Engineered t cell receptor against insulin producing cells

Abstract

Provided herein are compositions comprising engineered T cell receptor and T cells comprising the compositions for selectively targeting pancreatic beta cells.

Description

Docket No. 048536-808001 WOENGINEERED T CELL RECEPTOR AGAINST INSULIN PRODUCING CELLSRELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 717,217, filed November 6, 2024, which is incorporated herein by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. DK137140-01 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.INCORPORATION OF THE SEQUENCE LISTING

[0003] This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The accompanying Sequence Listing XML file, named “048536- 80800 IWO.xml” was created on November 3, 2025, and is 95,032 bytes.BACKGROUND

[0004] Type 1 diabetes (T1D) is an autoimmune condition marked by the immune system’s destruction of insulin-producing pancreatic beta cells. This destruction is central to disease progression and poses significant challenges in mitigating the autoimmune response.Regulatory T (Treg) cells have shown promise in modulating autoimmunity in T1D and preventing further destruction. However, effective application of Tregcell therapy in T1D presents substantial challenges. Traditional Tregtherapies have struggled to achieve the specificity required to focus their activity solely on the pancreas, leading to limited efficacy. Therefore, there remains a need for a therapeutic approach that enables precise modulation of autoimmune responses in the pancreas to protect insulin-producing cells.SUMMARY

[0005] Recognized herein is a need for precise modulation of Tregcells to selectively target autoimmune responses within pancreatic tissues in Type 1 diabetes (T1D). The presentDocket No. 048536-808001 WO disclosure relates to compositions comprising engineered T cell receptor (TCR) system and engineered Tregcells for selectively targeting pancreatic beta cells.

[0006] Provided herein is a composition, including: an engineered T cell receptor (eTCR) including alpha chain and beta chain, wherein the eTCR can include one or more mutations; and a chimeric co- stimulatory receptor comprising a target binder, a hinge, a transmembrane domain, and an intracellular domain of a co- stimulatory receptor.

[0007] Provided herein is a composition, including: an eTCR including alpha chain and beta chain, wherein the eTCR can include one or more mutations; a chimeric co-stimulatory receptor including a target binder, a hinge, a transmembrane domain, and an intracellular domain of a co-stimulatory receptor; and an ectopic CD8 co-receptor. In some embodiments, the ectopic CD8 co-receptor can include CD8a and CD8p. In some embodiments, the ectopic CD8 co-receptor can include a linker. In some embodiments, the linker can be selected from T2A, P2A, E2A, F2A, and IRES. In some embodiments, the linker can be P2A. In some embodiments, the ectopic CD8 co-receptor can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 25. In some embodiments, the ectopic CD8 co-receptor can include the polypeptide sequence set forth in SEQ ID NO: 25.

[0008] Also provided herein is a composition, including: an eTCR comprising alpha chain and beta chain; a chimeric co-stimulatory receptor including a target binder, a hinge, a transmembrane domain, and an intracellular domain of a co-stimulatory receptor; and an engineered Linker of Activation of T cells (eLAT). In some embodiments, the eTCR can include one or more mutations. In some embodiments, the eLAT can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 22. In some embodiments, the eLAT can include the polypeptide sequence set forth in SEQ ID NO: 22. In some embodiments, the eLAT can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 59-65. In some embodiments, the eLAT can include the polypeptide sequence set forth in any one of SEQ ID NOs: 59-65. In some embodiments, the eLAT can include at least one mutation. In some embodiments, the at least one mutation can be at position 131 in the polypeptide sequence set forth in SEQ ID NO: 21. In some embodiments, the at least one mutation can be at position 131 in the polypeptide sequence set forth in SEQ ID NO: 22. In some embodiments, the at least one mutation can be G131D. In some embodiments, the eLAT can be linked to theDocket No. 048536-808001 WO eTCR via a glycine- serine linker. In some embodiments, the glycine-serine linker can include a polypeptide sequence set forth in SEQ ID NO: 20.

[0009] In some embodiments, the composition further includes an LCK binding sequence. In some embodiments, the LCK binding sequence includes a palmitoylation site from a CD4 co-receptor. In some embodiments, the LCK binding sequence is from a CD4 receptor and / or from a CD8 receptor. In some embodiments, the LCK binding sequence comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 55-58. In some embodiments, the LCK binding sequence comprises a polypeptide sequence set forth in any one of SEQ ID NOs: 55-58.

[0010] In any of the embodiments of the compositions provided above, the eTCR can recognize insulin producing cells. In some embodiments, the eTCR can recognize preproinsulin (PPI): 15-24 presented by HLA-A*0201. In some embodiments, the eTCR can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the one or more mutations can be in complementary determining region 3 (CDR3) of the betachain. In some embodiments, the one or more mutations can be at positions 96 and 98 in the polypeptide sequence set forth in SEQ ID NO: 1. In some embodiments, the one or more mutations can be L96T and E98G. In some embodiments, the eTCR can include a polypeptide sequence set forth in SEQ ID NO: 2. In some embodiments, the one or more mutations can be L96N and E98G. In some embodiments, the eTCR can include a polypeptide sequence set forth in SEQ ID NO: 3.

[0011] In any of the embodiments of the compositions provided above, the target binder of the chimeric co-stimulatory receptor can bind to a dipeptidyl peptidase-like protein 6b (DPP6). In some embodiments, the target binder can be encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the target binder can be encoded by a polynucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the target binder can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 12. In some embodiments, the target binder can include a polypeptide sequence set forth in SEQ ID NO: 12.

[0012] In any of the embodiments of the compositions provided above, the hinge of the chimeric co-stimulatory receptor can be selected from IgGl, IgG2, IgG3, IgG4, IgE, IgM,Docket No. 048536-808001 WOIgA, CD8a, CD28, CD3e, and DAP10. In some embodiments, the hinge can be an IgG4 hinge. In some embodiments, the IgG4 hinge can be encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the IgG4 hinge can be encoded by a polynucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, the IgG4 hinge can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the IgG4 hinge can include a polypeptide sequence set forth in SEQ ID NO: 15.

[0013] In any of the embodiments of the compositions provided above, the transmembrane of the chimeric co-stimulatory receptor can be selected from CD28, CD8a, CD3(^, ICOS, 4- 1BB, DAP10, and NKG2D. In some embodiments, the transmembrane domain can be CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain can be encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the CD28 transmembrane domain can be encoded by a polynucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the CD28 transmembrane domain can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17. In some embodiments, the CD28 transmembrane domain can include a polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the CD28 transmembrane domain can include C14L, Y15L, S16L, or T20L amino acid substitutions in the polypeptide sequence of SEQ ID NO: 17.

[0014] In any of the embodiments of the compositions provided above, the intracellular domain of the chimeric co-stimulatory receptor can be an intracellular domain of 4- IBB (CD137), CD27 (TNFRSF7), CD28, CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), ICOS, LFA-1 (CD1 la / CD18), DAP10, and DAP 12. In some embodiments, the intracellular domain can be an intracellular domain of CD28. In some embodiments, the CD28 intracellular domain can be encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the CD28 intracellular domain can be encoded by a polynucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, the CD28 intracellular domain can include a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17.Docket No. 048536-808001 WOIn some embodiments, the CD28 intracellular domain can include a polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the CD28 intracellular domain can include one or more modifications in the polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the one or more modifications can be at positions 12, 14, 17, 20, and / or 29 in the polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the one or more modifications can be Y12F, Y14F, P17A, P20A, and / or P29A in the polypeptide sequence set forth in SEQ ID NO: 17.

[0015] Further provided herein is a T cell comprising any of the compositions provided herein.

[0016] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0017] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0019] FIG. 1 is schematics illustrating the process of T cell receptor substitution in human T cells.

[0020] FIG. 2A is schematics of different engineered TCR (eTCR) constructs used to transduce T cells in Example 1. Clone 32 (c32) TCR (positive control) is a native human TCR cloned from a subject with type 1 diabetes and reacts to HLA-A*0201 in a peptide independent manner. 1E6 TCR is a native TCR and recognizes preproinsulin (PPI): 15-24Docket No. 048536-808001 WO peptide. TWG TCR is an engineered TCR originated from 1E6 TCR comprising L96T and E98G mutations. NWG TCR is an engineered TCR originated from 1E6 TCR comprising L96N and E98G mutations. FIG. 2B is a schematic showing different co-culturing experiments with either K562 cells, SC-P cells, or human islets.

[0021] FIG. 3 is schematics illustrating the process of human Tregcell isolation from peripheral blood.

[0022] FIG. 4A is a histogram of the expression of CD3e on T cells as a marker of TCR reexpression after CRISPR deletion of the endogenous TCR alpha and beta chains and lend viral transduction of eTCRs.

[0023] FIG. 4B is a bar graph illustrating quantification of the editing efficiency. Plotted is percentage of CD3e+T cells, serving as a proxy for TCR surface expression.

[0024] FIG. 4C is flow cytometry contour plots illustrating the expression of GFP and CD3e. These two markers were followed by CD4 or CD8, which were used to sort T cells.

[0025] FIG. 5A is schematics illustrating the target cells co-cultured with CD8+T cells comprising eTCR.

[0026] FIG. 5B is schematics illustrating the CD80+target cells co-cultured with CD8+T cells comprising eTCR.

[0027] FIGS. 6A-6G collectively illustrate that high-affinity insulin eTCRs activate only CD8+T cells at low peptide concentrations and induce human p cell death. The eTCR- expressing T cells were co-cultured with target HLA-A2+K562 cells at different PPI15-24 concentrations (FIGS. 6A-6B) and with CD80+HLA-A2+K562 cells at different PPI15-24 concentrations (FIGS. 6C-6D). All co-cultures were performed on a round-bottom 96-well plate and incubated in the absence of IL-2 for 72 hours. Data is expressed a percentage of T cells that are double positive for the activation markers CD25 and CD71. FIG. 6E shows a schematic of P cell differentiation and representative images showing GFP expression after co-culturing T cells and SC-P cells expressing GFP. FIG. 6F shows SC-P cell killing assay. FIG. 6G shows the percent of CD25 and CD71 positive cells after co-culture of CD8+ T cells and SC-P cells.

[0028] FIG. 6A is a graph illustrating percentage of activated CD8+T cells after co-culture with A2+K562 cells at different PPI15-24 concentrations.

[0029] FIG. 6B is a graph illustrating percentage of activated CD4+T cells after co-culture with A2+K562 cells at different PPI15-24 concentrations.

[0030] FIG. 6C is a graph illustrating percentage of activated CD8+T cells after co-culture with CD80+A2+K562 cells at different PPI15-24 concentrations.Docket No. 048536-808001 WO

[0031] FIG. 6D is a graph illustrating percentage of activated CD4+T cells after co-culture with CD80+A2+K562 cells at different PPI15-24 concentrations.

[0032] FIG. 7 A is a schematic of ectopic CD8 co-receptor construct.

[0033] FIG. 7B is a histogram illustrating the expression of the co-receptor CD8 in Jurkat cells after lentivirus transduction containing the alpha and beta subunits.

[0034] FIG. 7C is a second histogram illustrating the expression of the co-receptor CD8 in Jurkat cells after lentivirus transduction containing the alpha and beta subunits

[0035] FIGS. 8A-8D collectively illustrates that ectopic CD8 co-receptor is required for eTCR activation in CD4+T cells.

[0036] FIG. 8A is schematics illustrating the target K562 cells co-cultured with CD4+T cells comprising eTCR and ectopic CD8 co-receptor.

[0037] FIG. 8B illustrates quantification of the eTCR and ectopic CD8-expressing CD4+T cell activation after co-culture with A2+K562 target cells.

[0038] FIG. 8C is schematics illustrating the target human islet cells co-cultured with CD4+T cells comprising eTCR, ectopic CD8 co-receptor, and DPP6 chimeric co- stimulatory receptor.

[0039] FIG. 8D is schematics illustrating the target huma islet cells co-cultured with CD4+T cells comprising eTCR and ectopic CD8 co-receptor.

[0040] FIG. 9A is schematics of DPP6 chimeric co- stimulating receptor structure.

[0041] FIG. 9B is a flow cytometry plot confirming surface expression of DPP6 chimeric co-stimulatory receptor in jurkat cells.

[0042] FIGS. 10A-10E collectively illustrate that CD4+T cells comprising eTCR, ectopic CD8 co-receptor, and a DPP6 chimeric co-stimulatory receptor showed significantly higher activation, particularly in the presence of the PPI15-24 peptide when co-cultured with target K562 cells. Each dot represents an independent experiment. Statistics included One-Way Anova with Tukey’s multiple comparisons test or Two-Way ANOVA with Sidak’s multiple comparisons.

[0043] FIG. 10A depicts a schematic of the chimeric co-stimulatory receptor and the signaling within a T cell upon binding to a P cell.

[0044] FIG. 10B is a graph illustrating percentage of activated eTCR CD4+T cells after co-culture with A2+K562.

[0045] FIG. 10C is a graph illustrating percentage of activated eTCR CD4+T cells after co-culture with human islet cells.Docket No. 048536-808001 WO

[0046] FIG. 10D is a graph illustrating percentage of activated eTCR CD4+T cells after co-culture with stem cell-derived beta-like cells (SC-beta).

[0047] FIG. 10E depicts different co-stimulatory receptors and the CD69 mean fluorescence intensity (MFI) for T cells expressing each of the co-stimulatory receptors after co-culture with K562 cells expressing A2 and PPI.

[0048] FIG. 11A is a density flow cytometry plot indicating the percentage of cells expressing Tregmarkers FOXP3 and HELIOS transcription factors with quantification of results from three donors.

[0049] FIG. 1 IB is a graph illustrating the percentage of cells expressing Tregmarkers FOXP3 and HELIOS transcription factors from three donors.

[0050] FIG. 12A is schematics illustrating the A2+K562 target cells without costimulation ligand CD80 co-cultured with CD4+Tregcells comprising eTCR and ectopic CD8 co-receptor.

[0051] FIG. 12B is schematics illustrating the A2+K562 target cells with co- stimulation ligand CD80 co-cultured with CD4+Tregcells comprising eTCR and ectopic CD8 coreceptor.

[0052] FIG. 13A is a graph illustrating the results of percentage of activated Tregafter coculture with K562 without CD80 co- stimulation. Each dot represents Tregs from a different donor. Statistical analysis included Two-Way Anova with Sidak’s multiple comparisons tests.

[0053] FIG. 13B is a graph illustrating the results of percentage of activated Tregafter coculture with K562 with CD80 co-stimulation. Each dot represents Tregs from a different donor. Statistical analysis included Two-Way Anova with Sidak’s multiple comparisons tests.

[0054] FIG. 14A-14B shows testing different TCRs with CD8 intracellular domains or fragments of CD8 intracellular domains added. FIG. 14A depicts schematics of a TCR without a CD8 intracellular domain added, a TCR with a CD8 intracellular domain added, and a TCR with a CD8 intracellular domain added but the LCK binding sequence was removed and FIG. 14A shows the percentage of T cells expressing CD25 and CD71 after exposure to K562 cells in co-culture experiments. FIG. 14B shows a diagram with TCRs that have different LCK binding sequences added and the percent of T cells that express the different TCRs that express CD25 and CD71 after co-culturing the T cells and K562 cells together.Docket No. 048536-808001 WO

[0055] FIG. 15A-15B shows testing different TCRs with various eLATs. FIG. 15A shows diagrams of the different TCRs with either GFP, LAT or mutLAT and the results of coculturing the different TCRs with K562 cells. FIG. 15B shows diagrams for TCRs that have different fragments of LAT included and the results of co-culturing T cells expressing each of the TCRs with K562 cells.

[0056] FIG. 16 shows a diagram of different TCRs that include either cLAT or mcLAT and a palmitoylation site from a CD4 co-receptor (CD4PL) and the results of co-culturing T cells expressing each of the TCRs with K562 cells.

[0057] FIG. 17A is a schematic illustrating a non-limiting exemplary composition of the present disclosure.

[0058] FIG. 17B is a schematic illustrating a non-limiting exemplary composition of the present disclosure.

[0059] FIG. 18 is a schematic illustrating a non-limiting exemplary composition of the present disclosure.

[0060] FIG. 19A is a schematic illustrating a non-limiting exemplary composition of the present disclosure.

[0061] FIG. 19B is a schematic illustrating a non-limiting exemplary composition of the present disclosureDETAILED DESCRIPTION

[0062] The present disclosure pertains to compositions for precise modulation of Tregcells to selectively target autoimmune responses within pancreatic islet tissues in Type 1 diabetes (T1D).

[0063] The following descriptions and examples illustrate embodiments of the present disclosure in detail. Although the present disclosure has been described in some details by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0064] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0065] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in theDocket No. 048536-808001 WO context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.I. DEFINITION

[0066] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0067] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0068] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0069] The term “treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof. With regard to diabetes, “treating” or “treatment” can refer to inhibiting or slowing the autoimmune destruction of insulin-producing beta cells, reducing blood glucose levels, improving insulin sensitivity, enhancing beta cell function or survival, preventing or delaying the onset of diabetes, or some combination thereof. With regard to diabetic complications, “treating” or “treatment” can include reducing or delaying theDocket No. 048536-808001 WO development of complications such as neuropathy, nephropathy, retinopathy, or cardiovascular disease, alleviating symptoms associated with these complications, or some combination thereof.

[0070] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.IL COMPOSITIONS OF THE DISCLOSURE

[0071] The present disclosure relates to compositions including engineered T cell receptors (eTCRs) designed for therapeutic applications, such as targeted modulation of immune responses. The compositions can further include eTCRs and chimeric co- stimulatory receptors. The compositions disclosed herein incorporate various structural and functional elements that work synergistically to enhance T cell activation, specificity, and efficacy.

[0072] In one embodiment, the composition includes an eTCR that includes an alpha chain and a beta chain, with one or more mutations introduced to increase its affinity or specificity toward a target antigen. This eTCR can be combined with a chimeric co-stimulatory receptor, which consists of a target-binding domain, a hinge region, a transmembrane domain, and an intracellular domain derived from a co-stimulatory receptor (FIGS. 14-15). In another embodiment, the composition can further include an ectopic CD8 co-receptor (FIG. 14), which can enhance T cell activation by assisting in recognition of antigens presented by class I major histocompatibility complex (MHC) molecules. The ectopic CD8 co-receptor can include both the CD8a and CD8P subunits. In some embodiments, the ectopic CD8 co-receptor can include a linker, such as, but not limited to, P2A.

[0073] In some embodiments, the composition can include a native TCR, such as, but is not limited to, c32 TCR or 1E6 TCR (FIG. 2A), linked to a Linker of Activation of T cells (LAT) protein with or without a chimeric co-stimulatory receptor. In some embodiments, the composition can include an eTCR with one or more mutations, such as but is not limited toDocket No. 048536-808001 WOTWG TCR or NWG TCR (FIG. 2A), linked to a LAT protein (FIG. 15) with or without a chimeric co- stimulatory receptor. The LAT protein can either be in its wildtype form or include mutations to further modulate T cell signaling. In some embodiments, the LAT protein can contain a mutation at position 131 of its amino acid sequence, with a G131D substitution being a preferred mutation. This mutation can enhance the T cell activation response. In some embodiments, the LAT protein can be linked to the eTCR through a linker, such as, but not limited to, glycine- serine linker.

[0074] The following sections describe in more detail embodiments and specific components of the presently described compositions.A. Engineered T Cell Receptor

[0075] T cell receptor (TCR) is a heterodimeric cell surface protein of the immunoglobulin superfamily that participate in the activation of T cells in response to the binding of an antigen. The TCR complex can consist of TCRa / p chains and CD3y / 6 / e / ^ subunits, which can associate through hydrophobic interactions. Somatic VDJ recombination enables the generation of distinct TCRa and TCRP chains, and TCRaP heterodimers are generally responsible for antigen recognition by binding to peptide-MHC complexes. CD3 can transmits the TCR-triggered signal through immunoreceptor tyrosine-based activation motifs (IT AMs) in its cytoplasmic tail, but it is generally not directly involved in antigen recognition.

[0076] TCRs are capable of eliciting an immunogenic response from antigens that are indicative of a disease and a critical component of T cell function, enabling T cells to recognize and respond to specific antigens presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. In the present disclosure, the TCR is engineered to have enhanced binding affinity while retaining specificity for a target antigen, through mutations introduced in the beta chains. This engineered TCR (eTCR) can be composed of an alpha chain and a beta chain that work in conjunction to form a heterodimeric structure capable of recognizing peptide-MHC complexes with high specificity. By modifying the amino acid sequences within the TCR, particularly in the complementarity-determining regions (CDRs), the eTCR’s binding properties can be optimized for specific therapeutic applications, such as targeting disease-associated antigens.

[0077] In some embodiments, the eTCR of the present disclosure can include a linker, such as, but is not limited to, T2A, P2A, E2A, F2A, or an IRES element, between alpha chain and beta chain (e.g., c32 TCR or 1E6 TCR of FIG. 2A). In some embodiments, the linker can beDocket No. 048536-808001 WOP2A. In some embodiments, the eTCR of the present disclosure can include one or more mutations in complementary determining region 3 (CDR3) of the beta-chain. In some embodiments, the eTCR comprises one or more mutations at positions 96 and 98 in the polypeptide sequence of SEQ ID NO: 1 (e.g., TWG TCR or NWG TCR of FIG. 2A).

[0078] In some embodiments, the eTCR of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the one or more mutations can be L96T and E98G. In some embodiments, the one or more mutations can be L96N and E98G. In some embodiments, the eTCR comprises a polypeptide sequence set forth in SEQ ID NO: 2. In some embodiments, the eTCR of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the eTCR comprises a polypeptide sequence set forth in SEQ ID NO: 3. In some embodiments, the eTCR of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the eTCR of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 30. In some embodiments, the eTCR of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the eTCR of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the eTCR of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 33.

[0079] The eTCRs disclosed herein can also be designed to work synergistically with other components, such as a chimeric co-stimulatory receptors (e.g., DPP6 chimeric co- stimulatory receptor), an ectopic CD8 co-receptors, and / or a Linker of Activation of T cells (LAT) protein. These additional components can further amplify the signaling pathways initiated by the TCR upon antigen recognition, thereby enhancing the overall efficacy of the immune response.Docket No. 048536-808001 WOB. Chimeric Co-Stimulatory Receptor

[0080] A chimeric co- stimulatory receptor is an engineered receptor construct designed to enhance the activation and functionality of T cells by providing additional signaling beyond that of the conventional T cell receptor (TCR). In natural immune responses, T cells require two signals to achieve full activation: antigen recognition through the TCR and a secondary, or co-stimulatory, signal provided by interactions with co- stimulatory ligands on antigen- presenting cells (APCs). Without this secondary signal, T cells can become anergic (non- responsive) or fail to proliferate effectively, limiting the immune response. The chimeric costimulatory receptor of the present disclosure is designed to mimic this co-stimulatory signal artificially to ensure robust and sustained activation of engineered T cells.

[0081] In an embodiment, a chimeric co-stimulatory receptor can include a target binder. In some embodiments, a chimeric co-stimulatory receptor can further include a hinge, a transmembrane domain, and an intracellular domain of a co-stimulatory receptor.

[0082] In some embodiments, a chimeric co-stimulatory receptor can be encoded by a polynucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the polynucleotide sequence encoding a chimeric co-stimulatory receptor is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, a chimeric co-stimulatory receptor of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 11. In some embodiments, a chimeric co-stimulatory receptor of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11.

[0083] In some embodiments, a polynucleotide sequence encoding a chimeric co- stimulatory receptor can include a signal peptide and an expression tag. In some embodiments, the signal peptide can be selected from, but is not limited to, CD8a signal peptide, CD28 signal peptide, IL-2 signal peptide, granzyme B signal peptide, CD4 signal peptide, TCR alpha (TRAC) signal peptide, GM-CSF signal peptide, and CCR7 signal peptide. In a preferred embodiment, the signal peptide is a CD8a signal peptide encoded by a polynucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the polynucleotide sequence encoding a CD8a signal peptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the signal peptide of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 13. In some embodiments, the signal peptide of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%,Docket No. 048536-808001 WO88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13.

[0084] In some embodiments, the expression tag can be selected from, but is not limited to, Myc tag, HA tag, FLAG tag, V5 tag, Strep tag, Twin-Strep tag, polyhistidine tag, biotin tag, EGFP, mCherry, c-Myc-PE and HA-PE conjugate, and NE tag. In a preferred embodiment, the expression tag is Myc tag encoded by a polynucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the polynucleotide sequence encoding a Myc is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the expression tag of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 14. In some embodiments, the expression tag of the present disclosure comprise a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14.

[0085] . In some embodiments, the chimeric co- stimulatory receptor of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 50-54. In some embodiments, the chimeric co- stimulatory receptor of the present disclosure comprises a polypeptide sequence set forth in any one of SEQ ID NOs: 50-54. In some embodiments, the chimeric co-stimulatory receptor of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 50. In some embodiments, the chimeric co-stimulatory receptor of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NOs: 50. In some embodiments, the chimeric co-stimulatory receptor of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 51. In some embodiments, the chimeric co-stimulatory receptor of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NOs: 51. In some embodiments, the chimeric co-stimulatory receptor of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 52. In some embodiments, the chimeric co-stimulatory receptor of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NOs: 52. In some embodiments, the chimeric co-stimulatory receptor of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Docket No. 048536-808001 WO94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 53. In some embodiments, the chimeric co- stimulatory receptor of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NOs: 53. In some embodiments, the chimeric costimulatory receptor of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 54. In some embodiments, the chimeric costimulatory receptor of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NOs: 54.1. Target Binder

[0086] The target binder of the chimeric co- stimulatory receptor disclosed herein can have a binding affinity for one or more target antigens.

[0087] In some embodiments, the target binder can include an antigen-binding moiety capable of binding to one or more antigens on the surface of a cell. In some embodiments, the antigen-binding moiety can include one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof. A functional fragment or variant of an antibody can be one which retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment or functional variant was derived. For instance, an antibody capable of binding to an epitope of a cell surface receptor can be truncated at the N-terminus and / or C-terminus. In some embodiments, the antigen-binding moiety can be selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, an F(ab’)2 fragment, an F(ab)v fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and a functional fragment of any thereof. In some embodiments, the antigen-binding moiety of the target binder includes a nanobody.

[0088] The target binder can include naturally- occurring amino acid sequences or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g., binding affinity. Generally, the binding affinity of the target binder can be calculated by any methods currently known in the art, such as, but not limited to, a competition radioimmunoassay, surface plasmon resonance (SPR), solution-affinity ELISA, or flow cytometry. The target binder that selectively binds a target antigen (such as DPP6) can be a moiety that can bind the target antigen with high affinity and does not significantly bind other unrelated antigens but binds the antigen with high affinity, e.g., with a dissociation constant (Kd) of about 10 nM or less, such as about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, or about 1 nM, or about 100 nM or less, or in theDocket No. 048536-808001 WO range of about 10 nM to about 100 nM, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less.

[0089] In some embodiments of the disclosure, the antigen-binding moiety of the target binder can be specific for an epitope present in an antigen that is expressed by a pancreatic beta cell.

[0090] Non-limiting examples of the target antigen expressed by a pancreatic beta cell can include dipeptidyl peptidase-like protein 6 (DPP6), islet antigen 2 (IA-2), zinc transporter 8 (ZnT8), phogrin (IA-2 ), glucose transporter 2 (GLUT2), and glucagon-like peptide- 1 receptor (GLP-1R). In a preferred embodiment, the target antigen is DPP6 encoded by a polynucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding DPP6 is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0091] In some embodiments, the target binder of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 12. In some embodiments, the target binder of the present disclosure comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12.2. Hinge

[0092] A chimeric co- stimulatory receptor can include a hinge sequence between the target binder and a transmembrane domain. A hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some embodiments, the length of the hinge sequence can be optimized based on the desired length of the extracellular portion of a chimeric co- stimulatory receptor, which can be based on the location of the epitope within the target molecule it binds. For example, if the epitope is in the membrane proximal region within the target molecule, longer hinges can be optimal.

[0093] In some embodiments, the hinge can be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In some embodiments, the hinge can include at least a portion of an IgGl, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. In some embodiments, the hinge can also include at least a portion of a corresponding immunoglobulin hinge region. In some embodiments, the hinge can be derived from or include at least a portion of a modified immunoglobulin Fc region, forDocket No. 048536-808001 WO example, a modified IgGl Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The modified immunoglobulin Fc region can have one or more mutations (e.g., point mutations, insertions, deletions, duplications) resulting in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the hinge to an Fe receptor (FcR). In some embodiments, the modified immunoglobulin Fe region can be designed with one or more mutations which result in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the hinge to one or more FcR including, but not limited to, FcyRI, FcyR2A, FcyR2Bl, Fcy2B2, Fcy3A, Fcy3B, FceRI, FceR2, FcaRI, Fca / pR, or FcRn.

[0094] In some embodiments, a hinge sequence can be derived from CD8a molecule, CD28 molecule, CD3e molecule, or DAP 10 molecule. In a preferred embodiment, the hinge sequence is derived from IgG4 encoded by a polynucleotide set forth in SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding CD8 hinge is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.

[0095] In some embodiments, the hinge domain of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 15. In some embodiments, the hinge of the present disclosure comprises a polypeptide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the hinge of the present disclosure comprises a polypeptide sequence having at least 58% sequence identity to SEQ ID NO: 15. In some embodiments, the hinge of the present disclosure comprises a polypeptide sequence having at least 67% sequence identity to SEQ ID NO: 15. In some embodiments, the hinge of the present disclosure comprises a polypeptide sequence having at least 75% sequence identity to SEQ ID NO: 15. In some embodiments, the hinge of the present disclosure comprises a polypeptide sequence having at least 83% sequence identity to SEQ ID NO: 15. In some embodiments, the hinge of the present disclosure comprises a polypeptide sequence having at least 92% sequence identity to SEQ ID NO: 15.3. Transmembrane domain (TMD)

[0096] A chimeric co-stimulatory receptor of the present disclosure can include a transmembrane domain derived from, for example, but not limited to, CD28, CD8a, CD3(^, ICOS, 4-1BB, DAP10, or NKG2D.Docket No. 048536-808001 WO

[0097] In some embodiments, the chimeric co-stimulatory receptor of the present disclosure can include a CD28 TMD. In some embodiments, the CD28 TMD can be a human CD28 TMD. In some embodiments, the CD28 TMD can be from different mammalian species, e.g., non-human mammals such as, mouse CD28 or non-human primate CD28. In some embodiments, the TMD can include one or more amino acid substitutions within a transmembrane dimerization motif of the CD28 TMD.

[0098] In a preferred embodiment, the CD28 TMD sequence is encoded by a polynucleotide set forth in SEQ ID NO: 9. In some embodiments, the polynucleotide sequence encoding CD28 TMD is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9.

[0099] In some embodiments, the CD28 TMD of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 16. In some embodiments, the CD28 TMD of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16.

[0100] In some embodiments, the CD28 TMD can include one or more amino acid substitutions at a position corresponding to positions 14, 15, 16, or 20 of SEQ ID NO: 16.

[0101] In some embodiments, the CD28 TMD can includes the polypeptide sequence of SEQ ID NO: 16, and further includes one or more amino acid substitutions at an amino acid residue selected from the group consisting of C14, Y15, S16, and T20 of SEQ ID NO: 16. In some embodiments, the CD28 TMD can include the sequence of SEQ ID NO: 16, and can further include one, two, three, four, or five amino acid residues in the sequence of SEQ ID NO: 16 optionally substituted by a different amino acid residue. In some embodiments, one, two, three, four, or five amino acid residues in the sequence of SEQ ID NO: 16 can be optionally substituted by an equivalent amino acid residue. In some embodiments, the CD28 TMD can include the sequence of SEQ ID NO: 16.

[0102] In some embodiments, the one or more amino acid substitutions can be independently selected from the group consisting of a leucine substitution, an alanine substitution, an arginine substitution, an aspartic acid substitution, a histidine substitution, a glutamic acid substitution, a lysine substitution, a serine substitution, a tryptophan substitution, and combinations of any thereof. In some embodiments, at least one of the one or more amino acid substitutions can be a nonpolar-to-polar amino acid substitution. In someDocket No. 048536-808001 WO embodiments, at least one of the one or more amino acid substitutions can be a nonpolar-to- polar amino acid substitution.

[0103] In some embodiments, the amino acid substitution at position 14 is a Cys-to-Leu substitution (C14L). In some embodiments, the amino acid substitution at position 15 is a Tyr-to-Leu substitution (Y15L). In some embodiments, the amino acid substitution at position 16 is a Ser-to-Leu substitution (S16L). In some embodiments, the amino acid substitution at position 20 is a Thr-to-Leu substitution (T20L). In some embodiments, the CD28 TMD can include the polypeptide sequence set forth in SEQ ID NO: 16, and further includes the following amino acid substitutions: C14L, Y15L, S16L, and T20L.4. Intracellular domain (ICD)

[0104] The intracellular domain (ICD) of a chimeric co- stimulatory receptor of the present disclosure can include one or more costimulatory domains. Generally, the costimulatory domain suitable for the chimeric co-stimulatory receptor disclosed herein can be any one of the costimulatory domains and functional variants thereof known in the art. Examples of suitable costimulatory domains that can enhance cytokine production include, but are not limited to, costimulatory polypeptide sequences derived from 4-1BB (CD137), CD27 (TNFRSF7), CD28, CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), ICOS, LFA-1 (CD1 la / CD18), DAP10, and DAP 12.

[0105] In a preferred embodiment, the ICD of the chimeric co-stimulatory receptor can be derived from CD28 co-stimulatory receptor.

[0106] In some embodiment, the CD28 ICD sequence can be encoded by a polynucleotide set forth in SEQ ID NO: 10. In some embodiments, the polynucleotide sequence encoding CD28 ICD can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

[0107] In some embodiments, the CD28 ICD of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the CD28 ICD of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17.

[0108] In some embodiments, the ICD of the chimeric co-stimulatory receptor disclosed herein can include a costimulatory sequence derived from CD28. In some embodiments, the chimeric co-stimulatory receptor sequence can be derived from CD28 protein and can includeDocket No. 048536-808001 WO a polypeptide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 17. In some embodiments, the ICD can include two costimulatory domains.

[0109] In some embodiments, the CD28 ICD can include one or more modifications in the polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the one or more modifications can be at positions 12, 14, 17, 20, and / or 29 in the polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the one or more modifications can be Y12F, N14F, P17A, P20A, and / or P29A amino acid substitutions in the polypeptide sequence set forth in SEQ ID NO: 17. In some embodiments, the one or more modifications can be Y12F. In some embodiments, the one or more modifications can be Y14F. In some embodiments, the one or more modifications can be P17A. In some embodiments, the one or more modifications can be P20A. In some embodiments, the one or more modifications can be P29A.C. CD8 Co-Receptor

[0110] In some embodiments, the compositions of the present disclosure can include an ectopic CD8 co-receptor (FIG. 7A). The CD8 co-receptor, composed of two subunits - CD8a and CD8P - can facilitate the interaction of T cells with antigen-presenting cells (APCs) by stabilizing the binding of the T cell receptor (TCR) to peptide-major histocompatibility complex (MHC) class I molecules, thereby reinforcing antigen recognition and subsequent T cell activation.

[0111] When used in combination with engineered TCRs (eTCRs), the ectopic CD8 co- receptor can enhance the binding affinity of the TCR to target antigens, particularly those presented on MHC class I molecules. This synergistic relationship between the ectopic CD8 co-receptor and the eTCR can allow for more effective targeting of specific antigen- presenting cells, thereby improving the sensitivity and specificity of the engineered T cells.

[0112] The CD8 co-receptor of the present disclosure can be expressed ectopically on T cells that are naturally CD4+, facilitating MHC class I recognition and enabling these engineered CD4+T cells to engage in activities typically associated with CD8+cytotoxic T cells. In this regard, the ectopic expression of CD8 in specific T cell subsets or its coexpression with a chimeric co- stimulatory receptor can enhance overall therapeutic outcomes.

[0113] In some embodiments, the ectopic CD8 co-receptor can include one or more linkers between CD8a and CD8p. In some embodiments, the linker can be a 2A peptide linker, suchDocket No. 048536-808001 WO as, but not limited to, T2A (SEQ ID NO: 26), P2A (SEQ ID NO: 27), E2A (SEQ ID NO: 28), F2A (SEQ ID NO: 29), IRES, an internal ribosome entry site (IRES) element, a flexible and cleavable linker, such as, but not limited to, Gly-Ser linker ((GGGGS)n), thrombin cleavage site, TEV protease cleavage site, or other cleavable linkers, such as, but not limited to, 3C protease cleavage site or sortase A recognition site. In some embodiments, the linker can be P2A.

[0114] In some embodiment, the ectopic CD8 co-receptor can be encoded by a polynucleotide set forth in SEQ ID NO: 24. In some embodiments, the polynucleotide sequence encoding the ectopic CD8 co-receptor can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24.

[0115] In some embodiments, the ectopic CD8 co-receptor of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 25. In some embodiments, the ectopic CD8 co-receptor of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25.

[0116] In some embodiments, the ectopic CD8 co-receptor of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 34. In some embodiments, the ectopic CD8 co-receptor of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34.

[0117] In some embodiments, the ectopic CD8 co-receptor of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 35. In some embodiments, the ectopic CD8 co-receptor of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35.D. Linker of Activation of T Cells (LAT) and LCK Binding Site

[0118] The engagement of TCR can be rapidly followed by the activation of protein tyrosine kinases (PTKs) that phosphorylate a number of downstream substrates, of which a prominent example is linker of activation of T cell (LAT), a transmembrane adapter protein.Docket No. 048536-808001 WOPhosphorylated tyrosines on LAT serve as docking sites for multiple proteins containing Src homology 2 domains, including adapters such as Gads and Grb2, which in turn can be associated with other signaling proteins. For example, SLP-76 is recruited to LAT through association with Gads. The LAT-Gads-SLP-76 complex can create a platform for the recruitment of numerous other signaling molecules, including phospholipase C-yl (PLC-yl), the Rho family GTPase exchange factor Vav, and the ubiquitin ligase Cbl. Thus, TCR engagement can induce the formation of LAT-based signaling complexes that can initiate intracellular signals required for T-cell activation.

[0119] Lymphocyte- specific protein tyrosine kinase (LCK) is a crucial enzyme in T cells that play a role in T cell receptor signaling and activation. CD4 and CD8 receptors have LCK binding sites to help facilitate signal transduction upon activation of the receptors.Incorporation of a LCK binding site into the TCR may help recruit LCK to the TCR and improve T cell activation.

[0120] In some embodiments, the composition of present disclosure can include an engineered LAT (eLAT). In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 22. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22.

[0121] In some embodiments, the eLAT of the present disclosure can have at least one mutation. In some embodiments, the at least one mutation can be at position 131 in the polypeptide sequence set forth in SEQ ID NO: 22. In some embodiments, the at least one mutation can be G131D. In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 23. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23.

[0122] In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 59. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,Docket No. 048536-808001 WO90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 59.

[0123] In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 60. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60.

[0124] In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 61. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 61.

[0125] In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 62. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 62.

[0126] In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 63. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 63.

[0127] In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 64. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 64.

[0128] In some embodiments, the eLAT of the present disclosure comprise a polypeptide sequence set forth in SEQ ID NO: 65. In some embodiments, the eLAT of the presentDocket No. 048536-808001 WO disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 65.

[0129] In some embodiments, the eLAT includes a linker. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence set forth in any one of SEQ ID NOs: 40-49. In some embodiments, the eLAT of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 40- 49.

[0130] In some embodiments, the composition of the present disclosure can include a minimal LCK binding sequence. In some embodiments, the composition of the present disclosure can include a minimal LCK binding sequence from either CD4 receptor and / or CD8 receptor. In some embodiments, the composition of the present disclosure can include a minimal LCK binding sequence and a palmitoylation site from a CD4 co-receptor. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 55-58. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence set forth in any one of SEQ ID NOs: 55-58. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 55. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NO: 55. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 56. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NO: 56. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%,Docket No. 048536-808001 WO72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 57. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NO: 57. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 58. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence set forth in SEQ ID NO: 58.

[0131] In some embodiments, the LCK binding sequence includes a linker sequence. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence having at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 36- 39. In some embodiments, the LCK binding sequence of the present disclosure comprises a polypeptide sequence set forth in any one of SEQ ID NOs: 36-39.E. Engineered T Cells

[0132] The compositions of the present disclosure can be introduced and expressed in a T cell. The methods of introducing and expressing the compositions of the present disclosure are well known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. In some embodiments, the expression vector can be a viral vector. In some embodiments, the viral vector can be a lend viral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector. In some embodiments, the viral vector can be a lentiviral vector.1. Regulatory T cells

[0133] Prior to expansion and genetic modification, a source of cells can be obtained from a subject through a variety of non-limiting methods well known in the art. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and disease sites. In some embodiments,Docket No. 048536-808001 WO any number of T cell lines available and known to those skilled in the art, can be used. In some embodiments, cells can be derived from a healthy donor or from a patient diagnosed with a disease or condition, such as Type 1 diabetes.

[0134] Accordingly, the cells, in some embodiments, are primary cells, e.g., primary human cells. The samples can include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation.

[0135] In some embodiments, the sample from which the cells can be derived or isolated is blood or a blood-derived sample or is or is derived from an apheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, neural tissue, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0136] In some embodiments, regulatory T cells (Treg) cells can be isolated, expanded, and transduced as follows: T cells can be isolated from human PBMCs by any of the known protocol. Treg cells can be enriched from PBMCs using a CD25 enrichment kit followed by fluorescent activated cell sorting (FACS) using CD4+CD25+and CD127low / _gating. The isolated and enriched Tregcells can be cryopreserved and aliquoted for a later use. After thawing, Tregcells can be subjected to TCR substitution with eTCR using e.g. CRISPR KO of TRAC and TRBC followed by lentiviral transduction of eTCRs of the present disclosure.The eTCRs can be introduced via lentiviral transduction, and at day 9, the cells can be sorted for CD3 expression. Thereafter, the CD4+cells and CD8+cells can be separated.2. T Cell Purification

[0137] In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells can be washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells can be separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.Docket No. 048536-808001 WO

[0138] In some embodiments, the blood cells collected from the subject can be washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells can be washed with phosphate buffered saline (PBS). In some embodiments, the wash solution can lack calcium and / or magnesium and / or many or all divalent cations. In some embodiments, a washing step can be accomplished a semi-automated “flow-through” centrifuge according to the manufacturer’s instructions. In some embodiments, a washing step can be accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells can be resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++free PBS. In certain embodiments, components of a blood cell sample can be removed, and the cells can be directly resuspended in culture media.

[0139] In some embodiments, the isolation methods can include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers. In some embodiments, the surface maker can be CD3. In some embodiments, the surface maker can be CD4+, CD25+, and / or CD127low / _. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation can be affinity- or immunoaffinity-based separation. For example, the isolation can include separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0140] Such separation steps can be based on positive selection, in which the cells having bound the reagents can be retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner can be retained. In some examples, both fractions can be retained for further use. In some embodiments, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation can be best carried out based on markers expressed by cells other than the desired population.

[0141] In some embodiments, multiple rounds of separation steps can be carried out, where the positively or negatively selected fraction from one step can be subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such asDocket No. 048536-808001 WO by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0142] In some embodiments, isolation can be carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection can be accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker*) at a relatively higher level (marker111811) on the positively or negatively selected cells, respectively.

[0143] In some embodiments, a cell population described herein can be collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers can be carried in a fluidic stream. In some embodiments, a cell population described herein can be collected and enriched (or depleted) via preparative scale (FACS)-sorting. In some embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system.3. T Cell Preparation and Expansion

[0144] The purified T cell population can be incubated in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. In some embodiments, the cells can be incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation.

[0145] In some embodiments, the cells can be incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions can include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0146] Tregcells can be expanded in vitro or in vivo. In some embodiments, the isolated cells of the present disclosure can be expanded by co-culturing with tissue or cells. The cellsDocket No. 048536-808001 WO can also be expanded in vivo, for example in the subject’s blood after administrating the cell into the subject.

[0147] In some embodiments, the preparation methods can include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and / or engineering. In some embodiments, the freeze and subsequent thaw step can remove granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells can be suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some embodiments can be used.4. T Cell Receptor Substitution

[0148] In some embodiments, a nucleic acid construct encoding the engineered T cell receptor of the present disclosure can be delivered by viral or non- viral delivery vehicles known in the art. The nucleic acid construct can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression. Accordingly, in some embodiments of the disclosure, the nucleic acid can be maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid can be stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA directed CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid can be present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression.

[0149] In some embodiments, the nucleic acids can be encapsulated in a viral capsid or a lipid nanoparticle or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells can be achieved by viral transduction.

[0150] In some embodiments, lend viral-derived vector systems can be useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors can offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy- target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences;Docket No. 048536-808001 WO(vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0151] In some embodiments, a native TCR can be substituted with the eTCR of the present disclosure using e.g., CRISPR KO of TRAC and TRBC followed by lentiviral transduction of the eTCR. In some embodiments, a native TCR can be substituted with the eTCR of the present disclosure using a CRISPR base editor. In some embodiments, the TRBC gene can be knocked out and the eTCR can be inserted into the TRAC locus. In this process, both TCRa and TCRP genes, with or without the eLAT, can be integrated, optionally along with the chimeric costimulatory receptor. In some embodiments, the multiple genes introduced into the TRAC locus can be separated by a linker, such as, but is not limited to, T2A, P2A, E2A, F2A, or an IRES element.

[0152] In some embodiments, a chimeric co- stimulatory receptor of the present disclosure can be introduced to T cells in addition to the eTCR. In some embodiments, ectopic CD8 coreceptor can be introduced in addition to the chimeric co-stimulatory receptor and the eTCR. In some embodiments, the engineered LAT (eLAT) can be introduced in addition to the chimeric co-stimulatory receptor and the eTCR.III. TREATMENTS WITH THE ENGINEERED T CELLS

[0153] The engineered T cells of the present disclosure can be used as a medicament in the treatment of a disease, disorder, or condition in a subject. In some embodiments, such a medicament can be used for treating type 1 diabetes.

[0154] The engineered T cells administered can be allogeneic or autologous to the subject. In instances wherein the cells are allogeneic, preferably the cells are MHC or HLA histocompatible relative to the subject to be treated and / or are modified to impair or eliminate expression or functionality of the cells’ endogenous TCRs and / or MHCs. In some instances, allogeneic Tregcells can be preferred, especially if the Tregcells of the subject to be treated are diseased and / or possess some property that renders them less than ideal for therapeutic use.In some instances, allogeneic Tregcells can be preferred, especially if the Tregcells are obtained from healthy donors as they may better migrate or traffic to desired sites.

[0155] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, can be carried out by autologous transfer, in which the cells can be isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some embodiments, the cells can be derivedDocket No. 048536-808001 WO from a subject, e.g., patient, in need of a treatment, and the cells, following isolation and processing can be administered to the same subject.

[0156] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, can be carried out by allogeneic transfer, in which the cells can be isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then can be administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects can be genetically identical. In some embodiments, the first and second subjects can be genetically similar. In some embodiments, the second subject can express the same HLA class or supertype as the first subject.

[0157] The subject referred to herein can be any living subject. In a preferred embodiment, the subject can be a mammal. The mammal referred to herein can be any mammal. In some embodiments, the mammal can be a human.

[0158] In some embodiments, the subject, to whom the cells, cell populations, or compositions can be administered is a primate, preferably a human. In some embodiments, the primate can be a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, the patient or subject can be a validated animal model for disease, adoptive cell therapy, and / or for assessing toxic outcomes, such as cytokine release syndrome (CRS).

[0159] In some embodiments, the subject has an autoimmune disease. In some embodiments, the autoimmune disease is type 1 diabetes.

[0160] In some embodiments, the cells, populations, and compositions can be administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions can be administered to the subject, such as a subject having or at risk for the disease or condition. In some embodiments, the methods thereby treat, e.g., ameliorate one or more symptoms of the disease or condition.

[0161] In some embodiments, the engineered Tregcells can undergo in vivo expansion and can persist for an extended amount of time.

[0162] Once the engineered Tregcells are administered to a subject (e.g., a human), the biological activity of the engineered Tregcells populations can be measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural Tregcell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In some embodiments, the biological activity can be measured byDocket No. 048536-808001 WO assessing clinical outcomes, such as the reduction in disease symptoms, e.g., destruction of pancreatic beta cells.

[0163] The engineered T cells of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired. In the case of adoptive cell therapy, methods for administration of cells for adoptive cell therapy are known and can be used in connection with the provided compositions.

[0164] Formulations comprising populations of engineered T cells of the present disclosure can include pharmaceutically acceptable excipient(s). Excipients included in the formulations can have different purposes depending, for example, on the engineered T cells, the subpopulation of engineered Tregcells used, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water- for-inf ection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents. The formulations comprising populations of engineered Tregcells of the present disclosure can typically have been prepared and cultured in the absence of any nonhuman components, such as animal serum e.g., bovine serum albumin).

[0165] The cells or population of cells can be administrated in one or more doses. In some embodiments, an effective amount of cells can be administrated as a single dose. In some embodiments, an effective amount of cells can be administrated as more than one dose over a period of time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The dosage administrated can be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In some embodiments, administration can be an intravenous administration.

[0166] In some embodiments, the methods of the disclosure involve administering an effective amount or number of the recombinant cells provided here to a subject in need thereof. This administering step can be accomplished using any method of implantation delivery in the art. For example, the recombinant cells can be infused directly in the subject’s bloodstream or otherwise administered to the subject.

[0167] For use in the various embodiments described herein, an effective amount of engineered T cells as disclosed herein, can be, but not limited to, at least 102cells, at least 5xl02cells, at least 103cells, at least 5xl03cells, at least 104cells, at least 5xl04cells, at least 105cells, at least 2xl05cells, at least 3xl05cells, at least 4xl05cells, at least 5xl05cells, at least 6xl05cells, at least 7xl05cells, at least 8xl05cells, at least 9xl05cells, at leastDocket No. 048536-808001 WO106cells, at least 2xl06cells, at least 3xl06cells, at least 4xl06cells, at least 5xl06cells, at least 6xl06cells, at least 7xl06cells, at least 8xl06cells, at least 9xl06cells, or multiples thereof.EXAMPLES

[0168] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.EXAMPLE 1. Engineering Insulin Specific TCR Tree Cells

[0169] This Example illustrates the process of engineering human T cells with insulinspecific TCRs and assessing their activation in response to pancreatic antigens.

[0170] T Cell Receptor Substitution in Human T Cells'. Human peripheral blood mononuclear cells (PBMCs) were collected from healthy donors, and T cells were isolated using a negative selection kit. On Day 0, the T Cell Receptor Alpha Constant (TRAC) and T Cell Receptor Beta Constant (TRBC) genes in the isolated T cells were knock-out using CRISPR-Cas9 ribonucleoprotein (RNP) system. The T cells were activated with Immunocult and supplemented with 300 U / mL of IL-2. This process is summarized in FIG. 1.

[0171] Co-Culture Assay Between T Cells and Target Cells'. After two days, engineered TCR (eTCR) of the present disclosure (FIG. 2A) were introduced into the T cells through lentiviral transduction. On Day 9, successfully transduced cells expressing CD3 were sorted using flow cytometry to isolate both CD4+and CD8+T cell subsets (FIGS. 4A-4C). These sorted cells were then rested for an additional 3-5 days. Cells were incubated with various target cells, including K562 cells, SC-P cells, and human islet cells to test T cell activation after engineering (FIG. 2B).Human Tree Isolation from Peripheral Blood: Tregcells were isolated from peripheral blood mononuclear cells (PBMCs) from three different healthy donors. Tregcells were enriched from the PBMCs using a CD25 enrichment kit followed by FACS sorting using CD4+CD25+and CD127low / _gating. Tregcells were cryopreserved and aliquoted for each experiment (FIG. 3). After thawing, TCRs were substituted with eTCRs in the Treg cells as described above.EXAMPLE 2. Activation of CD4+or CD8+T Cells with Engineered TCR

[0172] For the co-culture assay, the T cells comprising engineered (eTCR) and ectopic CD8 co-receptor were co-cultured with target cells (K562 cells, human islets, or stem cell-Docket No. 048536-808001 WO derived beta-like cells (SC-beta) expressing HLA-A2). The target cells were seeded in a round-bottom 96-well plate at 50,000 cells / well (FIGS. 5A-5B). When co-culturing with K562 cells, the preproinsulin: 15-24 peptide (PPI15-24: ALWGPDPAAA) (SEQ ID NO: 4) was added to the media at different molar concentrations and incubated for 3-4 hours. When coculturing with the islet or SC-beta cells, no PPI15-24 peptide was added to the media. The cocultures were incubated for 72 hours in a 1:1 ratio with T cells bearing different TCRs. T cell activation was analyzed via flow cytometry by measuring the percentage of cells positive for activation markers CD25 and CD71. As demonstrated in FIGS. 6A-6D, high-affinity insulin eTCR activated only CD8+T cells at low peptide concentrations.

[0173] Additionally, co-culture of TWG CD8+ T cells and human stem cell-derived P-like cells (SC-P) was performed using GFP positive SC-P cells (FIG. 6E). The co-culture resulted in CD8+ T cell activation and P cell destruction (FIG. 6F and FIG. 6G).

[0174] These data show that TWG and NWG mutations in the 1E6 TCR P chain increase the ability of CD8+ T cells, but not CD4+T cells, to respond to cells presenting the PPI: 15- 24 p cell antigen. These results also indicate that eTCRs can activate CD8+T cells at lower PPI15-24 concentrations, but they are not sufficient to induce conventional CD4+T cell activation.EXAMPLE 3. Activation of eTCR CD4+T Cells

[0175] To assess the activation of CD4+T cells comprising engineered TCR (eTCR) in different co-culture conditions, Jurkat cells were transduced with a lentiviral construct encoding the CD8 alpha and beta subunits to evaluate co-receptor expression and activation response. Flow cytometry analysis confirmed the expression of the CD8 co-receptor on Jurkat cells (FIGS. 7A-7C).

[0176] To further evaluate activation of CD4+T cell, CD4+T cells comprising eTCR or eTCR + ectopic CD8 were co-cultured with K562 cells expressing HLA-A2 (A2+K562 cells, pre-incubated with PPI15-24) (FIG. 8A), and CD4+T cells comprising eTCR or eTCR + ectopic CD8 + chimeric co-stimulatory receptor of the present disclosure (COSTR / DPP6) were co-cultured with human islet cells expressing HLA-A2 (A2+human islets) (FIG. 8C). T cell activation was analyzed via flow cytometry by measuring the percentage of cells positive for activation markers CD25 and CD71. As shown in FIG. 8A, ectopic CD8 co- receptor was required for activation of CD4+T cells comprising eTCR. CD4+T cells comprising eTCR and ectopic CD8 dimply recognized human islet cells (FIG. 8B).Docket No. 048536-808001 WO

[0177] These results indicate that CD8 co-receptor is sufficient to induce activation on CD4+T cells bearing the eTCRs in an PPI-dependent manner.EXAMPLE 4. Activation of eTCR CD4+T Cells with Chimeric Co-Stimulatory Receptor

[0178] To enhance the activation of engineered TCR (eTCR) CD4+T cells, a DPP6- specific chimeric co-stimulatory receptor was introduced. This COSTR was designed to target DPP6 on islet cells and included a nanobody specific to DPP6, followed by CD28 transmembrane and intracellular domains (FIG. 9A). Surface expression of the DPP6 chimeric co-stimulatory receptor on transduced cells was confirmed by flow cytometry, with positive signals for both the Myc and mCherry markers indicating successful expression (FIG. 9B).

[0179] An islet- specific chimeric co-stimulatory receptor (COSTR) comprised of a nanobody against the islet antigen DPP6. This nanobody was then fused with a CD8 signal peptide, a myc tag, a IgG4 hinge, a CD28 transmembrane and intracellular domain was generated (FIG. 10A). To identify transduced cells a GFP sequence was also added to this construct separated by a P2A sequence (SEQ ID NO: 50). This sequence was then introduced into a lentiviral vector and transduced into CD4+ T cells bearing the TWG TCR and CD8 coreceptor (which is discussed more in Example 6). To test the functional impact of the DPP6 chimeric co-stimulatory receptor on eTCR CD4+T cell activation, activated CD4+cells comprising eTCR, ectopic CD8 co-receptor, and DPP6 chimeric co-stimulatory receptor were co-cultured with K562 cells expressing HLA-A2 (FIG. 10B), human islets (FIG. 10C), and stem cell-derived beta-like cells (SC-beta) (FIG. 10D).

[0180] Activation of CD4+T cells was measured by flow cytometry based on CD25 and CD71 expression, and results indicated that cells co-expressing the DPP6 chimeric co- stimulatory receptor and ectopic CD8 co-receptor showed significantly higher activation levels compared to other groups, particularly in the presence of the PPI15-24 peptide (FIGS. 10B-10D).

[0181] These results indicate that DPP6-mediated co- stimulation is sufficient to induce activation of CD4+T cells bearing eTCR when co-cultured with human islet cells and human stem cell-derived beta-like cells.

[0182] The intracellular signaling domains of CD28 was further modified to modulate costimulation signaling on T cells. These signaling sequences can be separated into 3 main domains “YMNM”, “PRRPGP”, and “PYAAP”. These sequences were then mutated into 4Docket No. 048536-808001 WO different new sequences; “FMNM” (included in SEQ ID NO: 51), “YMFM” (included in SEQ ID NO: 52), “ARRAGP” (included in SEQ ID NO: 53), and “A YAAP” (included in SEQ ID NO: 54). These sequences were then introduced into TWG-CD8 Jurkat cells and coculture with A2-PPI+ DPP6+ K562 cells for 1 day. CD69 surface expression on Jurkat cells was used to indicate cell activation. The results show that DPP6 COSTR ml “FMNM” (SEQ ID NO: 51) can induce higher CD69 expression compared to WT DPP6 COSTR (SEQ ID NO: 50) (FIG. 10E). This result suggests that mutating the signaling domains of this COSTR molecule allows for signal modulation on cells.EXAMPLE 5. Insulin- Specific Engineered Tree Cells

[0183] To examine the activation of insulin-specific engineered regulatory T (Treg) cells in the presence of ectopic CD8 co-receptor, Tregcells were first isolated from human peripheral blood mononuclear cells (PBMCs) as described in Example 1. Following leukapheresis, Tregcells were enriched through CD25+selection and further sorted using fluorescence-activated cell sorting (FACS) based on CD4+, CD25Ugh, and CD127low / _markers. The identity of the isolated Tregcells was confirmed by flow cytometry (FIG. 11A) with high expression of FOXP3 and HELIOS, two key transcription factors indicative of functional Treg.Quantification from three different donors showed over 90% purity of FOXP3+and HELIOS+cells (FIG. 11B).

[0184] The functionality of these insulin-specific engineered Tregcells was tested in coculture experiments with K562 cells either lacking (FIG. 12A) or expressing ectopic CD80 co-receptor (FIG. 12B). When engineered Tregs were co-cultured with K562 cells without CD80 co-stimulation (FIG. 12A), flow cytometry analysis demonstrated limited activation, as indicated by low percentages of CD25+CD71+Tregacross different peptide concentrations (with and without the PPI15-24 peptide), suggesting that activation was minimal in the absence of co-stimulation (FIG. 13A). When engineered Tregswere co-cultured with CD80 (FIG. 12B), there was a significant increase in Tregactivation, particularly when the CD8 co- receptor was included alongside the insulin- specific eTCR. The CD25+ CD71+ activation marker levels were considerably higher in the presence of both CD80 co-stimulation and the PPI15-24 peptide (FIG. 13B).

[0185] These findings demonstrate that eTCRs with CD8 co-receptor can induce Tregactivation when co-cultured with K562 presenting PPI15-24.Docket No. 048536-808001 WOEXAMPLE 6. Engineering CD8 co-receptor signaling in CD4+ T cells

[0186] Since neither TWG nor NWG TCRs were able to induce CD4+ T cell activation in response to the P cell antigen PPI: 15-24, further modifications on the TWG TCR were explored.

[0187] First round of modifications involved adding the co-receptor CD8a and CD8P chains into TWG TCR CD4+ T cells. Without being bound by theory, the premise is that the CD8 co-receptor may enhance TWG TCR signaling by increasing avidity of the TCR:MHC interaction via its extracellular MHC binding domain and by recruiting LCK to the TCR via its intracellular LCK-binding domain. Full-length WT CD8a and CD8P was cloned downstream of the TWG TCR after a T2A sequence in the same plasmid (SEQ ID NO: 34). The CD8a and CD8P sequences were separated by a P2A sequence. A mutant construct with deletion of the LCK-binding region in CD8a was also generated to determine if LCK recruitment is necessary for CD8 co-receptor function in the context of TWG TCR activation in CD4+ T cells (SEQ ID NO: 35).

[0188] Lentivirus particles were produced using these two plasmids and transduced into CD4+ T cells in conjunction with TWG TCR. These cells were sorted for TWG and CD8 expression and co-culture with K562 as previously described. 3-4 days after co-culture, CD4+ T cell activation was analyzed by surface upregulation of T cell activation markers CD25 and CD71. Results show that upon addition to WT CD8a and CD8P chains, TWG TCR was able to induce T cell activation in a PPI: 15-24 antigen dose-dependent manner (FIG. 14A). Furthermore, compared to the WT CD8 co-receptor, CD8 co-receptor missing the LCK binding site (LKOA) was not as efficient at inducing T cell activation (FIG. 14A). This result demonstrates that the LCK-binding region in CD8a is important for CD8 co- receptor to enable TWG TCR activation in CD4+ T cells. Without being bound by theory, this may be due to the recruitment of the LCK kinase into the TWG TCR.

[0189] Based on these findings, it was thought that incorporating the minimal LCK binding sequence (CxCP) into the TWG TCR might help recruit LCK to the TCR and improve CD4+ T cell activation. To test this idea, different LCK-binding sequences from CD4 and CD8 coreceptors were added the C-terminus of the TCRP chain of TWG connected by a 3x GS linker. These sequences either contained the predicted LCK-binding site and palmitoylation site from the CD4 co-receptor (SEQ ID NO: 36), only the LCK binding site from CD4 (SEQ ID NO: 37), the LCK-binding site from CD8 (SEQ ID NO: 38), or LCK-binding sites from CD4 and CD8 in combination (SEQ ID NO: 39). These sequences were integrated into LVs and transduced into CD4+ T cells and tested in the co-culture assay described before. ResultsDocket No. 048536-808001 WO show that all sequences tested enhanced activation of TWG TCR activation in CD4+ T cells in response to the p cell antigen PPI: 15-24 (FIG. 14B) with comparable efficiency.Provision of co- stimulation via CD80 shifted the dose response by 1 log (FIG. 14B). These data shows that incorporation of the LCK-binding domain from co-receptors CD4 or CD8 increases TWG TCR activation in CD4+ T cells. However, 10X higher concentration of peptides is needed to activate CD4+ T cells using these constructs compared to co-expressing of CD8a and CD8P co-receptor.EXAMPLE 7. Addition of Intracellular Domains of the Scaffold Protein LAT

[0190] TCR signal transduction integrates scaffold proteins that allow for signal amplification and the recruitment of multiple signaling components. Without being bound by theory, incorporating the scaffold protein Linker of Activation of T cells (LAT) may increase TWG TCR induced CD4+ T cell activation. To achieve this, LAT was fused to the C- terminus of the TCRP chain of TWG after a 3x GS linker (SEQ ID NO: 40). In parallel, a mutated LAT (mutLAT) sequence with a mutation in the PLCyl -binding site (G131D) was also fused to the C-terminus of the TCRP chain of TWG (SEQ ID NO: 41). These sequences were introduced into CD8+, CD4+ Tconv and CD4+ Tregs cells and the transduced T cells were co-cultured with K562 as described before. Results showed that activation of CD8+ T cells with either TWG-LAT or TWG-mutLAT were comparable to TWG TCR alone control (FIG. 15A). CD4+ Tconv and CD4+ Treg cells expressing TWG-LAT or TWG-mutLAT were able to be activated by the P cell antigen PPI: 15-24 in contrast of using TWG alone which did not induce activation.

[0191] To determine what domains from LAT that are important for inducing TWG TCR activation in CD4+ T cells, the intracellular domain of mutLAT (G131D) was divided into 3 regions and those regions individually or in combination were fused with the TCRP of TWG with a 3x GS linker. Using this strategy, 6 different new sequences were generated named after the region (N-region, M-region, C-region) of LAT that was incorporated (FIG. 15B). TWG-nLAT (SEQ ID NO: 42), TWG-mLAT (SEQ ID NO: 43), TWG-cLAT (SEQ ID NO: 44), TWG-nmLAT (SEQ ID NO: 45), TWG-mcLAT (SEQ ID NO: 46), TWG-ncLAT (SEQ ID NO: 47). All these new sequences were transduced into CD4+ T cells and co-culture with K562 as described before. Only TWG-mutLAT with full-length mutant LAT (SEQ ID NO: 41) and TWG-cLAT (SEQ ID NO: 44) were able to induce CD4+ T cell activation using A2+ K562 cells with high dose of antigen. However, all constructs except TWG-nLAT, could enable CD4+ T cell activation by A2+ K562 cells expressing the co-stimulatory ligand CD80Docket No. 048536-808001 WO in the presence of high PPI: 15-24 concentrations (FIG. 15B). Altogether, these results suggest that addition of the intracellular domain of the scaffold protein LAT to the TCRP chain of TWG enhances antigen specific cell activation on CD4+ T cells in the absence of CD8 co-receptor. Moreover, most regions of the mutLAT contributed to TWG TCR activation whereas the n-region alone was not sufficient. However, a high antigen dose was needed to elicit a response with all of these constructs.EXAMPLE 8. Combination of TCR Signaling Modules into TWG Enhances CD4+ T cell activation

[0192] The n-region of mutLAT might aid the interaction between LCK and ZAP70. This differs from the LCK-binding domain in CD8 or CD4 co-receptors that directly binds to LCK. Thus, without being bound by theory, the n-region of LAT may not be sufficient to replace the function of CD4 and CD8 co-receptors. A fusion of CD4PL sequence (SEQ ID NO: 36) with the c-region or the me -region of mutLAT (SEQ ID NO: 44 and SEQ ID NO: 46) was generated. Without being bound by theory, these sequences may be able to incorporate palmitoylation and LCK-binding domain with the signal amplification capability of mc-LAT. This resulted in TWG-CD4PL-cLAT (SEQ ID NO: 48) and TWG-CD4PL- mcLAT (SEQ ID NO: 49). These sequences were then introduced into CD4+ T cells, and the transduced cells were co-cultured with K562 as previously described. Results showed that TWG-CD4PL-cLAT induce T cell activation irrespectively of PPI: 15-24 concentration which suggest that this TWG TCR fusion complex signaled constitutively (FIG. 16). In contrast, TWG-CD4PL-mcLAT shows CD4+ T cell activation in a PPI: 15-24 dependent manner (FIG. 16). While engaging CD80 co-stimulation increased the PPI: 15-24 dependent activation of TWG-CD8 bearing CD4+ T cells, this co- stimulatory signal seems to constitutively induce low levels of CD25 and CD71 (FIG. 16) This data shows adding the CD4PL-mcLAT enabled TWG TCR activation to the same extend as that enabled by coexpressing CD8 receptors.

[0193] Overall, recruitment of LCK via the addition of palmitoylation and LCK-binding sequences from CD4 intracellular domain to the TCR, TCR-CD4PL, can partially bypass the requirement for a co-receptor. Addition of subdomains of the scaffold protein LAT to the TCR-CD4PL fusion amplify TCR signal transduction to enable high sensitivity antigen response similar to that achieved by expressing full-length co-receptors. This data shows the potential of these added sequences to be incorporated into any TCR to direct CD8+, CD4+Docket No. 048536-808001 WOTconv and CD4+Treg activation. FIG. 17A, FIG. 17B, FIG. 18, FIG. 19A, and FIG. 19B depict representative configurations of the present disclosure.

[0194] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.Docket No. 048536-808001 WOSEQUENCE LISTING

[0195] Exemplary polynucleotides and amino acid sequences of one or more genes described in the present disclosure are shown in Table 1.Table 1. Exemplary nucleotides and amino acids sequencesDocket No. 048536-808001 WODocket No. 048536-808001 WODocket No. 048536-808001 WODocket No. 048536-808001 WODocket No. 048536-808001 WODocket No. 048536-808001 WODocket No. 048536-808001 WO

Claims

Docket No. 048536-808001 WOCLAIMSWe claim:

1. A composition, comprising: an engineered T cell receptor (eTCR) comprising alpha chain and beta chain, wherein the eTCR comprises one or more mutations; and a chimeric co-stimulatory receptor comprising a target binder, a hinge, a transmembrane domain, and an intracellular domain of a co-stimulatory receptor.

2. A composition, comprising: an eTCR comprising alpha chain and beta chain, wherein the eTCR comprises one or more mutations; a chimeric co-stimulatory receptor comprising a target binder, a hinge, a transmembrane domain, and an intracellular domain of a co-stimulatory receptor; and an ectopic CD8 co-receptor.

3. The composition of claim 2, wherein the ectopic CD8 co-receptor comprises CD8a and CD8p.

4. The composition of any one of claims 2-3, wherein the ectopic CD8 co-receptor comprises a linker.

5. The composition of claim 4, wherein the linker is selected from T2A, P2A, E2A, F2A, and IRES.

6. The composition of claim 5, wherein the linker is P2A.

7. The composition of any one of claims 2-6, wherein the ectopic CD8 co-receptor comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 25.

8. The composition of claim 7, wherein the ectopic CD8 co-receptor comprises the polypeptide sequence set forth in SEQ ID NO: 25.

9. A composition, comprising: an eTCR comprising alpha chain and beta chain; a chimeric co-stimulatory receptor comprising a target binder, a hinge, a transmembrane domain, and an intracellular domain of a co-stimulatory receptor; and an engineered Linker of Activation of T cells (eLAT).

10. The composition of claim 9, wherein the eTCR comprises one or more mutations.Docket No. 048536-808001 WO11. The composition of any one of claims 9-10, wherein the eLAT comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 22.

12. The composition of claim 11, wherein the eLAT comprises the polypeptide sequence set forth in SEQ ID NO: 22.

13. The composition of any one of claims 9-10, wherein the eLAT comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 59-65.

14. The composition of claim 13, wherein the eLAT comprises the polypeptide sequence set forth in any one of SEQ ID NOs: 59-65.

15. The composition of any one of claims 9-14, the eLAT comprises at least one mutation.

16. The composition of claim 15, wherein the at least one mutation is at position 131 in the polypeptide sequence set forth in SEQ ID NO: 21.

17. The composition of claim 16, wherein the at least one mutation is G131D.

18. The composition of any one of claims 9-17, wherein the eLAT is linked to the eTCR via a glycine- serine linker.

19. The composition of claim 18, wherein the glycine- serine linker comprises a polypeptide sequence set forth in SEQ ID NO: 20.

20. The composition of any one of claims 1-19, wherein the composition further includes an LCK binding sequence.

21. The composition of claim 20, wherein the LCK binding sequence includes a palmitoylation site from a CD4 co-receptor.

22. The composition of claim 20 or claim 21, wherein the LCK binding sequence is from a CD4 receptor and / or from a CD8 receptor.

23. The composition of any one of claims 20-22, wherein the LCK binding sequence comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 55-58.

24. The composition of any one of claims 20-23, wherein the LCK binding sequence comprises a polypeptide sequence set forth in any one of SEQ ID NOs: 55-58.Docket No. 048536-808001 WO25. The composition of any one of claims 1-24, wherein the eTCR recognizes insulin producing cells.

26. The composition of claim 25, wherein the eTCR recognizes preproinsulin (PPI): 15-24 presented by HLA-A*0201.

27. The composition of any one of claims 1-26, wherein the eTCR comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1.

28. The composition of any one of claims 1-27, wherein the one or more mutations are in complementary determining region 3 (CDR3) of the beta-chain.

29. The composition of claim 28, wherein the one or more mutations are at positions 96 and 98 in the polypeptide sequence set forth in SEQ ID NO: 1.

30. The composition of claim 29, wherein the one or more mutations are L96T and E98G.

31. The composition of claim 30, wherein the eTCR comprises a polypeptide sequence set forth in SEQ ID NO: 2.

32. The composition of claim 29, wherein the one or more mutations are L96N and E98G.

33. The composition of claim 32, wherein the eTCR comprises a polypeptide sequence set forth in SEQ ID NO: 3.

34. The composition of any one of claims 1-33, wherein the target binder binds to a dipeptidyl peptidase-like protein 6b (DPP6).

35. The composition of claim 34, wherein the target binder is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 7.

36. The composition of claim 35, wherein the target binder is encoded by a polynucleotide sequence set forth in SEQ ID NO: 7.

37. The composition of claim 34, wherein the target binder comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 12.

38. The composition of claim 37, wherein the target binder comprises a polypeptide sequence set forth in SEQ ID NO: 12.Docket No. 048536-808001 WO39. The composition of any one of claims 1-33, wherein the hinge is selected from IgGl, IgG2, IgG3, IgG4, IgE, IgM, IgA, CD8a, CD28, CD3e, and DAP10.

40. The composition of claim 39, wherein the hinge is an IgG4 hinge.

41. The composition of claim 40, wherein the IgG4 hinge is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 8.

42. The composition of claim 41, wherein the IgG4 hinge is encoded by a polynucleotide sequence set forth in SEQ ID NO: 8.

43. The composition of claim 40, wherein the IgG4 hinge comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15.

44. The composition of claim 43, wherein the IgG4 hinge comprises a polypeptide sequence set forth in SEQ ID NO: 15.

45. The composition of any one of claims 1-33, wherein the transmembrane domain is selected from CD28, CD8a, CD3^, ICOS, 4-1BB, DAP10, and NKG2D.

46. The composition of claim 45, wherein the transmembrane domain is CD28 transmembrane domain.

47. The composition of claim 46, wherein the CD28 transmembrane domain is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 9.

48. The composition of claim 47, wherein the CD28 transmembrane domain is encoded by a polynucleotide sequence set forth in SEQ ID NO: 9.

49. The composition of claim 46, wherein the CD28 transmembrane domain comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17.

50. The composition of any one of claims 46-49, wherein the CD28 transmembrane domain comprises a polypeptide sequence set forth in SEQ ID NO: 17.

51. The composition of claim 46, wherein the CD28 transmembrane domain comprises C14L, Y15L, S16L, or T20L amino acid substitutions in the polypeptide sequence of SEQ ID NO: 17.Docket No. 048536-808001 WO52. The composition of any one of claims 1-51, wherein the intracellular domain is an intracellular domain of 4-1BB (CD137), CD27 (TNFRSF7), CD28, CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), ICOS, LFA-1 (CD1 la / CD18), DAP10, and DAP 12.

53. The composition of claim 52, wherein the intracellular domain is an intracellular domain of CD28.

54. The composition of claim 53, wherein the CD28 intracellular domain is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10.

55. The composition of claim 54, wherein the CD28 intracellular domain is encoded by a polynucleotide sequence set forth in SEQ ID NO: 10.

56. The composition of claim 53, wherein the CD28 intracellular domain comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17.

57. The composition of claim 56, wherein the CD28 intracellular domain comprises a polypeptide sequence set forth in SEQ ID NO: 17.

58. The composition of any one of claims 53-57, wherein the CD28 intracellular domain comprises one or more modifications in the polypeptide sequence set forth in SEQ ID NO: 17.

59. The composition of claim 58, wherein the one or more modifications is at positions 12, 14, 17, 20, and / or 29 in the polypeptide sequence set forth in SEQ ID NO: 17.

60. The composition of claim 59, wherein the one or more modifications is Y12F, Y 14F, P17A, P20A, and / or P29A in the polypeptide sequence set forth in SEQ ID NO: 17.

61. A T cell comprising the composition of any one of claims 1-60.

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