P or A, and X10 is Y or F (SEQ ID NO.: 177), optionally to the amino acid sequence set forth in SEQ ID NO.: 178 or to the amino acid sequence set forth in SEQ ID NO.: 179; the amino acid sequence set forth in SEQ ID NO.: 180; the amino acid sequence set forth in SEQ ID NO.: 181; the amino acid sequence set forth in SEQ ID NO.:246; the amino acid sequence set forth in SEQ ID NO.:247; or the amino acid sequence set forth in SEQ ID NO.: 142.

9. A fusion protein comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of:23.the amino acid sequence set forth in SEQ ID NO.: 182; the amino acid sequence set forth in SEQ ID NO.: 183, or a variant of SEQ ID NO.: 183 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 184; the amino acid sequence set forth in SEQ ID NO.: 185, or a variant of SEQ ID NO: 185 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 186; the amino acid sequence set forth in SEQ ID NO.: 187, or a variant of SEQ ID NO.: 187 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 188; the amino acid sequence set forth in SEQ ID NO.: 189, or a variant of SEQ ID NO: 189 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:253;24.the amino acid sequence set forth in SEQ ID NO.:252, or a variant of SEQ ID NO.:252 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 190; the amino acid sequence set forth in SEQ ID NO.: 191, or a variant of SEQ ID NO.: 191 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 192; the amino acid sequence set forth in SEQ ID NO.: 193, or a variant of SEQ ID NO: 193 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 194; the amino acid sequence set forth in SEQ ID NO.:195, or a variant of SEQ ID NO.:195 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 196; the amino acid sequence set forth in SEQ ID NO.: 197, or a variant of SEQ ID NO: 191 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.: 198; the amino acid sequence set forth in SEQ ID NO.: 199, or a variant of SEQ ID NO.: 199 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID N0.:200; the amino acid sequence set forth in SEQ ID NO.:201, or a variant of SEQ ID NO.:201 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:255;25.the amino acid sequence set forth in SEQ ID NO.:254 or a variant of SEQ ID NO.:254 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in26.SEQ ID NO.:202; or the amino acid sequence set forth in SEQ ID NO.:203 or a variant of SEQ ID NO.:203 wherein the amino acid sequence MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 126);27.the amino acid sequence set forth in SEQ ID NO:478, or a variant thereof comprising an alternative signal peptide; or28.the amino acid sequence set forth in SEQ ID NO:479.

10. A fusion protein comprising, consisting essentially of, or consisting of:30.an extracellular component comprising, consisting essentially of, or consisting of the extracellular domain of a CD3s, or a functional fragment or variant thereof;31.an intracellular component comprising, consisting essentially of, or consisting of (i) an intracellular signaling domain of the CD3s, or a functional fragment or variant thereof and (ii) a costimulatory portion, wherein the costimulatory portion optionally comprises, consists essentially of, or consists of a costimulatory domain, or a functional fragment or variant thereof, from 4-1BB, CD2, CD226, CD28, 0X40, CD27, CD38, CD3y, CD3< CD79A, CD79B, SLAMF1, ICOS, DAP10, CD25, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, LIGHT, CD30, Lek, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, R0R2, Ryk, Slp76, pTa, TCRa, TCRP, TRIM, Zap70, PTCH2, or any combination thereof; and a transmembrane component disposed between and connecting the extracellular component and the intracellular component, wherein, optionally, the transmembrane component comprises, consists essentially of, or consists of the transmembrane domain of the CD3s, or a functional fragment or variant thereof.

11. The fusion protein of claim 10, wherein the costimulatory portion comprises, consists essentially of, or consists of:33.(1) a costimulatory domain of a 4- IBB, or a functional fragment or variant thereof, optionally being linked or fused to an amino acid sequence that is not the intracellular signaling domain of the CD3s or a functional fragment or variant thereof; (2) a costimulatory domain of a CD226, or a functional fragment or variant thereof, wherein, optionally, the variant comprises one or more (e.g. substitution, e.g. non-conservative) mutation that (a) provides increased expression of the fusion protein in a T cell exposed to PVR, as compared to expression of endogenous CD226 by the T cell exposed to PVR, and / or (b) disrupts a Src kinase phosphorylation site on the fusion protein, and / or (c) reduces ubiquitination of the fusion protein by CBL-B, wherein, further optionally, the mutated CD226 costimulatory domain or a functional fragment or variant thereof comprises a substitution mutation at a position corresponding to one or more of positions K295, Y319, and K333, optionally comprising K295A, Y319F, and / or K333 A mutations; (3) a costimulatory domain of a CD28, or a functional fragment or variant thereof; (4) a costimulatory domain of a CD2, or a functional fragment or variant thereof; and / or (5) a costimulatory domain of an 0X40, or a functional fragment or variant thereof.

12. The fusion protein of claim 10 or 11, wherein the extracellular component, the transmembrane component, and the intracellular domain of a CD3s together comprise, consist essentially of, or consist of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO.:204, optionally preceded immediately by a signal peptide, wherein, further optionally, the signal peptide comprises, consists essentially of, or consists of the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) or the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 126) wherein, still further optionally, the fusion protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO.:206, or a variant of SEQ ID NO.:206 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126).

13. The fusion protein of claim 10 or 11, wherein, in the intracellular component, the intracellular domain of the CD3s (or a functional fragment or variant thereof) is disposed N-terminal to the costimulatory portion.

14. The fusion protein of claim 10 or 11, wherein, in the intracellular component, the intracellular domain of the CD3s (or a functional fragment or variant thereof) is disposed C-terminal to the costimulatory portion.

15. The fusion protein of any one of claims 11-14, wherein:37.(a) the functional variant or fragment of a costimulatory domain of a 4- IBB does not comprise a(n, e.g., native) basic rich motif (e.g., does not comprise the amino acid sequence KRGRKKLLYIFKQPF (SEQ ID NO.: 156)) and / or the amino acid sequence that is not the intracellular signaling domain of the CD3s comprises, consists essentially of, or consists of DYHNPGYLVVLPDSTP (Ml-GS; SEQ ID NO: 157), EELDENYVPMNPNSPP (M2; SEQ ID NO: 158), EEGAPDYENLQELNHP (M3; SEQ ID NO: 159), LGSNQEEAYVTMSSFYQNQ (M4; SEQ ID NO: 160), LPMDTEVYESPFADPEEIR (M5; SEQ ID NO: 161), KPMAESITYAAVARHSAG (M6; SEQ ID NO: 162), LPTWSTPVQPMALIVLG (M7; SEQ ID NO: 163), PAPSIDRSTKPPLDRSL (M8; SEQ ID NO: 164), GSNTAAPVQETLHGCQ (M9; SEQ ID NO.:165), DDSLPHPQQATDDSGHES (MIO; SEQ ID NO.:166), KAPHAKQEPQEINFPDDLP (Mil; SEQ ID NO: 167), GSGPGSRPTAVEGLALGSS (M12; SEQ ID NO: 168), SAGS AGS AGS AGS AGS AG (Ml 3; SEQ ID NO: 169);38.(GSDYHNPGYLVVLPDSTP)x, wherein X is any integer >1 and is preferably 1 or 2 (Ml; SEQ ID NO.: 170); or any combination of two or more of the foregoing;39.(b) the costimulatory domain of a CD226 or a functional fragment or variant thereof comprises a K295A mutation, a Y319F mutation, a K333 A mutation, K295A and Y319F mutations, K295A and K333A mutations, K333A and Y319F mutations, or K295A, Y319F, and K333A mutations, and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence NRRRRRERRD (SEQ ID NO: 171));40.(c) the costimulatory domain of a CD28, or a functional fragment or variant thereof, comprises a native dileucine (LL) or comprises a diglycine (GG) in place of the native LL, and / or comprises an amino acid substitution at any one or more of amino acids Y191, Y206, Y209, Y218, Pl 96, Pl 99, P208, and P211, as described in International Application No.41.PCT / US2019 / 019014 (e.g., comprises Y191F, Y206F, Y209F, Y218F, P196A, P199A, P208A, P211 A, or any combination thereof, and / or does not comprise a native basic rich motif (e.g., does not comprise the amino acid sequence RSKRSR (SEQ ID NO.: 172)); or42.(d) the costimulatory domain of a CD2, or a functional fragment or variant thereof does not comprise the amino acid sequence KRKKQRSRR (SEQ ID NO.: 173) and / or comprises a truncation of from 1 to 44 N-terminal amino acids of the CD2 intracellular domain.

16. The fusion protein of any one of claims 10-15, wherein the costimulatory portion of the intracellular component comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to:the amino acid sequence set forth in SEQ ID NO.: 174; the amino acid sequence set forth in SEQ ID NO.: 175; the amino acid sequence set forth in SEQ ID NO.:79; the amino acid sequence set forth in SEQ ID NO.: 176; the amino acid sequence set forth in SEQ ID NO.:348; an amino acid sequence according to the consensus sequence RSKRSRX1X2HSDX3MNMTX4RRX5GPTRKHX6QX7X8AX9PRDFAAX10RS wherein Xi is L or G, X2 is L or G, Xi and X2 preferably being L-L or G-G, X3 is Y or F, X4 is P or A, X5 is P or A, X6 is Y or F, X7 is P or A, X8 is Y or F, X9 is P or A, and X10 is Y or F (SEQ ID NO.: 177), optionally to the amino acid sequence set forth in SEQ ID NO.: 178 or to the amino acid sequence set forth in SEQ ID NO.: 179; the amino acid sequence set forth in SEQ ID NO.: 180; the amino acid sequence set forth in SEQ ID NO.:181;the amino acid sequence set forth in SEQ ID NO.:246; the amino acid sequence set forth in SEQ ID NO.:247; or the amino acid sequence set forth in SEQ ID NO.: 142.

17. A fusion protein comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of: the amino acid sequence set forth in SEQ ID NO.:207; the amino acid sequence set forth in SEQ ID NO.:208, or a variant of SEQ ID NO.:208 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:209; the amino acid sequence set forth in (SEQ ID NO.:210), or a variant of SEQ ID NO.:210 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126);45.the amino acid sequence set forth in SEQ ID NO.:211; the amino acid sequence set forth in SEQ ID NO.:212, or a variant of SEQ ID NO.:212 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:213; the amino acid sequence set forth in SEQ ID NO.:214, or a variant of SEQ ID NO.:214 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.:126); the amino acid sequence set forth in SEQ ID NO.:215; the amino acid sequence set forth in SEQ ID NO.:216, or a variant of SEQ ID NO.:216 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:217; the amino acid sequence set forth in SEQ ID NO.:218, or a variant of SEQ ID NO.:218 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 126); the amino acid sequence set forth in SEQ ID NO.:257; the amino acid sequence set forth in (SEQ ID NO.:256, or a variant of SEQ ID NO.:256 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126);46.the amino acid sequence set forth in SEQ ID NO.:219; the amino acid sequence set forth in SEQ ID NO.:220, or a variant of SEQ ID NO.:220 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:221; the amino acid sequence set forth in SEQ ID NO.:222, or a variant of SEQ ID NO.:222 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.:126); the amino acid sequence set forth in SEQ ID NO.:223; the amino acid sequence set forth in SEQ ID NO.:224, or a variant of SEQ ID NO.:224 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:225; the amino acid sequence set forth in SEQ ID NO.:226, or a variant of SEQ ID NO.:226 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 126; the amino acid sequence set forth in SEQ ID NO.:227; the amino acid sequence set forth in SEQ ID NO.:228, or a variant of SEQ ID NO.:228 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:229; the amino acid sequence set forth in SEQ ID NO.:230, or a variant of SEQ ID NO.:230 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 126); the amino acid sequence set forth in SEQ ID NO.:259; the amino acid sequence set forth in (SEQ ID NO.:258, or a variant of SEQ ID NO.:258 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126); the amino acid sequence set forth in SEQ ID NO.:231; or the amino acid sequence set forth in SEQ ID NO.:232, or a variant of SEQ ID NO.:232 wherein the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO.:205) is replaced by the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 126).

18. The fusion protein of any one of claims 3-17, wherein, when the fusion protein is expressed by a human T cell that further expresses a target (e.g., antigen)-binding protein (e.g, a a chTCR (also referred to as a chimeric TCR or a TCR / CAR), a TCR, a CAR, a scTCR, a Co-STAR receptor, a HIT receptor, a STAR receptor, or a mutSTAR receptor), the T cell has increased production of one or more cytokine (e.g, IL-2, IFN-^, or both) when in the presence of the target (e.g., antigen), and / or has increased proliferation when in the presence of the target, ascompared to a reference human T cell ( / .<., a T cell that is substantially identical to the human T cell) that does not express the fusion protein.

19. The fusion protein of any one of claims 3-9, wherein the intracellular component comprises a linker disposed between and connecting: (i) the intracellular domain of the CD3(^ or a functional fragment or variant thereof and the costimulatory portion; and / or (ii) the costimulatory domain or a functional fragment or variant thereof and the amino acid sequence that is not the CD3(^ intracellular signaling domain or a functional fragment or portion thereof, wherein, optionally, the linker comprises: a (GlyxSery)nsequence, wherein x, y, and n are each independently 1 or more), a Whitlow linker, a Townsend linker, an alanine linker (e.g., AAA), a proline-glycine linker (e.g., PG or GP or GPP or PGP), or any combination thereof.

20. The fusion protein of any one of claims 10-18, wherein the intracellular component comprises a linker disposed between and connecting: (i) the intracellular domain of the CD3s or a functional fragment or variant thereof and the costimulatory portion; and / or (ii) the costimulatory domain or a functional fragment or variant thereof and the amino acid sequence that is not the CD3s intracellular signaling domain or a functional fragment or portion thereof, wherein, optionally, the linker comprises: a (GlyxSery)nsequence, wherein x, y, and n are each independently 1 or more), a Whitlow linker, a Townsend linker, an alanine linker (e.g., AAA), a proline-glycine linker (e.g., PG or GP or GPP or PGP), or any combination thereof.

21. The fusion protein of any one of claims 3-9 and 19, wherein, when expressed by a host cell (e.g., a human T cell), two molecules of the fusion protein are capable of together forming a protein homodimer at the host cell membrane; a single molecule of the fusion protein is capable of forming a protein heterodimer with a single molecule of an endogenous CD3(^ protein at the host cell membrane, or both.

22. The fusion protein of any one of claims 10-18 and 20, wherein, when expressed by a host cell (e.g., a human T cell), a single molecule of the fusion protein is capable of forming a protein heterodimer with a single molecule of an endogenous CD3y or CD36 protein at the host cell membrane.

23. A fusion protein comprising an extracellular component, an intracellular component, and a transmembrane component disposed between and connecting the extracellular and intracellular components, wherein the intracellular component comprises, consists essentially of, or consists of: the amino acid sequence set forth in SEQ ID NO.:233;53.the amino acid sequence set forth in SEQ ID NO.:234; the amino acid sequence set forth in SEQ ID NO.:235; or54.the amino acid sequence set forth in SEQ ID NO.:236, wherein, optionally, the extracellular component of the fusion protein does not comprise a target-binding domain (e.g., a scFv, a VH, a VL, a Fab, a Fd, a scFab, a dsFv, a VHH, a centyrin, a TCR variable domain, or the like) and / or the extracellular component of the fusion protein does not comprise a hinge (e.g., an immunoglobulin hinge or a variant thereof, a CD8 hinge or a variant thereof, or the like) and / or the extracellular component of the fusion protein does not comprise an immunoglobulin constant domain and / or the extracellular component of the fusion protein does not comprise a linker amino acid sequence.

24. The fusion protein of claim 23, wherein the intracellular component further comprises an intracellular signaling domain of a CD3(^ or an intracellular signaling domain of a CD3s.

25. The fusion protein of any one of claims 3-24, wherein the fusion protein does not comprise, in an extracellular portion or extracellular component thereof, (1) a target-binding domain (e.g., a scFv, a VH, a VL, a Fab, a Fd, a scFab, a dsFv, a VHH, a centyrin, a TCR variable domain, or the like) and / or (2) a hinge (e.g., an immunoglobulin hinge or a variant thereof, a CD8 hinge or a variant thereof, or the like) and / or (3) a linker amino acid sequence.

26. The fusion protein of any one of claims 3-25, wherein the extracellular component of the fusion protein consists of a human CD3(^ extracellular domain or a human CD3s extracellular domain.

27. A fusion protein that comprises, consists essentially of, or consists of:59.one or more of the following constructs shown in Figure 45: CD3z_4-lBB; CD3z_4-1BB-BRM; CD3z_4-lBB-BRM_ 7; CD3z 4-IBB-BRM A7 / A7 / ; CD3z_CD226mut;60.CD3z_CD226mut-BRM; CD3z_CD28; CD3z_CD28-BRM; CD3z CD2; and CD3z_CD2tr, optionally with the CD3z signal peptide (MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154)) removed or comprising a GM-CSF signal peptide (SEQ ID NO.: 126) in place of the CD3z signal peptide; or61.one or more of the following constructs shown in Figure 47: CD3z-CD226 (BRM deletion); and CD3z-CD2(BRM deletion), optionally with the CD3z signal peptide removed or comprising a GM-CSF signal peptide in place of the CD3z signal peptide.

28. A polynucleotide encoding the fusion protein of any one of claims 3-27, optionally wherein the polynucleotide comprises DNA or RNA, further optionally wherein the RNA comprises mRNA, further optionally saRNA, circRNA, or any combination thereof.

29. The polynucleotide of claim 28, further encoding a target (e.g., antigen)-binding protein.

30. The polynucleotide of claim 29, wherein the target-binding protein is selected from a chTCR (also referred to as a chimeric TCR or a TCR / CAR)), a TCR, a scTCR, a CAR, a HIT receptor, a STAR receptor, and a mutSTAR receptor, and is preferably a chTCR.

31. The polynucleotide of claim 30, wherein the encoded target-binding protein comprises: a first polypeptide comprising a TCR a-chain constant domain; and a second polypeptide comprising a TCR P-chain constant domain.

32. The polynucleotide of claim 31, comprising, in 5’ to 3’ direction: [P1]-[CS]-[FP]-[CS]-[P2]; [P2]-[CS]-[FP]-[CS]-[P1]; [FP]-[CS]-[P1]-[CS]-[P2]; or [FP]-[CS]-[P2]-[CS]-[P1];66.wherein [Pl] is a a nucleotide sequence encoding the first polypeptide of the targetbinding protein, [P2] is a nucleotide sequence encoding the second polypeptide of the targetbinding protein, [FP] is a nucleotide sequence encoding the fusion protein, and each [CS] is independently a nucleotide sequence encoding a cleaving sequence or a skip sequence, wherein each cleaving sequence independently comprises a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A, or a variant thereof, and optionally comprising a short N-terminal link (e.g, GSG)), a protease recognition sequence (e.g, a furin consensus sequence of RX(KR)R, such as RAKR)), or a combination thereof.

33. A polynucleotide encoding (i) a chTCR and (ii) a CD3(^ or a functional fragment or variant thereof, wherein, optionally, the CD3(^ or a functional fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 155, or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 153, optionally wherein the polynucleotide comprises DNA or RNA, further optionally wherein the RNA comprises mRNA, saRNA, circRNA, or any combination thereof.

34. A polynucleotide encoding (i) a chTCR and (ii) a CD3s or a functional fragment or variant thereof, wherein, optionally, the CD3s or a functional fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:206 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acidsequence set forth in SEQ ID NO.:204, optionally wherein the polynucleotide comprises DNA or RNA, further optionally wherein the RNA comprises mRNA, saRNA, circRNA, or any combination thereof.

35. The polynucleotide of claim 33 or 34, wherein the chTCR comprises:70.(1) a first polypeptide comprising a single-chain variable fragment (scFv) linked or fused to a TCR a-chain constant domain (Ca), and (2) a second polypeptide comprising a TCR P-chain constant domain (CP) and, optionally, no antigen-binding domain;71.(1) a first polypeptide comprising a single-chain variable fragment (scFv) linked or fused to a CP, and (2) a second polypeptide comprising a Ca and, optionally, no antigen-binding domain;72.(1) a first polypeptide comprising a single-chain variable fragment (scFv) linked or fused to a CP, and (2) a second polypeptide comprising a scFv linked or fused to a Ca;73.(1) a first polypeptide comprising an antibody heavy chain variable domain (VH) linked or fused to a TCR a-chain constant domain (Ca), and (2) a second polypeptide comprising a cognate antibody light chain variable domain (VL) linked or fused to a TCR P-chain constant domain (CP); or74.(1) a first polypeptide comprising an antibody light chain variable domain (VL) linked or fused to a TCR a-chain constant domain (Ca), and (2) a second polypeptide comprising a cognate antibody heavy chain variable domain (VH) linked or fused to a TCR P-chain constant domain (CP).

36. The polynucleotide of any one of claims 30-35, wherein the chTCR specifically binds to a human CD19, a human BMC A, a human CD22, a human SLAMF7, or a combination of two or more of these, optionally wherein the chTCR specifically binds to a human CD19 and specifically binds to a human CD22.

37. The polynucleotide of claim 36, wherein the chTCR comprises a scFv comprising, or a VH and VL that together comprise, the six CDRs (e.g., as defined by Kabat, AbM, AHo, IMGT, Chothia, North, Martin, or any combination thereof), and optionally the VH and VL amino acid sequences, of anti-CD19 monoclonal antibody FMC63.

38. The polynucleotide of claim 37, wherein the chTCR comprises a scFv comprising the VH amino acid sequence and the VL amino acid sequence of monoclonal antibody FMC63.

39. The polynucleotide of claim 36, wherein chTCR comprises: a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 110; and a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 111.

40. The polynucleotide of claim 39, encoding the amino acid sequence set forth in SEQ ID NO.:245.

41. The polynucleotide of any one of claims 30-38, wherein the chTCR comprises a scFv comprising, or a VH and VL that together comprise, the six CDRs (e.g., as defined by Kabat, AbM, AHo, IMGT, Chothia, North, Martin, or any combination thereof), and optionally the VH and VL amino acid sequences, of anti-CD22 monoclonal antibody 9A8, or of anti-CD22 monoclonal antibody m971.

42. The polynucleotide of any one of claims 30-38 and 41, wherein the chTCR comprises a scFv comprising the amino acid sequence of SEQ ID NO.:353 and the amino acid sequence of SEQ ID NO.:354, or comprising the amino acid sequence of SEQ ID NO.:350 and the amino acid sequence of SEQ ID NO.:351.

43. The polynucleotide of any one of claims 30-38, 41, and 42, wherein the chTCR comprises the amino acid sequence of any one of SEQ ID NOs.:349-354, 363-391, 394-413, and 416-448, preferably of any one of SEQ ID NOs.:394-401, further preferably comprising a scFv comprising the VL and VH amino acid sequences (optionally in VL-linker-VH orientation) of anti-CD19 monoclonal antibody FMC63, fused to TRAC, and a scFv comprising the VL and VH (optionally in VL-linker-VH orientation) amino acid sequences of anti-CD22 monoclonal antibody 9A8, fused to TRBC.

44. A polynucleotide encoding (i) a chTCR and (ii) a fusion protein comprising, consisting essentially of, or consisting of: (1) an extracellular component comprising, consisting essentially of, or consisting of, the extracellular domain of a CD3(^, or a functional fragment or variant thereof; an intracellular component comprising an intracellular signaling domain of the CD3(^ and a costimulatory domain; and a transmembrane component, wherein the transmembrane component is optionally the transmembrane domain of the CD3(^; or (2) an extracellular component comprising, consisting essentially of, or consisting of, the extracellular domain of a CD3s, or a functional fragment or variant thereof; an intracellular component comprising an intracellular signaling domain of the CD3s and a costimulatory domain; and a transmembrane component, wherein the transmembrane component is optionally the transmembrane domain of the CD3s.

45. The polynucleotide of claim 44, wherein the costimulatory domain comprises a costimulatory domain, or a functional fragment or variant thereof, from 4- IBB, CD2, CD226, CD28, 0X40, CD27, CD3e, CD38, CD3y, CD79A, CD79B, SLAMF1, ICOS, DAP10, CD25, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, LIGHT, CD30, Lek, LAG3, LAT, LRP, NKG2D,N0TCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTa, TCRa, TCRp, TRIM, Zap70, PTCH2, or any combination thereof.

46. The polynucleotide of claim 44, wherein the costimulatory domain comprises a costimulatory domain, or a functional fragment or variant thereof, from 4-1BB.

47. The polynucleotide of claim 44, wherein the functional fragment of a 4- IBB costimulatory domain does not comprise a native basic rich motif.

48. The polynucleotide of claim 46, wherein the fusion protein comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or comprising, consisting essentially of, or consisting of: the amino acid sequence set forth in SEQ ID NO.:237; the amino acid sequence set forth in SEQ ID NO.:238; the amino acid sequence set forth in SEQ ID NO.:239; the amino acid sequence set forth in SEQ ID NO.:465; or the amino acid sequence set forth in any one of SEQ ID NOs.: 182-185 and 207-214.

49. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising:88.(i) in place of the amino acid sequence of SEQ ID NO.:262, an amino acid sequence according to SEQ ID NO.:263;89.(ii) in place of the amino acid sequence of SEQ ID NO.:264, an amino acid sequence according to SEQ ID NO.:265; and / or90.(iii) in place of the amino acid sequence of SEQ ID NO.:266, an amino acid sequence according to SEQ ID NO.:267.

50. The polypeptide of claim 49, wherein:92.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152), or a variant thereof having one, two, or three amino acid substitutions; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID No.:240) or an amino acid sequence comprising one, two, three, or four substitutions relative to SEQ ID NO.:240.

51. The polypeptide of claim 50, wherein:94.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152); and95.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID NO.:240).

52. The polypeptide of any one of claims 49-51, wherein the intracellular component comprises amino acid sequences according to SEQ ID NOs.: (A) 263, 264, and 266; (B)263, 264, and 267; (C) 263, 265, and 266; (D) 263, 265, and 267; (E) 262, 264, and 267; (F)262, 265, and 266; or (G) 262, 265, and 267.

53. The polypeptide of any one of claims 49-52, wherein the intracellular component comprises amino acid sequences according to SEQ ID NOs.: (A) 450, 264, and 266; (B) 450, 264, and 452; (C) 450, 451, and 266; (D) 262, 451, and 452; (E) 262, 451, and 266; (F) 262, 264, and 452; or (G) 450, 451, and 452.

54. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:284, an amino acid sequence according to SEQ ID NO.:285, preferably the amino acid sequence of SEQ ID NO.:453.

55. The polypeptide of claim 54, wherein:100.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or101.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

56. The polypeptide of claim 55, wherein:103.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and104.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID No.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

57. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD36 extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD36 transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD36 intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:296, an amino acid sequence according to any one of SEQ ID NOs.:297-298a, preferably the amino acid sequence of SEQ ID NO.:455, the amino acid sequence of SEQ ID NO.:456, or the amino acid sequence of SEQ ID NO.:457.

58. The polypeptide of claim 57, wherein:107.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:458, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or (2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:459 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459.

59. The polypeptide of claim 58, wherein:109.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:458, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and110.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:459 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:459.

60. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3y extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3y transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3y intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:290, an amino acid sequence according to SEQ ID NO.:291, preferably the amino acid sequence of SEQ ID NO.:454.

61. The polypeptide of claim 60, wherein:113.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:460, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or114.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:461 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:461.

62. The polypeptide of claim 61, wherein:(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:460, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and116.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO.:461 or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:461.

63. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising:118.(iv) SEQ ID NO.:262;119.(v) in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:262; and120.(vi) in place of the amino acid sequence of SEQ ID NO.:266, the amino acid sequence of SEQ ID NO.:262.

64. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising:122.(iv) in place of the amino acid sequence of SEQ ID NO.:262, the amino acid sequence of SEQ ID NO.:266;123.(v) in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:266; and124.(vi) the amino acid sequence of SEQ ID NO.:

266.

65. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3(^ extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3(^ transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3(^ intracellular domain comprising:125.(iv) in place of the amino acid sequence of SEQ ID NO.:262, the amino acid sequence of SEQ ID NO.:284;126.(v) in place of the amino acid sequence of SEQ ID NO.:264, the amino acid sequence of SEQ ID NO.:284; and127.(vi) in place of the amino acid sequence of SEQ ID NO.:266, the amino acid sequence of SEQ ID NO.:284.

66. The polypeptide of any one of claims 63-65, wherein:129.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152), or a variant thereof having one, two, or three amino acid substitutions; and / or130.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID No.:240) or an amino acid sequence comprising one, two, three, or four substitutions relative to SEQ ID NO.:240.

67. The polypeptide of claim 66, wherein:132.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence QSFGLLDPK (SEQ ID NO.: 152); and133.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID NO.:240).

68. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant ofa human CD3s intracellular domain comprising, in place of the amino acid sequence of SEQ ID NO.:284, the amino acid sequence of SEQ ID NO.:266.

69. A polypeptide comprising (a) an extracellular component comprising, consisting essentially of, or consisting of a(n, e.g., human) CD3s extracellular domain or a functional variant thereof, (b) a transmembrane component comprising, consisting essentially of, or consisting of, a(n, e.g., human) CD3s transmembrane domain or a functional variant thereof, and (c) an intracellular component comprising, consisting essentially of, or consisting of, a variant of a human CD3s intracellular domain comprising:136.in place of the amino acid sequence KNRKAKAK, a variant thereof wherein one or more lysine amino acid residue is independently substituted with a neutral amino acid residue and / or wherein the arginine amino acid is substituted with a neutral amino acid residue, optionally a serine, further optionally, the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence KNRKAKAK, the amino acid sequence SNSSASAS;137.in place of the amino acid sequence PPPVPNPDY, a variant thereof wherein one or more proline amino acid residue is independently substituted with an alanine amino acid residue, a serine amino acid residue, or a valine amino acid residue, preferably an alanine amino acid residue, optionally wherein the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence PPPVPNPDY, the amino acid sequence PAPVANPDY; or138.in place of the amino acid sequence of SEQ ID NO.:284, a variant thereof wherein the first arginine amino acid residue and the lysine residue are each independently substituted with a neutral amino acid residue, preferably an alanine, optionally wherein the variant of a human CD3s intracellular domain comprises, in place of the amino acid sequence of SEQ ID NO.:284, the amino acid sequence YEPIAAGQRDLYSGL.

70. The polypeptide of claim 68 or 69, wherein:140.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and / or141.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

71. The polypeptide of claim 70, wherein:143.(1) the extracellular component comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO.:242, or a variant thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and144.(2) the transmembrane component comprises, consists essentially of, or consists of the amino acid sequence VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO.:243) or an amino acid sequence comprising one, two, three, four, or 5 substitutions relative to SEQ ID NO.:243.

72. A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:268-281, 286, 287, 292, 293, 299-316, 478, and 479.

73. A polypeptide comprising, consisting essentially of, or consisting of any one of the following constructs shown in Figure 47: CD3z_12X; CD3z_lX3; CD3z_X23; CD3z_lXX; CD3z_X2X; CD3z XX3; CD3z XXX or 6F; CD3e_2F; CD3g_2F; CD3d_2F; CD3d_lF; CD3z_lll; CD3z_333; CD3z_CD3elelel; CD3e_CD3zl; CD3e_BRS; CD3e_PRS; CD3 KR, optionally with the respective CD3(^ signal peptide or CD3s signal peptide removed or comprising a GM-CSF signal peptide in place of the respective CD3(^ signal peptide or CD3s signal peptide.

74. The polypeptide of any one of claims 49-73, wherein the polypeptide does not comprise, in an extracellular portion or extracellular component thereof, (1) a target-binding domain (e.g., a scFv, a VH, a VL, a Fab, a Fd, a scFab, a dsFv, a VHH, a centyrin, a TCR variable domain, or the like) and / or (2) a hinge (e.g., an immunoglobulin hinge or a variant thereof, a CD8 hinge or a variant thereof, or the like) and / or (3) a linker amino acid sequence.

75. The polypeptide of any one of claims 49-74, wherein an extracellular portion or extracellular component of the polypeptide consists essentially of or consists of the extracellular domain of a human CD3(^ or the extracellular domain of a human CD3s.

76. A polynucleotide encoding the polypeptide of any one of claims 49-75, optionally wherein the polynucleotide comprises DNA or RNA, further optionally wherein the RNA comprises mRNA, further optionally saRNA, circRNA, or any combination thereof.

77. The polynucleotide of claim 76, further encoding a target (e.g., antigen)-binding protein.

78. The polynucleotide of claim 77, wherein the target-binding protein is selected from a chTCR (also referred to as a chimeric TCR or a TCR / CAR)), a TCR, a scTCR, a CAR, a HIT receptor, a STAR receptor, a Co-STAR receptor, and a mutSTAR receptor, and is preferably a chTCR.

79. The polynucleotide of claim 78, wherein the encoded target-binding protein comprises: a first polypeptide comprising a TCR a-chain constant domain; and a second polypeptide comprising a TCR P-chain constant domain.

80. The polynucleotide of claim 79, comprising, in 5’ to 3’ direction: [P1]-[CS]-[FP]-[CS]-[P2]; [P2]-[CS]-[FP]-[CS]-[P1]; [FP]-[CS]-[P1]-[CS]-[P2]; or [FP]-[CS]-[P2]-[CS]-[P1];153.wherein [Pl] is a a nucleotide sequence encoding the first polypeptide of the targetbinding protein, [P2] is a nucleotide sequence encoding the second polypeptide of the targetbinding protein, [FP] is a nucleotide sequence encoding the fusion protein, and each [CS] is independently a nucleotide sequence encoding a cleaving sequence or a skip sequence, wherein each cleaving sequence independently comprises a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A, or a variant thereof, and optionally comprising a short N-terminal link (e.g, GSG)), a protease recognition sequence (e.g, a furin consensus sequence of RX(KR)R, such as RAKR)), or a combination thereof.

81. The polynucleotide of any one of claims 78-80, wherein the chTCR comprises: (1) a first polypeptide comprising a single-chain variable fragment (scFv) linked or fused to a TCR a-chain constant domain (Ca), and (2) a second polypeptide comprising a TCR P-chain constant domain (CP) and, optionally, no antigen-binding domain;155.(1) a first polypeptide comprising a single-chain variable fragment (scFv) linked or fused to a CP, and (2) a second polypeptide comprising a Ca and, optionally, no antigen-binding domain;156.(1) a first polypeptide comprising a single-chain variable fragment (scFv) linked or fused to a CP, and (2) a second polypeptide comprising a scFv linked or fused to a Ca;157.(1) a first polypeptide comprising an antibody heavy chain variable domain (VH) linked or fused to a TCR a-chain constant domain (Ca), and (2) a second polypeptide comprising a cognate antibody light chain variable domain (VL) linked or fused to a TCR P-chain constant domain (CP); or158.(1) a first polypeptide comprising an antibody light chain variable domain (VL) linked or fused to a TCR a-chain constant domain (Ca), and (2) a second polypeptide comprising a cognate antibody heavy chain variable domain (VH) linked or fused to a TCR P-chain constant domain (CP).

82. The polynucleotide of claim 78 or 81, wherein the chTCR specifically binds to a human CD19, a human BMC A, a human CD22, a human SLAMF7, or a combination of two or more of these, optionally wherein the chTCR specifically binds to a human CD19 and specifically binds to a human CD22.

83. The polynucleotide of claim 82, wherein the chTCR comprises a scFv comprising, or a VH and VL that together comprise, the six CDRs (e.g., as defined by Kabat, AbM, AHo, IMGT, Chothia, North, Martin, or any combination thereof), and optionally the VH and VL amino acid sequences, of anti-CD19 monoclonal antibody FMC63.

84. The polynucleotide of claim 83, wherein the chTCR comprises a scFv comprising the VH amino acid sequence and the VL amino acid sequence of anti-CD19 monoclonal antibody FMC63.

85. The polynucleotide of claim 84, wherein chTCR comprises: a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 110; and a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 111.

86. The polynucleotide of claim 85, encoding the amino acid sequence set forth in SEQ ID NO.:245.

87. The polynucleotide of any one of claims 76-84, wherein the chTCR comprises a scFv comprising, or a VH and VL that together comprise, the six CDRs (e.g., as defined by Kabat, AbM, AHo, IMGT, Chothia, North, Martin, or any combination thereof), and optionally the VH and VL amino acid sequences, of anti-CD22 monoclonal antibody 9A8, or of anti-CD22 monoclonal antibody m971.

88. The polynucleotide of any one of claims 76-84 and 87, wherein the chTCR comprises a scFv comprising the amino acid sequence of SEQ ID NO.:353 and the amino acid sequence of SEQ ID NO.:354, or comprising the amino acid sequence of SEQ ID NO.:350 and the amino acid sequence of SEQ ID NO.:351.

89. The polynucleotide of any one of claims 76-84, 87, and 88, wherein the chTCR comprises the amino acid sequence of any one of SEQ ID NOs.:349-354, 363-391, 394-413, and 416-448, preferably of any one of SEQ ID NOs.:394-401, further preferably comprising a scFv comprising the VL and VH amino acid sequences (optionally in VL-linker-VH orientation) of anti-CD19 monoclonal antibody FMC63, fused to TRAC, and a scFv comprising the VL and VH amino acid sequences (optionally in VL-linker-VH orientation) of anti-CD22 monoclonal antibody 9A8, fused to TRBC.

90. A vector comprising the polynucleotide of any one of claims 28-48.

91. A vector comprising the polynucleotide of any one of claims 76-89.

92. The vector of claim 90 or 91, which is a viral vector.

93. The vector of claim 92, which is a lentiviral vector.

94. The vector of claim 92, with is a retroviral vector.

95. A host cell expressing the fusion protein of any one of claims 3-27 and / or expressing a fusion protein that comprises one or more of the following constructs shown in Figure 45: CD3z_4-lBB; CD3z_4-lBB-BRM; CD3z_4-lBB-BRM_ 7; CD3z_4-lBB-BRMJV77JW7; CD3z_CD226mut; CD3z_CD226mut-BRM; CD3z_CD28; CD3z_CD28-BRM; CD3z_CD2; and CD3z_CD2tr, optionally with the CD3z signal peptide (MKWKALFTAAILQAQLPITEA (SEQ ID NO: 154)) removed or comprising a GM-CSF signal peptide (SEQ ID NO.: 126) in place of the CD3z signal peptide; or that comprises one or more of the following constructs shown in Figure 47: CD3z-CD226 (BRM deletion); and CD3z-CD2(BRM deletion), optionally with the CD3z signal peptide removed or comprising a GM-CSF signal peptide in place of the CD3z signal peptide, and / or comprising the polynucleotide of any one of claims 28-48 and / or comprising the vector of claim 90.

96. A host cell expressing the polypeptide of any one of claims 49-75 and / or comprising the polynucleotide of any one of claims 76-89 and / or comprising the vector of claim 91.

97. The host cell of claim 95 or 96, further expressing an antigen-binding protein, wherein, optionally, the antigen-binding protein comprises a chTCR, a TCR, a scTCR, a mutSTAR, a Co-STAR, a STAR, a HIT receptor, or a CAR, further optionally wherein the antigen-binding protein is a chTCR that binds to human CD19 and human CD22.

98. A host cell expressing (i) a chTCR and (ii) a CD3(^ or a functional fragment or variant thereof encoded by a polynucleotide that is heterologous to the host cell, wherein, optionally, the CD3(^ or a functional fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:155 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 153.

99. A host cell expressing (i) a chTCR and (ii) a CD3s or a functional fragment or variant thereof encoded by a polynucleotide that is heterologous to the host cell, wherein, optionally, the CD3s or a functional fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:206 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.:204.

100. The host cell of any one of claims 97-99, comprising a hematopoietic progenitor cell, a human immune system cell, an embryonic stem cell, a peripheral blood mononuclear cell, a hematopoietic stem cell, or an induced pluripotent stem cell.

101. The host cell of any one of claims 97-100, wherein the host cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yb T cell, a natural killer cell, a natural killer T cell, a monocyte, or any combination thereof.

102. The host cell of claim any one of claims 97-101, wherein the host cell comprises a T cell.

103. The host cell of claim 102, wherein the T cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

104. The host cell of any one of claims 97-103, comprising a chromosomal gene knockout or a mutation in: a CD3(^ gene locus, a CD3s gene locus, a TGFpRl gene locus, a TRAC gene locus, a TRBC gene locus, a TGFPR2 gene locus, a PD-1 gene locus, a CTLA4 gene locus, a LAT gene locus, a TIM-3 gene locus, a PD-L1 gene locus, a TIGIT gene locus, anA2AR gene locus, a Fas locus, a FasL gene locus, a B7-H3 gene locus, a B7-H4 gene locus, an IDO gene locus, a VISTA gene locus, a SIGLEC7 gene locus, a SIGLEC9 gene locus, a T cell receptor gene locus, a MHC (e.g. HLA) gene locus, a CBLB gene locus, a RASA2 gene locus, a UBASH3 A gene locus, a CISH gene locus, a CD4 gene locus, a CD8 gene locus, or any combination thereof, such as a a CD3(^ gene locus or a CD3s gene locus, TRAC gene locus, a TRBC gene locus, and / or one or both of a CD4 gene locus and a CD8 gene locus, wherein, optionally, the chromosomal gene knockout or mutation comprises a missense mutation, a nonsense mutation, a splice junction mutation, or any combination thereof, and / or wherein the chromosomal gene knockout or mutation was introduced using base editing, such as cytosine base editing.

105. The host cell of any one of claims wherein the host cell is modified (e.g., having a chromosomal knockout mutation and / or a chromosomal missense mutation and / or a chromosomal splice junction mutation; encoding an inhibitory nucleic acid such as an siRNA or an antisense oligonucleotide) to have reduced protein expression (including null expression), of an endogenous CD3(^, and endogenous CD3s, an endogenous TRAC, an endogenous TRBC, an endogenous TIGIT, endogenous CD4, endogenous CD8, or any combination thereof, as compared to the unmodified host cell, wherein, optionally, the host cell is or was modified using base editing, such as cytosine base editing.

106. A composition comprising: (i) the fusion protein of any one of claims 3-27; and / or (ii) the polynucleotide of any one of claims 28-48, optionally comprised in a lipid composition; and / or (iii) the vector of claim 90; and / or (iv) the host cell of claim 95, and a pharmaceutically acceptable carrier, excipient, or diluent.

107. A composition comprising: (i) the fusion protein of any one of claims 49-75; and / or (ii) the polynucleotide of any one of claims 76-89, optionally comprised in a lipid composition; and / or (iii) the vector of claim 91; and / or (iv) the host cell of claim 96, and a pharmaceutically acceptable carrier, excipient, or diluent.

108. The composition of claim 106 or 107, comprising (i) a composition comprising at least about 30% CD4+ T host cells, combined with (ii) a composition comprising at least about 30% CD8+ T host cells, in about a 1:1 ratio.

109. A method of treating a disease or condition in a subject, the method comprising administering to the subject an effective amount of: (i) the fusion protein of any one of claims 3-27; and / or (ii) the polynucleotide of any one of claims 28-48; and / or (iii) the vector of claim 90; and / or (iv) the host cell of claim 95 (wherein, optionally, the host cell is a hematopoietic progenitor cell, a human immune system cell, an embryonic stem cell, a peripheral bloodmononuclear cell, a hematopoietic stem cell, or an induced pluripotent stem cell T cell, wherein, further optionally, the host cell is T cell, wherein, still further optionally, the host cell is a CD8+ T cell or a CD4+ T cell), and / or (v) the composition of claim 106.

110. The fusion protein of any one of claims 3-27; the polynucleotide of any one of claims 28-48; the vector of claim 90; the host cell of claim 95 (wherein, optionally, the host cell is a hematopoietic progenitor cell, a human immune system cell, an embryonic stem cell, a peripheral blood mononuclear cell, a hematopoietic stem cell, or an induced pluripotent stem cell T cell, wherein, further optionally, the host cell is T cell, wherein, still further optionally, the host cell is a CD8+ T cell or a CD4+ T cell), and / or the composition of claim 106, for use in a method of treating a disease or condition in a subject.

111. The fusion protein of any one of claims 3-27; the polynucleotide of any one of claims 28-48; the vector of claim 90; the host cell of claim 95 (wherein, optionally, the host cell is a hematopoietic progenitor cell, a human immune system cell, an embryonic stem cell, a peripheral blood mononuclear cell, a hematopoietic stem cell, or an induced pluripotent stem cell T cell, wherein, further optionally, the host cell is T cell, wherein, still further optionally, the host cell is a CD8+ T cell or a CD4+ T cell), and / or the composition of claim 106, for use in the preparation of a medicament for treating a disease or condition in a subject.

112. A method of treating a disease or condition in a subject, the method comprising administering to the subject an effective amount of: (i) the polypeptide of any one of claims 49-75; and / or (ii) the polynucleotide of any one of claims 76-89; and / or (iii) the vector of claim 91; and / or (iv) the host cell of claim 96 (wherein, optionally, the host cell is a hematopoietic progenitor cell, a human immune system cell, an embryonic stem cell, a peripheral blood mononuclear cell, a hematopoietic stem cell, or an induced pluripotent stem cell T cell, wherein, further optionally, the host cell is T cell, wherein, still further optionally, the host cell is a CD8+ T cell or a CD4+ T cell), and / or188.(vii) the composition of claim 107.

113. The fusion protein of any one of claims 49-75; the polynucleotide of any one of claims 76-89; the vector of claim 91; the host cell of claim 96 (wherein, optionally, the host cell is a hematopoietic progenitor cell, a human immune system cell, an embryonic stem cell, a peripheral blood mononuclear cell, a hematopoietic stem cell, or an induced pluripotent stem cell T cell, wherein, further optionally, the host cell is T cell, wherein, still further optionally, the host cell is a CD8+ T cell or a CD4+ T cell), and / or the composition of claim 107, for use in a method of treating a disease or condition in a subject.

114. The fusion protein of any one of claims 28-48; the polynucleotide of any one of claims 76-89; the vector of claim 91; the host cell of claim 96 (wherein, optionally, the host cell is a hematopoietic progenitor cell, a human immune system cell, an embryonic stem cell, a peripheral blood mononuclear cell, a hematopoietic stem cell, or an induced pluripotent stem cell T cell, wherein, further optionally, the host cell is T cell, wherein, still further optionally, the host cell is a CD8+ T cell or a CD4+ T cell), and / or the composition of claim 107, for use in the preparation of a medicament for treating a disease or condition in a subject.

115. The method of claim 109 or 112, or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of any one of claims 110, 111, 113, and 114, wherein the disease or condition comprises or is a hyperproliferative disease, a proliferative disease, an autoimmune disease, a neurodegenerative disease, or an infection.

116. The method of claim 109, 112, or 115 or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of any one of claims 110, 111, 113, 114, and 115, wherein the disease or condition is a cancer, such as a hematological cancer or a solid cancer, wherein, optionally:192.(1) the cancer comprises a myeloma (e.g. multiple myeloma), a carcinoma, a sarcoma, a glioma, a lymphoma, a leukemia, a myeloma, or any combination thereof; and / or (2) the cancer comprises a cancer of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer such as triple-negative breast cancer (TNBC), gastric cancer, non-small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, or any combination thereof; and / or193.(3) the cancer comprises Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, linitis plastic, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz tumor, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optice nerve glioma, a mixed glioma, Hodgkin’s lymphoma, a B-cell lymphoma, non-Hodgkin’s lymphoma (NHL), Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma, Waldenstrom's macroglobulinemia, CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extra-nodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, adult T-cell lymphoma, extranodal NK / T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, Sezary syndrome, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, or any combination thereof.

117. The method of claim 116 or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of claim 116, wherein the cancer comprises a solid tumor, wherein, optionally, the solid tumor is a sarcoma or a carcinoma, wherein, further optionally, the solid tumor is selected from: chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma;195.Ewing’s sarcoma; a gastrointestinal stromal tumor; Leiomyosarcoma; angiosarcoma (vascular sarcoma); Kaposi’s sarcoma; liposarcoma; pleomorphic sarcoma; or synovial sarcoma.

118. The method of claim 117 or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of claim 117, wherein the solid tumor is selected from a lung carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma; Adenosquamous carcinoma; anaplastic carcinoma; Large cell carcinoma; Small cell carcinoma; a breast carcinoma (e.g., Ductal Carcinoma in situ (non-invasive), Lobular carcinoma in situ (non-invasive), Invasive Ductal Carcinoma, Invasive lobular carcinoma, Non-invasive Carcinoma); a liver carcinoma (e.g., Hepatocellular Carcinoma, Cholangiocarcinomas or Bile Duct Cancer); Large-cell undifferentiated carcinoma, Bronchioalveolar carcinoma); an ovarian carcinoma (e.g., Surface epithelial-stromal tumor (Adenocarcinoma) or ovarian epithelial carcinoma (which includes serous tumor, endometrioid tumor and mucinous cystadenocarcinoma), Epidermoid (Squamous cell carcinoma), Embryonal carcinoma and choriocarcinoma (germ cell tumors)); a kidney carcinoma (e.g., Renal adenocarcinoma, hypernephroma, Transitional cell carcinoma (renal pelvis), Squamous cell carcinoma, Bellini duct carcinoma, Clear cell adenocarcinoma, Transitional cell carcinoma, Carcinoid tumor of the renal pelvis); an adrenal carcinoma (e.g., Adrenocortical carcinoma), a carcinoma of the testis (e.g., Germ cell carcinoma (Seminoma, Choriocarcinoma, Embryonal carciroma, Teratocarcinoma), Serous carcinoma); Gastric carcinoma (e.g., Adenocarcinoma); an intestinal carcinoma (e.g., Adenocarcinoma of the duodenum); a colorectal carcinoma; or a skin carcinoma (e.g., Basal cell carcinoma, Squamous cell carcinoma).

119. The method of claim 117 or 118, or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of claim 117 or 118, wherein the solidtumor is an ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.

120. The method of claim 116 or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of claim 116, wherein the disease or condition is multiple myeloma.

121. The method of any one of claims 109, 112, and 115-120, or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of any one of claims 110, 111, and 113-120, wherein the host cell is an allogeneic cell, a syngeneic cell, or an autologous cell.

122. The method of any one of claims 109, 112, and 115-120, or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of any one of claims 110, 111, and 113-121, wherein the method comprises administering a plurality of doses of the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition to the subject, wherein, optionally: (1) the plurality of doses are administered at intervals between administrations of about two, three, four, five, six, seven, eight, or more weeks; and / or (2) a dose of the host cell comprises about 105cells / m2to about 1011cells / m2.

123. The method of any one of claims 109, 112, and 115-122, or the fusion protein, polypeptide, polynucleotide, vector, host cell, or composition for use of any one of claims 110, 111, and 113-122, wherein the subject is receiving, has received, or will receive one or more of: (i) chemotherapy; (ii) radiation therapy; (iii) an inhibitor of an immune suppression component; (iv) an agonist of a stimulatory immune checkpoint agent; (v) RNAi; (vi) a cytokine; (vii) a surgery; (viii) a monoclonal antibody and / or an antibody-drug conjugate; or (ix) any combination of (i)-(viii), in any order.

124. A method comprising introducing into a host cell (i) the polynucleotide of any one of claims 28-48, (ii) the vector of claim 90, (iii) the polynucleotide of any one of claims 76-89, or (iv) the vector of claim 91, wherein, optionally,203.the host cell comprises a chromosomal gene knockout of CD3z, CD3e, TIGIT, CD226, TRAC, TRBC, CD8, CD4, or any combination thereof, and / or204.the host cell comprises a hematopoietic progenitor cell, an induced pluripotent stem cell, an embryonic stem cell, a hematopoietic stem cell, a peripheral blood mononuclear cell, or an immune system cell, optionally a human immune system cell; and / or the host cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y6 T cell, a natural killer cell, a natural killer T cell, a monocyte, or any combination thereof; and / or205.the host cell comprises a T cell, wherein, further optionally, the T cell comprises a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

125. The method of claim 124, further comprising introducing into the host cell, or wherein the host cell has received, a chromosomal gene knockout of CD3z, CD3e, TIGIT, CD226, TRAC, TRBC, CD8, CD4, or any combination thereof, wherein, optionally:207.the chromosomal gene knockout introducing a missense mutation, a splice junction mutation, a nonsense mutation, or any combination thereof; and / or208.introducing a chromosomal gene knockout comprises base editing, or wherein the chromosomal gene knockout was introduced by base editing, further optionally cytosine base editing.

126. An expression construct comprising (i) a polynucleotide encoding a first expression product of interest, and (ii) a polynucleotide encoding at least one additional expression product of interest, wherein:210.the expression construct comprises an EFla promoter (SEQ ID NO:7) operably linked to a polynucleotide encoding (i) and a MNDu3 promoter (SEQ ID NO: 8) operably linked to a polynucleotide encoding (ii); or211.the expression construct comprises a MNDu3 promoter (SEQ ID NO:8) operably linked 30 to a polynucleotide encoding (i) and an EFla promoter (SEQ ID NO:7) operably linked to a polynucleotide encoding (ii), wherein, optionally, the EFla promoter and the MNDu3 promoter are disposed on opposite strands of the expression construct and are preferably oriented in opposite directions.

127. An expression construct comprising (i) a polynucleotide encoding a first expression product of interest, and (ii) a polynucleotide encoding at least one additional expression product of interest, wherein:213.the expression construct comprises an EFla promoter (SEQ ID NO:7) or an EFla core promoter (SEQ ID NO:473) operably linked to a polynucleotide encoding (i) and a MNDu3 promoter (SEQ ID NO:8) or a minimal MND promoter (SEQ ID NO:473) operably linked to a polynucleotide encoding (ii); or214.the expression construct comprises an EFla promoter (SEQ ID NO:7) or an EFla core promoter (SEQ ID NO:473) operably linked to a polynucleotide encoding (ii) and a MNDu3 promoter (SEQ ID NO:8) or a minimal MND promoter (SEQ ID NO:473) operably linked to a polynucleotide encoding (i), wherein, optionally, the EFla promoter or EFla core promoter and the MNDu3 promoter or the MNDu minimal promoter are disposed on opposite strands of the expression construct and are preferably oriented in opposite directions.

128. An expression construct comprising (i) a polynucleotide encoding a first expression product of interest, and (ii) a polynucleotide encoding at least one additional expression product of interest, wherein:216.the expression construct comprises an EFla promoter (SEQ ID NO:7) operably linked to a polynucleotide encoding (i) and a MNDu3 promoter (SEQ ID NO: 8) operably linked to a polynucleotide encoding (ii),217.wherein the EFla promoter and the MNDu3 promoter are preferably disposed on opposite strands of the expression construct and are preferably oriented in opposite directions.

129. The expression construct of any one of claims 126-128, wherein the first expression product of interest and / or the at least one additional expression product of interest comprises an target-binding protein, optionally a chTCR, a CAR, a TCR, a scTCR, a mutSTAR receptor, a STAR receptor, a Co-STAR receptor, a HIT receptor, a cytokine receptor, or a TRuC.

130. The expression construct of claim 129, wherein the at least one additional expression product of interest comprises: any one of the fusion proteins or polypeptides of any one of the preceding claims, or any combination thereof.

131. The expression construct of claim 129, wherein the at least one additional expression product of interest comprises: an IT AM-engineered CD3 polypeptide as disclosed herein.

132. The expression construct of claim 129, wherein the at least one additional expression product of interest comprises: a tag (optionally an EGFRt tag, such as an EGFRtopttag); a CD8 co-receptor; a CD4 co-receptor; a CD8 co-receptor fusion protein; a CD4 coreceptor fusion protein; a cytokine (e.g., IL-2, GM-CSF, or IFN-y); cytokine fusion protein; a siRNA; an antisense oligonucleotide; a miRNA; an antibody; an antigen-binding fragment of an antibody; a transcription factor; or an enzyme.

133. The expression construct of any one of claims 126-128, wherein the first expression product of interest and the at least one additional expression product of interest are each an expression product of interest for expressing in a human T cell.

134. The expression construct of claim 129, wherein the first expression product of interest comprises a chTCR and the at least one additional expression product of interestcomprises a CD3 polypeptide, engineered CD3 polypeptide, or CD3 fusion protein of any one of the preceding claims, or wherein the at least one additional expression product of interest comprises a chTCR and the first expression product of interest comprises a CD3 polypeptide, engineered CD3 polypeptide, or CD3 fusion protein of any one of the preceding claims.

135. The expression construct of claim 129, wherein the first expression product of interest comprises a chTCR and the at least one additional expression product of interest comprises a CD3 polypeptide, engineered CD3 polypeptide, or CD3 fusion protein of any one of the preceding claims, or wherein the at least one additional expression product of interest comprises a chTCR and the first expression product of interest comprises a CD3 polypeptide, engineered CD3 polypeptide, or CD3 fusion protein of any one of the preceding claims.

136. The expression construct of claim 129, wherein the first expression product of interest comprises a chTCR and the at least one additional expression product of interest comprises a CD3 polypeptide, engineered CD3 polypeptide, or CD3 fusion protein of any one of the preceding claims, wherein the EFla promoter is operably linked to the polynucleotide encoding the first expression product of interest and the MNDu3 promoter is operably linked to the polynucleotide encoding the second at least one additional product of interest, wherein the EFla promoter and the MNDu3 promoter are preferably disposed on opposite strands of the expression construct and are preferably oriented in opposite direction.

137. The expression construct of claim 129, wherein the first expression product of interest comprises a chTCR and the at least one additional expression product of interest comprises a CD3 polypeptide, engineered CD3 polypeptide, or CD3 fusion protein of any one of the preceding claims, wherein the MNDu3 promoter is operably linked to the polynucleotide encoding the first expression product of interest and the EFla promoter is operably linked to the polynucleotide encoding the second expression product of interest, wherein the EFla promoter and the MNDu3 promoter are preferably disposed on opposite strands of the expression construct and are preferably oriented in opposite direction.

138. The expression construct of any one of claims 126-137, encoding 3 or fewer, 2 or fewer, 1, or zero self-cleaving peptides (e.g., 2A peptides), and / or encoding 3 or fewer, 2 or fewer, 1, or zero furin cleavage sequences (e.g., RAKR), and / or wherein the expression construct expresses the first expression product of interest and the at least one additional expression product of interest, and is more efficiently transduced into human CD8+ T cells, than a reference expression construct comprising a single promoter (e.g., EFla) encoding the first expression product of interest, the at least one additional expression product, and one or more self-cleaving peptides and / or furin cleavage sequences.

139. A vector comprising the expression construct of any one of claims 128-138.

140. The vector of claim 139, wherein the vector comprises a DNA plasmid.

141. The vector of claim 139, comprising a viral vector.

142. The vector of claim 139, comprising a lentiviral vector.

143. The vector of claim 139, comprising a retroviral vector.

144. The vector of claim 139, comprising an adenoviral vector.

145. A lipid composition (e.g., a lipid nanoparticle) comprising the expression construct of any one of claims 128-138 or the vector of any one of claims 139-144.

146. A T cell comprising the expression construct of any one of claims 128-138, the vector of any one of claims 140-144, or the lipid composition of claim 145, optionally wherein the T cell is a human T cell and / or a CD8+ T cell.

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