A multichain synthetic receptor that simultaneously mediates ligand-induced transcriptional regulation and membrane-proximal signal transduction.

Abstract

The present disclosure generally relates to multi-chain chimeric polypeptides or receptors having separate polypeptide chains that post-translationally assemble to allow simultaneous activation of a signaling domain and release of a transcriptional regulator upon binding to a ligand. The present disclosure also provides nucleic acid constructs, recombinant cells, vectors, pharmaceutical compositions, and methods of treatment comprising the multi-chain chimeric polypeptides of the present disclosure. The present disclosure further provides methods for simultaneously inducing T cell signaling and gene regulation in T cells. The multi-chain chimeric polypeptide allows simultaneous activation of a signaling domain and release of a transcriptional regulator upon binding to a ligand.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 398,185, filed August 15, 2022, the entire disclosure of which, including any drawings, is incorporated herein by reference.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. OD025751-01 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporating a sequence listing

[0001] This application contains a Sequence Listing, which is hereby incorporated by reference in its entirety. The attached Sequence Listing file entitled "2023-08-14 Sequence_Listing_ST26 048536-727001WO.xml" was created on August 14, 2023, and is 345,882 bytes.

[0004] Field The present disclosure generally relates to the fields of immunology and medicine. More particularly, the present disclosure relates to compositions and methods comprising multi-chain receptors. The present disclosure also relates to enhanced CAR T cell therapy comprising such receptors. [Background technology]

[0005] background There have been many important advances in the development of immunotherapies based on the adoptive transfer of lymphocytes (e.g., T cells), among which T cells expressing chimeric immune receptors, including chimeric antigen receptors (CAR-T cells), stand out. However, a key problem limiting these therapies is the inability to modulate or stop CAR-T activity when needed. Modulation of CAR-T activity to reduce or eliminate interactions that cause serious side effects to the administration of chimeric antigen receptor T cells is particularly important.

[0006] Unlike T cell receptors (TCRs), chimeric immune receptors, such as CARs, include molecules that combine tumor antigen recognition and intracellular activation. The structure and design of such immune receptors minimally comprise an extracellular antigen recognition domain linked to intracellular activation domain(s) via a TMD. An example of such an immune receptor is the synNotch receptor, which can bind to a custom-defined cell surface-displayed ligand, triggering proteolytic cleavage of the receptor and the release of transcriptional regulators that induce a custom transcriptional program in the cell. Other examples include receptors incorporating signaling (e.g., costimulatory, CD3 zeta) that can initiate T cell activation concomitant with custom transcriptional regulation. These immune receptor examples are engineered as a single protein chain with sequential fusion of the costimulatory domain, CD3 zeta domain, and transcriptional regulator domain.

[0007] There is also a need for receptors that can enable more efficient and more reliable induction of proximal T cell activation signals and simultaneous signal transduction and transcriptional regulation for gene regulation in primary T cells. Summary of the Invention [Means for solving the problem]

[0008] Abstract The present disclosure generally relates to multi-chain chimeric polypeptides (e.g., chimeric immunoreceptors) having configurations that enable transcriptional regulation and signal transduction using separate protein chains. In particular, the present disclosure relates to multi-chain chimeric polypeptides having separate polypeptide chains that post-translationally associate with each other (through residues located within the transmembrane domains) to enable simultaneous activation of a signaling domain and release of a transcriptional regulator upon ligand binding.

[0009] Provided herein, inter alia, are multi-chain chimeric polypeptides comprising: (a) a first polypeptide having (i) an extracellular ligand-binding domain having binding affinity for a selected ligand, (ii) a first transmembrane domain (TMD) comprising a first engineered interface, and (iii) a first intracellular domain (ICD) comprising a transcriptional regulator; and (b) a second polypeptide having (i) a second TMD having a second interface and (ii) a second ICD comprising a signaling domain, wherein the first and second engineered interfaces comprise oppositely charged amino acid residues, respectively, and the first polypeptide is coupled to the second polypeptide through the first engineered interface and the second interface, wherein binding of the selected ligand to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments, binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces activation of the signaling domain and release of the transcriptional regulator.

[0010] In some embodiments, the first polypeptide comprises, from the N-terminus to the C-terminus of the first polypeptide, (i) an extracellular ligand-binding domain, (ii) a first TMD, and (iii) a first intracellular domain. In some embodiments, the first TMD comprises (i) 10-25 consecutive valine residues or (ii) a Notch1 TMD.

[0011] In some embodiments of the multi-chain chimeric polypeptide, the second polypeptide comprises, in order from the N-terminus to the C-terminus of the second polypeptide, (i) a second TMD and (ii) a second intracellular domain.

[0012] In some embodiments of the multi-chain chimeric polypeptide, the first engineered interface comprises positively charged residues and the second interface comprises negatively charged residues, and the first polypeptide is bound to the second polypeptide via electrostatic forces between the first and second engineered interfaces. In some embodiments, the positively charged residue is lysine or arginine. In some embodiments of the multi-chain chimeric polypeptide of the present disclosure, the first TMD comprises SEQ ID NO:21 or a functional variant thereof, and the lysine or arginine residue is at a position selected from positions 10-14 of SEQ ID NO:21; or (ii) the first TMD comprises SEQ ID NO:18, and the lysine or arginine is at a position selected from positions 8-11 of SEQ ID NO:18. In some embodiments, the first transmembrane comprises SEQ ID NO:21, the lysine or arginine residue is at position 12 of SEQ ID NO:21. In some embodiments, the first transmembrane comprises SEQ ID NO:18, the lysine or arginine residue is at position 9 relative to SEQ ID NO:18.

[0013] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the extracellular domain comprises an antigen-binding moiety that binds (or is capable of binding) to a ligand on the surface of a cell. In some embodiments, the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, a F(ab')2 fragment, a F(ab)2 fragment, a single-chain variable fragment (scFv), a single-domain antibody (sdAb), and functional fragments thereof.

[0014] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the ligand comprises a protein or carbohydrate. In some embodiments, the ligand is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the ligand comprises a cell surface receptor, an adhesion protein, an integrin, a mucin, a lectin, a tumor-associated antigen, and a tumor-specific antigen. In some embodiments, the ligand is CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein alpha (SIRPα).

[0015] In some embodiments, the ligand is present on the surface of a cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a tumor cell.

[0016] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the transcriptional regulator comprises a transcriptional activator or a transcriptional repressor, hi some embodiments, the transcriptional regulator comprises Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, or HAP1-VP16.

[0017] In some embodiments of the multi-chain chimeric polypeptide of the present disclosure, the transcriptional regulator is a human or humanized transcriptional regulator. In some embodiments, the transcriptional regulator is HNF1a.

[0018] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the second polypeptide comprises a signaling domain comprising a CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, or CD66d signaling domain.

[0019] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the signaling domain comprises a DAP12, CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc.ε.RI, DAP10, DAP12, or CD66d signaling domain.

[0020] In some embodiments of the multi-chain chimeric polypeptide of the present disclosure, the first polypeptide further comprises one or more of the following: a hinge domain, a ligand-inducible proteolytic cleavage site, an autoproteolytic peptide sequence, a nuclear localization signal, or a juxtamembrane domain. In some embodiments, the juxtamembrane domain is a polybasic domain. In some embodiments, the polybasic domain comprises a Notch-1 or Notch-2 juxtamembrane domain. In some embodiments, the autoproteolytic peptide sequence is derived from porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), or a combination thereof.

[0021] In some embodiments of the multi-chain chimeric polypeptide of the present disclosure, the first polypeptide further comprises a hinge domain derived from CD8, CD28, OX40, or IgG4. In some embodiments, the hinge domain is derived from CD8. In some embodiments, the hinge domain comprises a truncated CD8α hinge domain (also referred to as CD8 hinge or CD8 hinge 2).

[0022] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the ligand-inducible proteolytic cleavage site is cleavable by gamma secretase.

[0023] Also provided herein in one aspect are multi-chain chimeric polypeptides comprising: (a) a first polypeptide having (i) an extracellular ligand-binding domain comprising CD19scFv; (ii) a first TMD comprising a contiguous stretch of valine residues containing a lysine or arginine residue; (iii) a Notch 2 juxtamembrane domain; and (iv) a first ICD comprising a Gal4VP64 transcriptional regulator; and (b) a second polypeptide comprising DNAX-activation protein 12 (DAP12), wherein the first polypeptide is linked to the second polypeptide through a lysine or arginine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain simultaneously induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments, the contiguous stretch of valine residues comprises 5-25 valine residues, and a contiguous stretch of 5-15 valine residues flanks the lysine residue. In some embodiments, the contiguous stretch of valine residues comprises 5-25 valine residues. In some embodiments, the first TMD comprises 15-20 valine residues. In some embodiments, the lysine residue is at position 9 or 10 of the first TMD.

[0024] Also provided herein in certain aspects is a multi-chain chimeric polypeptide comprising: (a) a first polypeptide having (i) an extracellular ligand-binding domain comprising a CD19scFv; and (ii) a first TMD comprising a contiguous stretch of valine residues that includes a lysine or arginine residue, and further comprising a first intracellular domain comprising a Notch 2 juxtamembrane domain and a Gal4VP64 transcriptional regulator; and a second polypeptide comprising a CD3z signaling domain, wherein the first polypeptide is linked to the second polypeptide via a lysine residue within the contiguous stretch of valine residues, and wherein binding of CD19 to the extracellular ligand-binding domain simultaneously induces activity of the signaling domain and release of the transcriptional regulator.

[0025] Also provided herein in certain aspects is a multi-chain chimeric polypeptide comprising: (a) a first polypeptide having (i) an extracellular ligand-binding domain comprising CD19scFv; and (ii) a first TMD comprising a contiguous stretch of valine residues that includes a lysine or arginine residue, the first polypeptide further comprising a Notch 2 juxtamembrane domain and a human or humanized transcriptional regulator; and a second polypeptide comprising DNAX-activation protein 12 (DAP12) or CD3z, wherein the first polypeptide is linked to the second polypeptide via a lysine residue within the contiguous stretch of valine residues, and wherein binding of CD19 to the extracellular ligand-binding domain simultaneously induces activation of the signaling domain and release of the transcriptional regulator.

[0026] In some embodiments, the transcriptional regulator is HNF1a.

[0027] In some embodiments, the multi-chain chimeric polypeptide of the present disclosure is an immunoreceptor. In some embodiments, the immunoreceptor is a chimeric antigen receptor.

[0028] In certain aspects, provided herein are multi-chain chimeric polypeptides comprising: (a) a first polypeptide comprising (i) an extracellular ligand-binding domain having binding affinity for a selected ligand, (ii) a first transmembrane domain (TMD) comprising a first engineered interface, and (iii) a first intracellular domain comprising a transcriptional regulator or signaling domain; and (b) a second polypeptide comprising (i) a second TMD comprising a second interface and (ii) a second intracellular domain comprising a signaling domain, wherein the first and second engineered interfaces comprise oppositely charged amino acid residues, respectively; and wherein the first polypeptide binds to the second polypeptide through the first engineered interface and the second interface; and wherein binding of the selected ligand to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator.

[0029] In some embodiments, the first intracellular domain comprises a transcriptional regulator. In some embodiments, the transcriptional regulator comprises Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, or HAP1-VP16. In some embodiments, the transcriptional regulator is a human or humanized transcriptional regulator. In some embodiments, the transcriptional regulator is HNF1a.

[0030] In some embodiments, the first intracellular domain comprises a signaling domain, hi some embodiments, the signaling domain comprises one or more of CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, CD66d, 4-1BB, or common gamma chain signaling domains.

[0031] In some embodiments, the signaling domain of the second polypeptide comprises a signaling domain comprising a CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, CD66d, or 4-1BB signaling domain.

[0032] In some embodiments, the signaling domain of the second polypeptide comprises a cytokine signaling domain, hi some embodiments, the cytokine signaling domain comprises an IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-13R, IL-15R, or IL-21R endodomain.

[0033] In some embodiments, binding of a selected ligand to the extracellular ligand-binding domain concomitantly induces activation of the signaling domain and release of the transcriptional regulator.

[0034] In some embodiments, the first polypeptide comprises, in order from the N-terminus to the C-terminus of the first polypeptide, (i) an extracellular ligand-binding domain, (ii) a first TMD, and (iii) a first intracellular domain.

[0035] In some embodiments, the first TMD comprises (i) 10-25 consecutive valine residues, or (ii) the Notch1 transmembrane domain.

[0036] In some embodiments, the second polypeptide comprises, in order from the N-terminus to the C-terminus of the second polypeptide, (i) a second TMD, and (ii) a second intracellular domain.

[0037] In some embodiments, the first modified interface comprises a positively charged residue and the second interface comprises a negatively charged residue, and the first polypeptide is bound to the second polypeptide via electrostatic forces between the first and second modified interfaces. In some embodiments, the positively charged residue is lysine or arginine.

[0038] In some embodiments, (i) the first TMD comprises SEQ ID NO: 21 or a functional variant thereof, and the lysine or arginine residue is at a position selected from positions 10-14 of SEQ ID NO: 21, or (ii) the first TMD comprises SEQ ID NO: 18, and the lysine or arginine is at a position selected from positions 8-11 of SEQ ID NO: 18. In some embodiments, the lysine or arginine residue is (i) at position 12 of SEQ ID NO: 21, or (ii) at position 9 of SEQ ID NO: 18.

[0039] In some embodiments, the extracellular domain comprises an antigen-binding portion capable of binding to a ligand on the surface of a cell, hi some embodiments, the antigen-binding portion is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, a F(ab')2 fragment, a F(ab)2 fragment, a single-chain variable fragment (scFv), a single-domain antibody (sdAb), and functional fragments thereof.

[0040] In some embodiments, the ligand comprises a protein or a carbohydrate.

[0041] In some embodiments, the ligand is a tumor-associated antigen or a tumor-specific antigen.

[0042] In some embodiments, the ligand comprises a cell surface receptor, an adhesion protein, an integrin, a mucin, a lectin, a tumor-associated antigen, or a tumor-specific antigen.

[0043] In some embodiments, the ligand is CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein a (SIRPα).

[0044] In some embodiments, the cells are human cells.

[0045] In some embodiments, the cell is a tumor cell.

[0046] In some embodiments, the first polypeptide further comprises one or more of the following: a hinge domain, a ligand-inducible proteolytic cleavage site, an autoproteolytic peptide sequence, a nuclear localization signal, or a juxtamembrane domain. In some embodiments, the juxtamembrane domain is a polybasic domain. In some embodiments, the polybasic domain comprises a Notch-1 or Notch-2 juxtamembrane domain. In some embodiments, the autoproteolytic peptide sequence is derived from porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), or a combination thereof.

[0047] In some embodiments, the first polypeptide further comprises a hinge domain derived from CD8, CD28, OX40, or IgG4. In some embodiments, the hinge domain comprises a truncated CD8α hinge domain.

[0048] In one aspect, provided herein is a recombinant nucleic acid construct comprising, in a 5' to 3' direction, a first cassette and a second cassette, wherein the first cassette and the second cassette are joined by an autoproteolytic peptide, and wherein the first cassette encodes a first polypeptide of a multi-chain chimeric polypeptide of the disclosure, and the second cassette encodes a second polypeptide of the multi-chain chimeric polypeptide of the disclosure.

[0049] In a further aspect, provided herein is a nucleic acid construct comprising, in a 5' to 3' direction, a first cassette and a second cassette, wherein the first cassette and the second cassette are joined by an autoproteolytic peptide, and wherein the first cassette encodes a second polypeptide of a multi-chain chimeric polypeptide of the disclosure, and the second cassette encodes a first polypeptide of a multi-chain chimeric polypeptide of the disclosure.

[0050] In a further aspect, provided herein is a nucleic acid construct encoding the first polypeptide of any one of the multi-chain chimeric polypeptides of the present disclosure.

[0051] In a further aspect, provided herein is a nucleic acid construct encoding the second polypeptide of any one of the multi-chain chimeric polypeptides of the present disclosure.

[0052] In some embodiments, the autoproteolytic peptide is a Thosea asigna virus 2A (T2A) peptide.

[0053] In some embodiments, the nucleic acid construct has a nucleic acid sequence that comprises 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or a functional variant thereof.

[0054] In a further aspect, the present disclosure provides a vector comprising the recombinant nucleic acid molecule of the present disclosure. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0055] In another aspect, provided herein is a recombinant cell comprising a) a multi-chain chimeric polypeptide of the present disclosure, or b) a recombinant nucleic acid molecule of the present disclosure, or c) a vector of the present disclosure. In some embodiments, the recombinant cell is a human cell. In some embodiments, the recombinant cell is a tumor cell. In some embodiments, the recombinant cell is an immune cell. In some embodiments, the immune cell is a B cell, monocyte, natural killer cell, basophil, eosinophil, neutrophil, dendritic cell, macrophage, regulatory T cell, helper T cell, cytotoxic T cell, and other T cell. In some embodiments, the T cell is a CD4+ T cell or a CD8+ T cell.

[0056] The present disclosure also provides a pharmaceutical composition comprising a recombinant cell of the present disclosure and a pharmaceutically acceptable excipient.

[0057] Also provided herein are methods for simultaneously inducing T cell signaling and gene regulation in a T cell, the methods comprising: (a) providing a T cell having a multi-chain chimeric polypeptide or an antigen receptor of the present disclosure; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of a transcriptional regulator.

[0058] In a further aspect, the present disclosure provides methods for simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising: (a) providing (i) a vector comprising a multi-chain chimeric polypeptide of the present disclosure or (ii) a first vector comprising a first polypeptide of the present disclosure and a second vector comprising a second polypeptide of the present disclosure; and (b) transducing a T cell with the one or more vectors, wherein binding of a selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of a transcriptional regulator. In some embodiments, the induced intracellular signaling in the T cell modulates expression of selected genes involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of molecules, cell adhesion, and / or cytolytic activity.

[0059] In a further aspect, the present disclosure provides a method of inducing enhanced T cell signaling in a T cell, the method comprising: (a) providing a T cell comprising a multi-chain chimeric polypeptide of the present disclosure; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain results in enhanced intracellular signaling.

[0060] In a further aspect, the present disclosure provides a method of inducing enhanced T cell signaling in a T cell, the method comprising: (a) providing a vector comprising a multi-chain chimeric polypeptide of the disclosure, or a vector comprising a first polypeptide and a second vector comprising a second polypeptide of any one of the multi-chain polypeptides of the disclosure; and (b) transducing a T cell with the one or more vectors, wherein binding of a selected ligand to the extracellular ligand-binding domain induces enhanced intracellular signaling.

[0061] In some embodiments, the released transcriptional regulator modulates expression of a payload in the T cell. In some embodiments, the payload includes a chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA-guided nuclease, a site-specific nuclease, a T cell receptor, a toxin, a toxin-derived protein, a transcriptional regulator, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immunoinhibitor, or an inhibitory immunoreceptor.

[0062] In another aspect, provided herein are methods for treating a condition in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant cell or pharmaceutical composition of the present disclosure to the subject, wherein the recombinant cell or pharmaceutical composition treats the condition in the subject. In some embodiments, the administered recombinant cell modulates the activity of a target cell in the individual. In some embodiments, the target cell activity includes expression of selected genes involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of molecules, cell adhesion, and cytolytic activity. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is a solid tumor cell or a hematological malignancy cell. In some embodiments, the hematological malignancy cell is a multiple myeloma cell.

[0063] Also provided herein is a method for modulating T cell activity, comprising: (a) providing an effective amount of any of the recombinant cells of the present disclosure; and (b) contacting the cell with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain (i) induces cleavage of the ligand-inducible proteolytic cleavage site, releasing a transcriptional regulator, and concomitantly (ii) activates T cell signaling, wherein the released transcriptional regulator modulates the activity of the recombinant cell.

[0064] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings and detailed description, and from the claims. [Brief explanation of the drawings]

[0065] [Figure 1]Figure 1 shows a schematic diagram of a multi-chain chimeric polypeptide according to the present disclosure, having a first chain / first polypeptide (on the right) that delivers transcriptional regulation via a transcription factor and a second chain / second polypeptide (on the left) that delivers a signaling function. The first polypeptide comprises an extracellular ligand-binding domain with binding affinity for a selected ligand, a first TMD with a first modified interface (positively charged amino acid residues), and a first ICD with a transcriptional regulator. The second polypeptide comprises a second TMD with a second interface (negatively charged amino acid residues) and a second ICD with a signaling domain. Binding between the first and second polypeptides is via interactions between positively and negatively charged amino acid residues. Activation of the multi-chain chimeric polypeptide by binding to a ligand (e.g., CD19) results in the release of the transcriptional regulator (e.g., Gal4VP64), which then enters the nucleus and activates transcription of a gene (in this example, BFP). A schematic diagram of the transcriptional regulator response cassette using the BFP gene is also shown.

[0066] [Figure 2A]Figures 2A-D graphically summarize the results from experiments conducted to evaluate the functionality of various chimeric multi-chain polypeptides / receptors to optimize a multi-chain chimeric receptor that induces activation of the signaling domain and release of the transcriptional regulator upon ligand binding to the extracellular ligand-binding domain. Figure 2A shows an embodiment of a multi-chain chimeric polypeptide (e.g., an immunoreceptor) designated 056. The 056 embodiment includes (i) a first polypeptide having a CD19scFv binding domain, TREM2, a first TMD with a positively charged first modified interface, and a Gal4VP64 translational regulator, and (ii) a second polypeptide chain comprising DAP12 with a negatively charged modified interface. The nucleic acid construct encoding the 056 embodiment (SEQ ID NO: 2) includes, from 5' to 3', a cassette encoding DAP12, a T2A linker, and a first polypeptide that is CD19scFV-TREM2-Gal4VP64. Figure 2B shows another design of a multi-chain chimeric polypeptide designated 056C. The 056C embodiment comprises: (i) a first polypeptide comprising a CD19scFv binding domain, a truncated CD8 α-hinge domain (denoted CD8hinge2), a first TMD with a positively charged first modified interface that is Notch1 with an L1747K (Uniprot P46531 numbering, Uniprot P46531 amino acids 1736-1757 with the L1747K mutation) substitution, a Notch2 juxtamembrane domain, and a Gal4VP64 translational regulator; and (ii) a second polypeptide chain comprising DAP12. A nucleic acid construct encoding the 056C embodiment (SEQ ID NO:4) includes, in the 5' to 3' direction, DAP12, a T2A linker, and a cassette encoding a first polypeptide having CD19scFV, a truncated CD8α hinge domain (CD8 hinge 2), Notch1 with an L1747K substitution (Uniprot P46531 amino acids 1736-1757 with the L1747K mutation), a Notch2 juxtamembrane domain, and a Gal4VP64 transcriptional regulator.Figure 2C shows a different design (056D) of a multi-chain chimeric polypeptide having (i) a first polypeptide chain comprising a CD19scFv binding domain, a TREM2 hinge, a first TMD with a positively charged first modified interface that is Notch1 with an L1747K substitution (Uniprot P46531 amino acids 1736-1757 with the L1747K mutation), a Notch2 juxtamembrane domain, and a Gal4VP64 translational regulator; and (ii) a second polypeptide chain comprising DAP12. The nucleic acid construct (SEQ ID NO: 5) encoding the 056D embodiment comprises, from 5' to 3', a cassette encoding DAP12, a T2A autoproteolytic peptide sequence, and a first polypeptide chain that is CD19scFV-TREM2-Notch1 with an L1747K substitution (Uniprot P46531 amino acids 1736-1757 with the L1747K mutation)-Gal4VP64. Figure 2D shows a multi-chain chimeric polypeptide designated 056B (056B) having (i) a first polypeptide having a first TMD with a positively charged first modified interface that is Notch1 containing a CD19scFv binding domain, a TREM2 hinge, and a gamma-secretase active site, a Notch2 juxtamembrane domain, and a Gal4VP64 translational regulator, and (ii) a second polypeptide chain that includes DAP12. The nucleic acid construct encoding the 056B embodiment (SEQ ID NO: 3) includes, from 5' to 3', DAP12, a T2A autoproteolytic peptide sequence, and a cassette encoding a first polypeptide that is CD19scFV-TREM2-Notch1 insert containing a gamma-secretase active site-TREM2 intracellular domain sequence-Gal4VP64. A comparison of the expression levels of the four multi-chain receptors, 056, 056C, 056D, and 056B, is also shown. Of the four designs, 056C has the strongest transcriptional activity. The bottom panel of each of Figures 2A-2D shows the relative expression of the four receptors (designated second chain) (y-axis) measured by anti-myc-tag staining versus reporter construct expression measured by GFP (x-axis).Specifically, primary human CD3+ T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Gibco) and transduced with one lentiviral construct (two lentiviral constructs could also be used, with one lentivirus encoding the first polypeptide of the multi-chain chimeric polypeptide and the second lentivirus encoding the second polypeptide of the multi-chain chimeric polypeptide). The DAP12 / TREM immunoreceptor (056) and DAP12 / hinge-Notch hybrid (056C) showed some expression, whereas the DAP12 / TREM-Notch hybrid 1 (056D) and DAP12 / TREM-Notch hybrid 2 (056B) showed no expression. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above.

[0067] [Figure 3-1] Figures 3A-C show a comparison of the design (Figure 3A), activation dynamics (Figure 3B), and killing activity (Figure 3C) of the 056 multi-chain chimeric polypeptide described in Figure 2A and the 056C multi-chain chimeric polypeptide. Figure 3B shows receptor transcriptional activation of an inducible BFP reporter gene (measured using a Fortessa X-50 (BD Sciences) in T cells expressing the anti-CD19 receptor using (i) no additional cells (top trace), (ii) K562 cells (middle trace), or (iii) CD19+ K562 cells (bottom trace)). Figure 3C also shows a comparison of the killing activity of the 056 receptor and the 056C receptor. The 056C receptor has higher transcriptional activity than the 056 receptor, but does not result in a potent killing response. [Figure 3-2] Same as above.

[0068] [Figure 4A]Figures 4A-4D illustrate additional multi-chain chimeric polypeptide designs (056, 056E, 056F, and 056G). Figure 4A shows the same receptor 056 as in Figure 2A, and Figure 4B shows a multi-chain chimeric polypeptide design (056E) in which the second polypeptide contains the DAP12 and Gal4VP64 transcriptional regulators, and the first polypeptide contains the CD19scFv ligand-binding domain, TREM2 transmembrane and intracellular domains. The 056E receptor is encoded by a nucleic acid construct containing a Gal4VP64 cassette linked (by a T2A linker) to a cassette containing DAP12, CD19scFV, and the TREM2 transmembrane and intracellular domains. Such designs have limited relative expression. Figure 4C shows an embodiment (056F) of a multi-chain chimeric polypeptide according to the present disclosure, in which a first polypeptide comprises a CD19scFV ligand-binding domain, a truncated CD8α hinge domain (CD8 hinge 2), a polyvaline TMD with a V9K substitution, a Notch 2 juxtamembrane domain, and a Gal4VP64 transcriptional regulator, and a second polypeptide comprises a DAP12 signaling domain. The 056F embodiment is encoded by a nucleic acid construct having, from 5' to 3', DAP12 followed by a T2A autoproteolytic peptide sequence and a cassette comprising a) a truncated CD8α hinge domain (CD8 hinge 2), b) a polyvaline TMD with a V9K substitution, c) a Notch 2 juxtamembrane domain, and d) a Gal4VP64 transcriptional regulator. Figure 4D shows an embodiment (056G) similar to the 056F embodiment, but with a polyvaline TMD with a V10K substitution. The 056F and 056G embodiments, which have a polyvaline TMD, show increased expression compared to the other embodiments. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above.

[0069] [Figure 5-1]Figures 5A-D show a comparison of the killing activity (target clearance) and activation dynamics of embodiments 056, 056E, 056F, and 056G of the multi-chain polypeptides described in Figures 4A-D. Embodiments 056 and 056E do not exhibit strong transcriptional activation, but can exhibit killing activity against target cells. Embodiments 056F and 056G exhibit strong transcriptional activation, but only 056F achieves target killing. [Figure 5-2] Same as above.

[0070] [Figure 6] FIG. 6 shows a schematic diagram of the multi-chain receptor modular engineering strategy using pRay056F as a prototype.

[0071] [Figure 7A] FIG. 7 shows testing of multichain receptor function with CD3z substitutions. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above.

[0072] [Figure 8A] FIG. 8 shows testing of multichain receptor function with human transcription factors. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above.

[0073] [Figure 9] FIG. 9 shows a schematic diagram of a multi-chain receptor using a modular engineering strategy using dual vector transduction.

[0074] [Figure 10-1] FIG. 10 shows the expression profile of each multi-chain receptor pair. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above. [Figure 10-7] Same as above. [Figure 10-8] Same as above.

[0075] [Figure 11] Figure 11 shows a screen for multi-chain receptors that enhance killing and survival. The graph shows T cell counts at days 5, 12, and 20 at an ET ratio of 1:1.

[0076] [Figure 12] Figure 12 shows a screen for multi-chain receptors that enhance killing and survival. The graph shows T cell counts at days 5, 12, and 20 at an ET ratio of 1:2.

[0077] [Figure 13] Figure 13 shows a screen for multi-chain receptors that enhance killing and survival. The graph shows T cell counts at days 5, 12, and 20 at an ET ratio of 1:4.

[0078] [Figure 14] Figure 14 shows a screen for multi-chain receptors that enhance killing and survival. The graph shows K562 cell counts at days 5, 12, and 20 at an ET ratio of 1:1.

[0079] [Figure 15] Figure 15 shows a screen for multi-chain receptors that enhance killing and survival. The graph shows K562 cell counts at days 5, 12, and 20 at an ET ratio of 1:2.

[0080] [Figure 16] Figure 16 shows a screen for multi-chain receptors that enhance killing and survival. The graph shows K562 cell counts at days 5, 12, and 20 at an ET ratio of 1:4. DETAILED DESCRIPTION OF THE INVENTION

[0081] Detailed Description of the Disclosure In the field of chimeric polypeptides (e.g., receptors), it is widely known that when adding a signaling domain, proximity to the membrane is important and affects how well the domain signals. The present disclosure provides chimeric polypeptides / receptors with configurations different from those generally known in the art, allowing for greater control over the location of signaling motifs. The membrane association of the chimeric polypeptides / receptors of the present disclosure is much more controllable than previously known.

[0082] Thus, the present disclosure provides, inter alia, multi-chain chimeric polypeptides having two separate polypeptides (chains) such that a transcriptional factor is located on a first polypeptide near the membrane and a signaling domain is located on a second polypeptide. The first and second polypeptides can associate such that binding of a selected ligand to one of the polypeptides simultaneously allows modulation of signaling (e.g., T cell signaling) by one chain and transcriptional regulation by the other chain in a ligand-dependent manner.

[0083] In particular, the present disclosure relates to multi-chain chimeric polypeptides having separate polypeptide chains post-translationally associated via residues located within the TMDs of the first and second polypeptides. Ligand binding to the extracellular ligand-binding domain (ECD) of the first polypeptide can activate cell signaling and cleave and release a transcriptional regulator from the first polypeptide. Thus, the cleaved transcriptional regulator can function independently of any of the other fused signaling domains, and vice versa.

[0084] In some embodiments, the multi-chain chimeric polypeptides provided herein may have the following unique configuration: A first polypeptide may include (i) an extracellular ligand-binding domain having binding affinity (specificity) for a selected ligand, (ii) a first TMD having a first modified interface, and (iii) a first intracellular domain having a transcriptional regulator. A second polypeptide may include (i) a second TMD having a second interface and (ii) a second intracellular domain having a signaling domain. The first and second polypeptides may associate through charged residues within the first modified interface and the second interface. Binding of a selected ligand to the extracellular ligand-binding domain may simultaneously induce activation of the signaling domain and release of the transcriptional regulator.

[0085] The present disclosure further provides compositions comprising the chimeric antigen receptors and nucleic acid constructs encoding same, host cells genetically modified with the nucleic acid constructs, pharmaceutical compositions and methods for modulating cellular immune responses or activity, methods for inducing T cell signaling, and methods for treating various conditions, e.g., diseases (e.g., cancer).

[0086] The multi-chain chimeric polypeptides of the present disclosure constitute a platform for engineering T cells that can not only detect and activate responses to tumors but also turn on one or more transcriptional programs, such as the production of various biological substances, cytokines, inflammatory factors, regulatory RNAs, etc. The multi-chain chimeric polypeptides of the present disclosure make it possible to link receptor capacity with defined accessory functions that are not typically encoded or triggered by the signaling domains incorporated into prior art synthetic receptors. Thus, the multi-chain chimeric polypeptides of the present disclosure may enable an expanded approach to positively modify T cell function in ways different from those previously known. The multi-chain chimeric polypeptides of the present disclosure can also be used to deliver payloads such as chemokines to recruit different immune cells for tumor control or to direct T cell fate toward more effective subtypes through the expression of transcription factors or other factors that control differentiation.

[0087] The design of the multi-chain polypeptides of the present disclosure facilitates and enables more immediate signaling to receptors where antigen recognition was previously coupled only to a transcriptional response (i.e., without other signaling effects). By incorporating signaling domains into the multi-chain formats of the present disclosure, one skilled in the art can effectively couple accessory functions to the transcriptional response, resulting in activation of both simultaneously and in response to the same antigen. Accessory functions can include, for example, cell destruction (by using immunoreceptor tyrosine-based activation motif (ITAM) domains in the signaling chain) or promoting survival signaling by using costimulatory domains (e.g., 41BB, CD28 costimulatory domain, etc.).

[0088] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like numerals generally identify like elements unless the context dictates otherwise. Illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects as generally described herein and illustrated in the drawings may be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are expressly contemplated and form a part of this application.

[0089] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly utilized by those skilled in the art using conventional methodology.

[0090] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. As used herein, "A and / or B" is used to include all of the following alternatives: "A," "B," "A or B," and "A and B."

[0091] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limits within the stated range. Where a stated range includes one or both limits, ranges excluding one or both of those included limits are also included within the disclosure. Certain ranges are presented herein with numerical values ​​preceded by the term "about," and as used herein, this term has its ordinary meaning of approximately. The term "about" is used to provide literal support for the exact number it precedes, as well as for numbers that are near or approximately the number preceded by the term. When determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number. If the degree of approximation is not clear from the context, "about" means within plus or minus 10% of the provided value in all cases inclusive of the provided value, or rounded to the nearest significant figure. In some embodiments, the term "about" refers to up to ±10%, up to ±5%, or up to ±1% of the specified value.

[0092] The terms "administration" and "administering," as used herein, refer to the delivery of a bioactive composition or formulation by a route of administration, including, but not limited to, oral, intranasal, transdermal, intravenous, intraarterial, intramuscular, intranodal, intraperitoneal, subcutaneous, and intramuscular administration, or a combination thereof. This term includes, but is not limited to, administration by a medical professional and self-administration.

[0093] Terms such as "cell," "cell culture," and "cell line" are understood to refer not only to the particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of culture transfers or passages. It is understood that not all progeny will be completely identical to the parent cell. This is because certain modifications may occur in subsequent generations, either due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that the progeny may not actually be identical to the parent cell, but are still within the scope of the term as used herein, so long as the progeny retain the same functionality as the original cell, cell culture, or cell line.

[0094] It is understood that aspects and embodiments of the present disclosure described herein include multiple aspects and embodiments "comprising," "consisting," and "consisting essentially of." As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising," particularly in a description of a component of a composition or a description of a step of a method, is understood to encompass compositions and methods that consist essentially of, and consist of, the recited components or steps.

[0095] The term "cancer" refers to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Some types of cancer cells may aggregate into a mass like a tumor, while some cancer cells may exist singly within a subject. A tumor may be a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term "cancer" also encompasses other types of non-tumor cancers. Non-limiting examples include blood cancers or hematological malignancies, such as leukemia, lymphoma, and myeloma. Cancers may include pre-malignant and malignant cancers.

[0096] The term "nucleic acid" is used herein in reference to either DNA or RNA, or molecules containing deoxynucleotides and / or ribonucleotides. Nucleic acids can be naturally occurring or synthetically produced, and thus include analogs of naturally occurring polynucleotides in which one or more nucleotides are modified relative to the naturally occurring nucleotides.

[0097] As used herein, the term "operably linked" refers to a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, permitting them to function in their intended manner.

[0098] As used herein in the context of two or more nucleic acid sequences or proteins, the term "percent identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides or amino acids (e.g., about 50% or more sequence identity over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region, e.g., about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) that are the same, as measured, for example, using the BLAST or BLAST 2.0 sequence comparison algorithms of The National Center for Biotechnology (NCBI) with the default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to or can be applied to the complement of a sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Sequence identity can be calculated using published techniques and publicly available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990, which is hereby incorporated by reference in its entirety). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with default parameters.For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the reference amino acid sequence, including all values ​​therebetween: For polypeptides, the length of comparison sequences is generally at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 50, at least 75, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, or at least 350 contiguous amino acids (e.g., full-length sequences), including all values ​​therebetween. For nucleic acids, the length of comparison sequences will generally be at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or 25 contiguous nucleotides, including all values ​​therebetween (e.g., full-length nucleotide sequences).

[0099] As used herein, a "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient under the care of a physician. Thus, a subject may be a human patient or individual who has, is at risk of, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject may also be an individual who has been diagnosed as having a risk of a condition of interest at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, such as mammals, e.g., rodents, e.g., mice, and non-mammals, e.g., non-human primates, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0100] When used in reference to nucleic acids, the term "recombinant" means that the nucleic acid has been altered or produced by human intervention, e.g., modified by or as a result of laboratory methods. Thus, for example, recombinant nucleic acids include viral genomes and nucleic acids produced by laboratory methods. Recombinant proteins or polypeptides produced by recombinant constructs may contain amino acid residues not found in the native (non-recombinant or wild-type) form of the protein or may contain amino acid residues that are modified, e.g., labeled. The term may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include: any chemical modification of a peptide, protein, or nucleic acid sequence, including one or more amino acids, deoxyribonucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; the creation of fusion proteins, e.g., fusion proteins including antibody fragments; and the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence.

[0101] As used herein, the term "recombinant" polypeptide refers to a polypeptide that has been altered by human intervention. As non-limiting examples, an engineered polypeptide can be: 1) one that has been synthesized or modified in vitro, e.g., using chemical or enzymatic techniques; 2) one that contains a linked polypeptide sequence that is not naturally linked; 3) one that has been engineered using molecular cloning techniques to lack one or more amino acids relative to a naturally occurring polypeptide sequence; and / or 4) one that has been engineered using molecular cloning techniques to have one or more sequence changes or rearrangements relative to a naturally occurring polypeptide.

[0102] As will be understood by those skilled in the art, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges within that range. Any recited range can be recognized as fully descriptive and allowing for that same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than," etc., is inclusive of the recited numbers and refers to a range that can be subsequently divided into subranges, as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0103] It is understood that certain features of the present disclosure that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0104] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0105] II. Composition A. Multi-chain Chimeric Polypeptides The present disclosure provides, among other things, multi-chain chimeric polypeptides comprising two polypeptide chains. A multi-chain polypeptide can be a functional polypeptide composed of two or more separate polypeptide elements (i.e., "chains") covalently or non-covalently linked by molecular associations other than peptide bonds. The chains of a multi-chain polypeptide can be structurally distinct. The chains of a multi-chain polypeptide can be functionally distinct. The present disclosure provides multi-chain chimeric polypeptides in which one of the polypeptides (a first polypeptide) comprises (i) an extracellular ligand-binding domain having binding affinity for a selected ligand, (ii) a first TMD, and (iii) a first intracellular domain having a transcriptional regulator. The other polypeptide (a second polypeptide) comprises an intracellular domain having (i) a TMD and (ii) a signaling domain. Ligand binding to the extracellular ligand-binding domain activates the first polypeptide, subsequently resulting in release of the transcriptional regulator from the first polypeptide and cellular signaling via the signaling domain on the second polypeptide. The two polypeptides are associated, for example, by oppositely charged residues in the TMD.

[0106] The multi-chain chimeric polypeptides disclosed herein can function as receptors under certain cellular and environmental conditions, facilitating amplified activation of cell signaling or cellular pathways. Such receptor activity can be exploited to enhance and modulate the production of therapeutic payloads by cells engineered / transduced to express the multi-chain chimeric polypeptides or receptors of the present disclosure.

[0107] Thus, in one aspect, provided herein is a multi-chain chimeric polypeptide comprising: (a) a first polypeptide comprising (i) an extracellular ligand-binding domain having binding affinity for a selected ligand, (ii) a first TMD having a first engineered interface, and (iii) a first intracellular domain having a transcriptional regulator; and (b) a second polypeptide comprising (i) a second TMD having a second interface and (ii) a second intracellular domain having a signaling domain, wherein the first and second engineered interfaces comprise oppositely charged amino acid residues, respectively, such that the first polypeptide binds to the second polypeptide through the first and second engineered interfaces, and binding of the selected ligand to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments of the multi-chain chimeric polypeptide of the present disclosure, the first engineered interface comprises positively charged residues and the second interface comprises negatively charged residues, and the first polypeptide binds to the second polypeptide through electrostatic forces between the first and second engineered interfaces.

[0108] In a further aspect, provided herein are multi-chain chimeric polypeptides that can allow for simultaneous transcriptional regulation and cellular activation (e.g., T cell activation) upon binding of a selected ligand to the extracellular ligand-binding domain of a first polypeptide of the multi-chain chimeric polypeptide of the present disclosure.

[0109] In some embodiments, the multi-chain chimeric polypeptide of the present disclosure can be an immunoreceptor. In some embodiments, the immunoreceptor is a chimeric antigen receptor (CAR).

[0110] 1. First Polypeptide The multi-chain polypeptides of the present disclosure include a first chimeric polypeptide having an extracellular ligand-binding domain (portion) having binding affinity for a selected ligand, a first TMD having a first modified interface, a first intracellular domain, and one or more transcriptional regulators. As used herein, "interface" refers to the interacting outward-facing residues in the transmembrane domain of a first polypeptide and the outward-facing residues in the transmembrane domain of another polypeptide. In some embodiments, the outward-facing residues in the transmembrane domain of the first polypeptide form a charged surface, thereby interacting with a partner interface from another protein presenting an oppositely charged surface.

[0111] In some embodiments, a first polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, (i) an extracellular ligand-binding domain, (ii) a first TMD, and (iii) a second TMD.

[0112] In some embodiments, the first polypeptide of the present disclosure comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8 or SEQ ID NO:10 or a functional variant thereof.

[0113] a. Extracellular ligand-binding domain (ECD) In some embodiments, the extracellular domain (ECD) of the first polypeptide of the multi-chain chimeric polypeptide disclosed herein has binding affinity for one or more target ligands. The target ligand may be expressed on the cell surface or may be tethered, immobilized, or constrained in other ways so that it can exert a mechanical force on the chimeric receptor. Thus, without being bound by any particular theory, binding of the ECD of the first polypeptide of the multi-chain chimeric polypeptide provided herein to a cell surface ligand does not necessarily remove the target ligand from the target cell surface, but rather exerts a mechanical attractive force on the chimeric receptor. For example, an otherwise soluble ligand can be targeted if it is bound to a molecule on the surface or in the extracellular matrix.

[0114] In some embodiments, the targeting ligand is a cell surface ligand. Non-limiting examples of suitable ligand types include cell surface receptors; adhesion proteins; surface-bound carbohydrates, lipids, glycolipids, lipoproteins, and lipopolysaccharides; integrins, mucins; and lectins. In some embodiments, the ligand is a protein. In some embodiments, the ligand includes tumor-associated antigens or tumor-specific antigens. In some embodiments, the ligand is a cluster of differentiation ligand (CD), including CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD These include, but are not limited to, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein a (SIRPα).

[0115] In some embodiments, the extracellular domain of the first polypeptide of the multi-chain chimeric polypeptide of the present disclosure comprises an antigen-binding portion. In some embodiments, the antigen-binding portion can bind to a ligand on the surface of a cell. In some embodiments, the antigen-binding portion can bind to one or more targets. In some embodiments, the antigen-binding portion comprises one or more antigen-binding determinants of an antibody or functional antigen-binding fragment thereof. After reading this disclosure, one of skill in the art will readily understand that the terms "functional fragment thereof" or "functional variant thereof" refer to a molecule that shares quantitative and / or qualitative biological activity with the wild-type molecule from which the fragment or variant is derived. For example, a functional fragment or functional variant of an antibody retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment or functional variant is derived. For example, an antibody capable of binding to an epitope of a cell surface receptor can be truncated at the N-terminus and / or C-terminus, and retention of its epitope-binding activity can be assessed using assays known to those of skill in the art. The antigen-binding portion can be, but is not limited to, an antibody, a nanobody, a diabody, a triabody, a minibody, a F(ab')2 fragment, a F(ab) fragment, a single-chain variable fragment (scFv), a single-domain antibody (sdAb), and any functional fragment thereof.

[0116] The antigen-binding portion may comprise a naturally occurring amino acid sequence or may be engineered, designed, or modified to provide desired and / or improved properties, such as binding affinity. Generally, the binding affinity of an antigen-binding portion, such as an antibody, to a target antigen (e.g., CD19 antigen) can be calculated by the Scatchard method described by Frankel et al., Mol. Immunol, 16: 101-06, 1979. In some embodiments, the binding affinity is measured by antigen / antibody dissociation rate. In some embodiments, the binding affinity is measured by competitive radioimmunoassay. In some embodiments, the binding affinity is measured by ELISA. In some embodiments, the antibody affinity is measured by flow cytometry.

[0117] An antibody that "selectively binds" to an antigen (e.g., CD19) is an antigen-binding portion that binds to that antigen with high affinity, e.g., with an equilibrium constant (KD) of 100 nM or less, e.g., 60 nM or less, e.g., 30 nM or less, e.g., 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less, but does not significantly bind to other antigens.

[0118] The ECD can be selected by those skilled in the art based on the desired localization or function of the cells genetically modified to express the multi-chain chimeric polypeptide or first polypeptide of the present disclosure. For example, a first polypeptide having an ECD comprising an antibody specific to the HER2 antigen can target cells to HER2-expressing breast cancer cells. In some embodiments, the ECD of the first polypeptide of the present disclosure can bind to a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). Those skilled in the art will understand that TAAs include molecules, such as proteins, that are present in tumor cells and normal cells, or that are present in many normal cells but at much lower concentrations than in tumor cells. In contrast, TSAs generally include molecules, such as proteins, that are present in tumor cells but not in normal cells.

[0119] In some cases, the antigen-binding portion is specific for an epitope present in an antigen expressed by tumor cells, i.e., a tumor-associated antigen. Tumor-associated antigens can be, for example, antigens associated with breast cancer cells, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma, glioblastoma, colorectal cancer cells, etc. It will also be understood that tumor-associated antigens can be expressed by non-cancerous cells. In some embodiments, the antigen-binding domain is specific for an epitope present in a tissue-specific antigen. In some embodiments, the antigen-binding domain is specific for an epitope present in a disease-associated antigen.

[0120] Non-limiting examples of suitable target antigens include CD19, B7H3 (CD276), BCMA (CD269), alkaline phosphatase, placenta-like 2 (ALPPL2), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein alpha (SIRPα), CD123, CD171, CD179 alpha, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, and CLECL1. , CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-11R alpha, KIT (CD117), MUC1, NCAM, PAP, PDGFR beta, PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.

[0121] In some embodiments, the target antigen is CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179.alpha., CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC 5D, HER2 (ERBB2 / neu), IGLL1, IL-11Ra, KIT (CD117), MUC1, NCAM, PAP, PDGFR-beta, PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, Axl, GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL-13R-alpha 1, IL-13R-alpha 2, alpha.Anti-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (Gross cystic disease fluid protein) Protein) (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (subunit of heterodimeric IL-2 receptor), CD3, CD4, CD5, IFN-alpha, IFN-gamma, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin alpha 4, integrin alpha 4 beta 7, LFA-1 (CD11 alpha), myostatin, OX-40, sclerostin, SOST, TGF beta 1, TNF- alpha, VEGF-A, pyruvate kinase isoenzyme M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBC1, TRBC2, BCMA, CD38, CD123, CD93, CD34, CD1a, SLAMF7 / CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, kappa light chain, lambda light chain, CD16 / Fc gamma.The antibody is selected from RIII, CD64, FITC, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and its isovariants, TEM-8, sperm protein 17 (Sp17), and mesothelin.

[0122] Further non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T-cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate six-transmembrane epithelial antigen 1), aberrant Ras proteins, aberrant p53 proteins, integrin (33 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), Ral-B, GPC2, CD276 (B7H3), or IL-13R.alpha. In some embodiments In some embodiments, the antigen is Her2. In some embodiments, the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen-binding portion of the ECD is specific for a reporter protein such as GFP and eGFP. Non-limiting examples of such antigen-binding portions include the LaG17 anti-GFP nanobody. In some embodiments, the antigen-binding portion of the ECD comprises an anti-BCMA fully humanized VH domain (FHVH). In some embodiments, the antigen is signal regulatory protein alpha (SIRP alpha).

[0123] Additional antigens suitable for targeting by the first chimeric polypeptide and multi-chain CAR disclosed herein include GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL-13R.alpha.1, IL-13R.alpha.2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA). Other suitable target antigens include, but are not limited to, tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), macroscopic cystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, and thyroid transcription factor-1.

[0124] Additional antigens suitable for targeting by the polypeptides disclosed herein include antigens associated with inflammatory diseases, such as AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (dimeric subunit of the IL-2 receptor), CD3, CD4, CD5, IFN-alpha, IFN-gamma, IgE, and IgE. These include, but are not limited to, Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin alpha 4, integrin alpha 4 beta 7, LFA-1 (CD11 alpha), myostatin, OX-40, sclerostin, SOST, TGF beta 1, TNF alpha, and VEGF-A.

[0125] Additional antigens suitable for targeting by the first chimeric polypeptide and multi-chain CAR disclosed herein include, but are not limited to, pyruvate kinase isozyme type M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBC1, TRBC2, BCMA, CD38, CD123, CD93, CD34, CD1a, SLAMF7 / CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, kappa light chain, lambda light chain, CD16 / Fc.gamma.RIII, CD64, FITC, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and its isovariants, TEM-8, sperm protein 17 (Sp17), and mesothelin. Further non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T-cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate six-transmembrane epithelial antigen 1), aberrant Ras protein, aberrant p53 protein, integrin beta 3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B. In some embodiments, the antigen is GPC2, CD19, Her2 / neu, CD276 (B7H3), IL-13R alpha 1, or IL-13R alpha 2. In some embodiments, the antigen is Her2. In some embodiments, the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen-binding portion of the ECD is specific for a reporter protein such as GFP and eGFP. Non-limiting examples of such antigen-binding portions include the LaG17 anti-GFP nanobody. In some embodiments, the antigen-binding portion of the ECD includes an anti-BCMA fully humanized VH domain (FHVH).

[0126] In some embodiments, the antigen may be HER2, which is produced by HER2-positive breast cancer cells. In some embodiments, the antigen may be CD19, which is expressed in B-cell leukemia. In some embodiments, the antigen may be EGFR, which is expressed in glioblastoma multiforme (GBM) but is expressed much less in healthy CNS tissue. In some embodiments, the antigen may be CEA, which is associated with cancer in adults, such as colon cancer.

[0127] In some embodiments, the antigen-binding portion of the ECD is specific for a cell surface target, non-limiting examples of which include CD19, CD30, Her2, CD22, ENPP3, EGFR, CD20, CD52, CD11α, and α-integrin. In some embodiments, the chimeric first polypeptide and multi-chain CAR disclosed herein comprise an extracellular domain having an antigen-binding portion that binds to CD19, CEA, HER2, MUC1, CD20, ALPPL2, BCMA, or EGFR. In some embodiments, the multi-chain CAR provided herein comprises an extracellular domain comprising an antigen-binding portion that binds to CD19. In some embodiments, the chimeric first polypeptide provided herein comprises an extracellular domain comprising an antigen-binding portion that binds to ALPPL2. In some embodiments, the chimeric first polypeptide provided herein comprises an extracellular domain comprising an antigen-binding portion that binds to BCMA. In some embodiments, the chimeric first polypeptide comprises an extracellular domain comprising an antigen-binding portion that binds to Her2.

[0128] In some embodiments, antigens suitable for targeting by a chimeric first polypeptide disclosed herein include ligands derived from pathogens.

[0129] b. The linking sequence between the ECD and the first transmembrane domain (TMD) The multi-chain chimeric polypeptides and receptors of the present disclosure can include a linking sequence disposed between the ECD and TMD. The linking sequence can be a natural or synthetic polypeptide. The linking sequence can be a flexible connector that provides structural flexibility and spacing to the flanking polypeptide regions. The linking sequence can be a hinge domain inserted N-terminal to the TMD. In some embodiments, the hinge domain is a CD8 hinge domain. In some embodiments, the CD8 hinge domain is a truncated CD8α hinge domain. In some embodiments, the truncated CD8α is encoded by an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17. In some embodiments, the truncated CD8α comprises an amino acid sequence comprising SEQ ID NO: 17. In some embodiments, the truncated CD8α is encoded by SEQ ID NO: 17.

[0130] Hinge polypeptide sequences suitable for the compositions and methods of the present disclosure may be naturally occurring hinge polypeptide sequences (e.g., derived from naturally occurring immunoglobulins). Alternatively, the hinge polypeptide sequence may be a synthetic sequence corresponding to a naturally occurring hinge polypeptide sequence, or may be a completely synthetic hinge sequence that has been engineered, designed, or modified to provide desired and / or improved properties, such as transcriptional modulation. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as the IgG1 hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or functional variants thereof. In some embodiments, the hinge polypeptide sequence contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence contains one or more CPPC motifs. Additional information in this regard can be found, for example, in a recent review by Vidarsson G. et al., Frontiers Immunol. Oct. 20, 2014, which is incorporated herein by reference in its entirety.

[0131] Thus, in some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from an IgG1 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from an IgG2 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from an IgG3 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from an IgA hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from an IgD hinge domain or a functional variant thereof.

[0132] Additional hinge polypeptide sequences suitable for the compositions and methods disclosed herein include, but are not limited to, hinge polypeptide sequences derived from the CD8α hinge domain, CD28 hinge domain, CD152 hinge domain, PD-1 hinge domain, CTLA4 hinge domain, OX40 hinge domain, Fcγ RIIIα hinge domain, and functional variants thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the CD8.alpha hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the OX40 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from the IgG4 hinge domain or a functional variant thereof.

[0133] In principle, there are no particular limitations on the length and / or amino acid composition of the hinge domain, other than the flexibility provided by the hinge domain. However, it will be readily apparent to those skilled in the art that the orientation and / or proximity of the ECD and TMD relative to each other, and the orientation and / or proximity of the first and second polypeptides relative to each other, can be varied to optimize the length and amino acid composition of the hinge polypeptide sequence to achieve the desired activity of the chimeric multi-chain polypeptide of the present disclosure. In some embodiments, any single peptide chain containing about 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues, etc.) can be used as the hinge domain. In some embodiments, the hinge domain comprises about 5-50, about 10-60, about 20-70, about 30-80, about 40-90, about 50-100, about 60-80, about 70-100, about 30-60, about 20-80, or about 30-90 amino acid residues. In some embodiments, the hinge domain comprises about 1-10, about 5-15, about 10-20, about 15-25, about 20-40, about 30-50, about 40-60, or about 50-70 amino acid residues. In some embodiments, the hinge domain comprises about 40-70, about 50-80, about 60-80, about 70-90, or about 80-100 amino acid residues. In some embodiments, the hinge domain comprises about 1-10, about 5-15, about 10-20, or about 15-25 amino acid residues. In some embodiments, the hinge domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 17, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity.

[0134] c. first transmembrane domain The multi-chain chimeric polypeptides of the present disclosure include a transmembrane domain (e.g., a first TMD) located within a first polypeptide. In some embodiments of the first polypeptide of the present disclosure, the first TMD is completely synthetic. In some embodiments, the first TMD includes a contiguous stretch of valine residues. In some embodiments, the contiguous stretch of valine residues includes 5-25 valine residues. In some embodiments, the first TMD includes a contiguous stretch of 5-25 valine residues. In some embodiments, the first TMD includes a contiguous stretch of 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25 consecutive valine residues. An example of a first TMD having a contiguous stretch of valine residues (referred to as a poly-V TMD) is shown in Figures 4C and 4D, which show embodiments 056F and 056G of the multi-chain chimeric polypeptide of the present disclosure, respectively.

[0135] In some embodiments, the first TMD comprises a positively charged residue. In some embodiments, the positively charged residue is lysine or arginine. In some embodiments, the positively charged residue is present within a contiguous stretch of valine residues. In some embodiments, the positively charged residue is located after the first valine residue from the N-terminus. In some embodiments, the positively charged residue is located after the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-first, twenty-second, or twenty-third valine residue from the N-terminus. In some embodiments, the sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth valine residue from the N-terminus is replaced by a lysine or arginine residue. In some embodiments, the TMD of the first polypeptide comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the TMD is SEQ ID NO: 18. In some embodiments, the lysine or arginine residue is at or is at a position corresponding to residue 8, 9, 10, or 11 of SEQ ID NO: 18.

[0136] The first TMD of the multi-chain chimeric polypeptide of the present disclosure can comprise a TMD derived from a Notch receptor. In some embodiments, the TMD comprises a Notch1 receptor (see Figures 2B-2D, which show embodiments 056C, 056D, and 056B of the multi-chain chimeric polypeptide of the present disclosure). In some embodiments, the Notch1 receptor is a human Notch1 receptor.

[0137] The first TMD can include one or more ligand-inducible proteolytic cleavage sites. Examples of ligand-inducible proteolytic cleavage sites (e.g., S2 or S3) in Notch receptors are described in U.S. Patent No. 11,202,801, the entire contents of which are incorporated herein by reference. In some embodiments, the ligand-inducible proteolytic cleavage site is cleavable by gamma secretase, a multiprotein enzyme complex, as in embodiment 056B (FIG. 2D).

[0138] The first polypeptide of the multi-chain chimeric polypeptide of the present disclosure can comprise a juxtamembrane domain (JMD). The JMD can be located C-terminal to the TMD. The JMD can comprise a highly charged domain. In some embodiments, the JMD is a Notch2 JMD.

[0139] In principle, there are no particular limitations regarding the length and / or amino acid composition of the JMD. Any single-chain peptide containing about 4 to about 40 amino acid residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues) can be used as a JMD. In some embodiments, the JMD contains about 4 to 15, about 6 to 20, about 8 to 25, about 10 to 30, about 12 to 35, about 14 to 40, about 5 to 40, about 10 to 35, about 15 to 30, about 20 to 25, about 20 to 40, about 10 to 30, about 4 to 20, or about 5 to 25 amino acid residues. In some embodiments, the JMD comprises about 4 to 10, about 5 to 12, about 6 to 14, about 7 to 18, about 8 to 20, about 9 to 22, about 10 to 24, or about 11 to 26 amino acid residues. In some embodiments, the JMD comprises about 4 to 10 residues, e.g., 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0140] In some embodiments, the juxtamembrane domain is a polybasic domain. In some embodiments, the polybasic domain comprises Notch-1 or Notch-2. In some embodiments, the polybasic domain comprises an amino acid sequence in which a majority of the residues (i.e., at least 55%, at least 60%, at least 70%, at least 75%, 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%) are lysine and / or arginine and / or histidine and / or any combination thereof.

[0141] The JMD can comprise a sequence having at least 70% sequence identity, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity, to a JMD sequence derived from Notch1, Notch2, Notch3, Notch4, CLSTN1, CLSTN2, CSF1R, CXCL16, DAG1, GHR, PTPRF, AGER, KL, NRG1, LRP1B, Jag2, EPCAM, KCNE3, CDH2, CDH5, NRG2, PTPRK, BTC, EPHA3, EPHA4, IL1R2, or PTPRM. In some embodiments, the JMD comprises a sequence in which the first four residues are exclusively Lys (K) or Arg (R). In some embodiments, the JMD includes 1, 2, 3, 4, 5, or more basic residues, hi some embodiments, the JMD includes 5, 4, 3, 2, 1, or no aromatic residues or residues with hydrophobic and / or bulky side chains.

[0142] d. First intracellular domain and transcriptional regulator The intracellular domain of the first polypeptide of the multi-chain chimeric polypeptide of the present disclosure may comprise a transcriptional regulator. The transcriptional regulator of the present disclosure may be a polypeptide element that acts to activate or inhibit transcription of a DNA sequence driven by a promoter. Transcriptional regulators suitable for the compositions and methods of the present disclosure may be naturally occurring transcriptional regulators, or may be engineered, designed, or modified to provide desired and / or improved properties, such as transcription modulation. As described above, the engineered receptors of the present disclosure are advantageous in that they can provide the ability to induce custom transcriptional programs in engineered cells. In some embodiments, the transcriptional regulators of the present disclosure are custom transcriptional regulators that prevent transcription of specific sequences that only occur once in the engineered cells. In some embodiments, the transcriptional regulators of the present disclosure comprise human or humanized sequences.

[0143] In some embodiments, the transcriptional regulator directly regulates cell differentiation. In some embodiments, the transcriptional regulator indirectly modulates (e.g., regulates) cell differentiation by modulating the expression of a second transcription factor. Those skilled in the art will understand that the transcriptional regulator can be a transcriptional activator or a transcriptional repressor. In some embodiments, the transcriptional regulator is a transcriptional repressor. In some embodiments, the transcriptional regulator is a transcriptional activator. In some exemplary embodiments, the transcriptional regulator of the chimeric receptor of the present disclosure comprises a transcription factor DNA-binding domain (DBD). Exemplary DBDs can include Gal4, tetR, ZFHD1, Zif268, and HAP1. In some exemplary embodiments, at least one transcriptional regulator of the chimeric receptor of the present disclosure further comprises a transactivation domain (TAD). In some embodiments, the transcriptional regulator can further comprise a nuclear localization signal.

[0144] In some embodiments, the transcriptional regulator of a chimeric receptor of the present disclosure comprises one or more zinc finger proteins or zinc finger-containing transcriptional effectors (ZTEs) containing zinc finger motifs (ZFs). For example, the transcriptional regulator of a chimeric receptor of the present disclosure may comprise a DBD containing one or more ZFs. A ZF is a protein finger-like fold that allows a protein to interact with nucleic acid sequences such as DNA and RNA. Such finger-like folds are well known in the art. This fold is generally created by specific amino acids of the protein binding to a zinc atom and is stabilized by the coordination of the zinc ion between four largely invariant Cys and / or His residues (depending on the type of zinc finger framework). Exemplary zinc finger proteins may include ZF3, ZF6, ZF10, etc.

[0145] The term "motif" as used herein refers to a structural motif. The ZF motif is a relatively small polypeptide domain with a supersecondary structure, containing approximately 30 amino acids, that folds to form an α-helix adjacent to an antiparallel β-sheet (known as a ββα fold) and is stabilized by zinc ions. ZF domains recognize and bind to nucleic acid triplets or overlapping quadruplets (described below) within double-stranded DNA target sequences. Naturally occurring zinc finger domains (also known as ZF proteins) have been well studied and described in the literature. Natural ZF proteins can regulate gene expression as well as nucleic acid recognition, reverse transcription, and virus assembly. Additional information on this point can be found, for example, in U.S. Pat. No. 10,138,493.

[0146] C2H2 zinc fingers (C2H2-ZFs) are one of the most widespread types of vertebrate DNA-binding domains, commonly occurring in tandem arrays (ZFAs), where a series of C2H2-ZFs each contact a series of three (or more) bases. C2H2-ZFs can assemble in a modular manner. Given a set of modules with defined trinucleotide specificities, modular assembly also offers a method for constructing artificial proteins with specific DNA-binding preferences.

[0147] ZF-containing proteins generally contain a stretch or chain of ZF motifs that form a ZF array (ZFA). Thus, ZF proteins can contain two or more ZFs, for example, ZFAs contain two or more ZF motifs, which can be directly adjacent to each other (e.g., separated by a short linker sequence) or separated by a longer, flexible, or structured polypeptide sequence. For example, ZFAs can have six ZF motifs (six-finger ZFAs), seven ZF motifs (seven-finger ZFAs), or eight ZF motifs (eight-finger ZFAs) arranged in tandem. Directly adjacent ZF domains are generally expected to bind to contiguous nucleic acid sequences, such as adjacent trinucleotides / triplets. In some cases, cross-linking can occur between adjacent ZFs and their respective target triplets, which can serve to strengthen or enhance target sequence recognition, resulting in the binding of overlapping quadruplet sequences. In comparison, distantly separated ZF domains within the same protein may recognize and / or bind to non-contiguous nucleic acid sequences or even different molecules (eg, proteins rather than nucleic acids).

[0148] In some embodiments, the multi-stranded receptors of the present disclosure comprise zinc finger-containing transcriptional effectors (ZTEs) having a DNA-binding zinc finger protein domain (ZF protein domain) and another domain (effector domain) through which the protein exerts its effect. As described in more detail below, exemplary effector domains suitable for multi-stranded receptors of the present disclosure include, but are not limited to, transcriptional activation domains (e.g., TADs), epigenetic effector domains, and DNA-modifying enzymes.

[0149] In some embodiments, the multi-stranded receptors of the present disclosure comprise a transcriptional effector having a DNA-binding domain other than a zinc finger (e.g., a DNA-binding domain without a zinc finger structure) and another domain (effector domain) through which the protein exerts its effect. Exemplary non-zinc finger DNA-binding domains include those derived from PAX6. As described in more detail below, exemplary effector domains suitable for multi-stranded receptors of the present disclosure include, but are not limited to, transcriptional activation domains (e.g., TADs), epigenetic effector domains, and DNA-modifying enzymes. In some embodiments, the multi-stranded receptors of the present disclosure comprise from one to about ten DNA-binding domains, each of which independently comprises a sequence having at least about 90% identity to the sequence of SEQ ID NO: 61 (PAX6). In some embodiments, the DNA binding domain of the transcriptional effector has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of SEQ ID NO: 61 (PAX6).

[0150] In some embodiments, the multi-chain receptor of the present disclosure comprises a ZTE having two or more, e.g., three or more, e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more (e.g., up to about 30 or 32) ZF motifs arranged adjacent to one another in tandem to form an array of ZF motifs or ZFAs. In some embodiments, the ZTE comprises at least 3 ZF motifs, at least 4 ZF motifs, at least 5 ZF motifs, or at least 6 ZF motifs, at least 7 ZF motifs, at least 8 ZF motifs, at least 9 ZF motifs, at least 10 ZF motifs, at least 11 or at least 12 ZF motifs; and in some cases at least 18 ZF motifs. In some embodiments, the ZTE of the engineered Notch receptor disclosed herein contains up to 6, 7, 8, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35, 36, 40, 41, 42, 46, 47, 48, 54, 55, 56, 58, 59, or 60 ZF motifs. In some embodiments, the ZTE of the present disclosure binds to an orthogonal target nucleic acid binding site. That is, the ZF or ZFA in the ZF domain of the ZTE binds to an orthogonal target nucleic acid sequence. In some embodiments, the orthogonal target nucleic acid binding sites are contiguous. In some embodiments, the ZTE of the engineered Notch receptor disclosed herein binds to a specific orthogonal target DNA sequence, for example, with reduced or minimal potential for functional binding in a eukaryotic genome.

[0151] In some embodiments of the present disclosure, a ZTE comprises (a) a first domain (ZF protein domain) comprising a DNA-binding zinc finger protein domain, and (b) a second domain (effector domain) through which the ZTE exerts its effect, wherein the ZTE has the following formula I: [Effector domain]a-[ZF protein domain]-[Effector domain]b (Formula I) (wherein a and b each independently represent an integer from 0 to 5, and at least one of a and b is not 0). wherein the ZF protein domain comprises from 1 to about 10 zinc finger arrays (ZFAs), the ZFAs having the formula II (from N-terminus to C-terminus): XcCXdCXe-(helix)-HXfH-L2 (Formula II) wherein L2 is a linker peptide having about 4 to 6 amino acid residues, C is Cys, H is His, each X is independently any amino acid, c is an integer from 0 to 3, d is an integer from 1 to 5, e is an integer from 2 to 7, f is an integer from 3 to 6, and (helix) is a peptide domain of about 6 amino acids that forms an α-helix), and the ZFA is capable of binding to a specific nucleic acid sequence.

[0152] In some embodiments, the ZF protein domain of the multi-chain receptors disclosed herein comprises 1 to about 10 ZFAs, each of which independently comprises a sequence having at least about 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 62-67. In some embodiments, the ZFA comprises a sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 62-67. In some embodiments, the ZFA sequence has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of SEQ ID NO: 62 (ZF3). In some embodiments, the ZFA sequence has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of SEQ ID NO: 63 (ZF6). In some embodiments, the ZFA sequence has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of SEQ ID NO: 64 (ZF6 / SV40NLS). In some embodiments, the ZFA sequence has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of SEQ ID NO: 65 (ZF6 / Notch1NLS). In some embodiments, the ZFA sequence has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of SEQ ID NO: 66 (ZF10 / SV40NLS). In some embodiments, the ZFA sequence has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of SEQ ID NO: 67 (ZF10 / Notch1NLS).In some embodiments, the ZFA sequence has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence of ZF2, ZF4, or ZF11.

[0153] In some embodiments, the ZF protein domain of the multi-chain receptors disclosed herein comprises from 1 to about 10 ZFAs, each of which independently comprises a sequence having about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 62-67. In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 62 (ZF3). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 63 (ZF6). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 64 (ZF6 / SV40NLS). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 65 (ZF6 / Notch1NLS). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 66 (ZF10 / SV40NLS). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 67 (ZF10 / Notch1NLS). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 67 (ZF10 / Notch1NLS). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of ZF2, ZF4, or ZF11.

[0154] In some embodiments, the ZF protein domain comprises multiple ZFAs that have the same amino acid sequence. In some embodiments, the ZF protein domain comprises multiple ZFAs that differ from each other in amino acid sequence.

[0155] In some embodiments, the ZF protein domain of a multi-chain receptor disclosed herein comprises one or more ZFAs independently capable of specifically binding to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 62-67. In some embodiments, at least one ZFA is capable of specifically binding to a target nucleic acid sequence having the sequence of SEQ ID NO: 62. In some embodiments, at least one ZFA is capable of specifically binding to a target nucleic acid sequence having the sequence of SEQ ID NO: 63. In some embodiments, at least one ZFA is capable of specifically binding to a target nucleic acid sequence having the sequence of SEQ ID NO: 64. In some embodiments, at least one ZFA is capable of specifically binding to a target nucleic acid sequence having the sequence of SEQ ID NO: 65. In some embodiments, at least one ZFA is capable of specifically binding to a target nucleic acid sequence having the sequence of SEQ ID NO: 66. In some embodiments, at least one ZFA is capable of specifically binding to a target nucleic acid sequence having the sequence of SEQ ID NO: 67. In some embodiments, the ZF protein domain of an engineered Notch receptor disclosed herein comprises one or more ZFAs independently capable of specifically binding to ZF2, ZF4, or ZF11.

[0156] As described herein, the zinc finger-containing transcriptional effector (ZTE) of the multi-chain receptor disclosed herein comprises a second domain (effector domain) through which the ZTE exerts its effect. Exemplary effector domains suitable for the multi-chain receptor of the present disclosure include, but are not limited to, transcriptional activation domains (e.g., TADs), epigenetic effector domains, and DNA-modifying enzymes. Non-limiting examples of transcriptional activation domains (TADs) suitable for use in the compositions and methods disclosed herein include the herpes simplex virus protein 16 (HSV VP16) activation domain; the activation domain consisting of four tandem copies of VP16 (VP64); the p65 activation domain of NFκB; the Epstein-Barr virus R transactivator activation domain (Rta); the tripartite activator (VPR) consisting of the VP64 and Rta activation domains; and the histone acetyltransferase core domain of human E1A-associated protein p300 (p300 HAT core activation domain). In some embodiments, the effector domain of ZTE comprises the p65 activation domain of NFκB.

[0157] In some embodiments, the transcriptional regulator is derived from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16. In some embodiments, the transcriptional regulator is Gal4-VP64. In some embodiments, the transcriptional regulator has a sequence derived from or selected from the group consisting of VP64, p65, KRAB transactivation variant, and VP16. In some exemplary embodiments, the transcriptional regulator has a sequence derived from a TAD of human or humanized p65. In some exemplary embodiments, the transcriptional regulator has a sequence derived from a TAD of human or humanized HNF1α, HSF-1, GATA3, HIF1a, GR Tau1, ATF6, ELF3, p53, MIER3, MLXIPL, NFE2L1, or PTF1A. In other exemplary embodiments, the transcriptional regulator has a sequence derived from human or humanized HNF1α, HSF-1, GATA3, HIF1a, GR Tau1, ATF6, ELF3, p53, MIER3, MLXIPL, NFE2L1, or PTF1A.

[0158] In some embodiments, the human or humanized sequence contains a linear amino acid sequence motif. In some embodiments, the transcriptional regulators described herein are directly fused, with or without a linker sequence.

[0159] In some embodiments, the transcriptional regulator comprises a sequence having at least 80% sequence identity to one or more of SEQ ID NOs: 68-75, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs: 68-75. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 95% sequence identity to one or more of SEQ ID NOs: 68-75. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 100% sequence identity to one or more of SEQ ID NOs: 68-75. In some embodiments, the transcriptional regulator comprises one or more amino acid sequences of SEQ ID NOs: 68-75, wherein one, two, three, four, or five of the amino acid residues in one or more of SEQ ID NOs: 68-75 are substituted with a different amino acid residue.

[0160] In some embodiments, the transcriptional regulator comprises a sequence having at least 80% sequence identity to one or more of SEQ ID NOs:76-90, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs:76-90. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 95% sequence identity to one or more of SEQ ID NOs:76-90. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 100% sequence identity to one or more of SEQ ID NOs:76-90. In some embodiments, the transcriptional regulator comprises one or more amino acid sequences of SEQ ID NOs:76-90, wherein one, two, three, four, or five of the amino acid residues in one or more of SEQ ID NOs:76-90 are substituted with a different amino acid residue.

[0161] In some embodiments, the transcriptional regulator of the multi-chain receptor of the present disclosure comprises ZF3, a TAD derived from a TAD of human or humanized p65, and a TAD derived from a TAD of human or humanized HSF1. In some embodiments, the transcriptional regulator of the multi-chain receptor of the present disclosure comprises ZF3, human or humanized p65, and human or humanized HSF1. In other exemplary embodiments, the transcriptional regulator of the multi-chain receptor of the present disclosure comprises ZF3, human or humanized p65, and human or humanized GR Tau1. In some exemplary embodiments, the transcriptional regulator of the multi-chain receptor of the present disclosure comprises ZF3, human or humanized p65, and human p53 or humanized p53. In other exemplary embodiments, the transcriptional regulator of the multi-chain receptor of the present disclosure comprises ZF3, human or humanized p65, and human or humanized ATF6.

[0162] In some embodiments, the transcriptional regulator of the multi-chain receptor of the present disclosure comprises a TAD derived from a TAD of human or humanized p65 and a TAD derived from a TAD of human or humanized HNF1α. In some embodiments, the transcriptional regulator of the multi-chain receptor of the present disclosure comprises human or humanized p65 and human or humanized HNF1α.

[0163] In some embodiments, the intracellular domain of the first polypeptide of the present disclosure does not comprise a transcription factor. In some embodiments, the intracellular domain can be involved in the propagation of downstream signal transduction when the first polypeptide chain binds to a ligand. The intracellular signal transduction domain can have at least two distinct domains, at least one costimulatory domain and an activation domain.

[0164] In some embodiments, the costimulatory domain comprises a sequence derived from a signaling molecule. The signaling molecule can be a protein selected from class 1 or class 3 human membrane proteins. In some embodiments, the signaling molecule is CD28, 4-1BB, OX40, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, CRTAM, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Ly108, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIR1, LAG3, CD160, CD27, GITR, CD30, TNFR1, TNFR2, HVEM, LT_R, DR3, DCR3, FAS, CD40, RANK, OPG, TRAILR1, TACI, BAFFR, BCMA, or TWEAK. Selected from R, EDAR, XEDAR, RELT, DR6, TROY, NGFR, CD22, SIGLEC-3, SIGLEC-5, SIGLEC-7, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR3DL1, CD94-NKG2A, CD300b, CD300e, TREM1, TREM2, ILT7, ILT3, ILT4, TLT-1, CD200R, CD300a, CD300f, DC-SIGN, B7-2, allergin-1, LAT, BLNK, LAYN, SLP76, EMB-LMP1, HIV-NEF, HVS-TIP, HVS-ORF5, and HVS-stpC. In some exemplary embodiments, the signaling molecule is selected from the list consisting of OX40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27, and CD226, and derivatives, mutants, variants, fragments, and combinations thereof. In other embodiments, the signaling molecule is selected from the list consisting of OX40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27, and CD226, and derivatives, mutants, variants, fragments, and combinations thereof.In some embodiments, the signaling molecule is selected from the group consisting of 4-1BB, BAFF-R, BCMA, BTLA, CD2, CD200R, CD244, CD28, CD300a, CD300f, CD40, CD7, CD72, CD96, CRACC, CRTAM, CTLA4, CXADR, DC-SIGN, GITR, HAVCR2, ICOS, ILT2, ILT3, ILT4, KIR2DL1, KIR3DL1, KLRG1, LAG3, LAIR1, NKG2D, NKR-P1A, NTB-A, PD1, Siglec-3, TACI, TIGIT, TLT-1, and TNR8 (CD30), and derivatives, mutants, variants, fragments, and combinations thereof. In other embodiments, the signaling molecule is CD28 or 4-1BB. In an exemplary embodiment, the costimulatory domain comprises a sequence derived from CD28. In another exemplary embodiment, the costimulatory domain comprises a sequence derived from 4-1BB.

[0165] In some embodiments, the activation domain comprises one or more conserved amino acid motifs that serve as substrates for phosphorylation, such as immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the activation domain comprises at least one, at least two, at least three, at least four, or at least five specific tyrosine-based motifs selected from ITAM motifs, ITIM motifs, or related intracellular motifs that serve as substrates for phosphorylation. In some embodiments of the present disclosure, the activation domain of the intracellular signaling domain comprises at least one, at least two, at least three, at least four, or at least five ITAMs. Generally, any activation domain containing an ITAM can be suitably used in constructing the multi-chain receptors described herein. ITAMs generally comprise a conserved protein motif often present in the tails of signaling molecules expressed in many immune cells. This motif can comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid that results in the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs in signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues within the ITAM after activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in signaling pathways.

[0166] In some embodiments, the activation domain comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the activation domain is derived from CD3ζ, CD3σ, CD3γ, and CD3ε. For example, in some embodiments, the ITAM is derived from CD3ζ, CD3σ, CD3γ, and CD3ε. In an exemplary embodiment, the ITAM is derived from CD3ζ. In certain embodiments, the ITAM comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a CD3ζ ITAM. In some embodiments, the activation domain comprises at least one, at least two, at least three, at least four, or at least five ITAMs independently selected from ITAMs derived from CD3ζ, FcRγ, and combinations thereof. In some embodiments, the activation domain comprises a CD3ζ ITAM.

[0167] In some embodiments, the intracellular domain of the first polypeptide of the present disclosure comprises a signaling chain derived from the IL-2 receptor common gamma chain. In some embodiments, the signaling chain derived from the IL-2 receptor common gamma chain comprises the amino acid sequence of ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 91).

[0168] Various embodiments of the first polypeptide of the present disclosure may include the following non-limiting examples of components: The first polypeptide may include an extracellular domain, a TMD, an intracellular domain, and other sequences derived from a known receptor, such as triggering receptor 2 (TREM2; Figures 2A, 2B, 2C, 2D, 3A, 4A, and 4B) expressed on myeloid cells. TREM2 can undergo proteolytic cleavage between amino acids H157 and S158 by members of the disintegrin and metalloproteinase (ADAM) family. This cleavage results in the release of a soluble TREM2 ectodomain (sTREM2) into the extracellular fluid and the generation of a membrane-tethered C-terminal fragment (CTF). The TREM2 CTF represents a substrate for intramembrane proteolysis by γ-secretase (Figure 2A). In some embodiments of the present disclosure, the first polypeptide may include the TREM2 TMD without the cytoplasmic domain. In some embodiments, the first polypeptide may comprise a TREM2 extracellular domain and a TMD having a Notch1 γ-secretase cleavage site and / or a Notch-2 JMD (FIG. 2D).

[0169] The first polypeptide of the multi-chain chimeric polypeptide of the present disclosure can include a first modified interface through which the first polypeptide and the second polypeptide associate. The first modified interface can be derived from the Notch1 TMD. The first modified interface can have a positively charged residue through which the first polypeptide of the multi-chimeric polypeptide of the present disclosure associates with the second polypeptide. The positively charged residue can be lysine or arginine. In some embodiments, the lysine or arginine residue can substitute for the leucine located at position 12 of the Notch2 JMD (with respect to SEQ ID NO:21, corresponding to residue 1747 in the entire construct comprising SEQ ID NO:8). In some embodiments, the first polypeptide comprises a truncated CD8α hinge domain ECD, a Notch1 TMD with an L12K mutation, a Notch2 JMD, and a Gal4VP64 transcriptional regulator (FIG. 2B). In some embodiments, the first polypeptide comprises a TREM2 ECD, a Notch1 TMD with an L1747K mutation (Uniprot P46531 amino acids 1736-1757 with an L1747K mutation), a Notch2 JMD, and a Gal4VP64 transcriptional regulator (Figure 2C). In some embodiments, the first polypeptide comprises a TREM2 ECD, a Notch1 TMD with a gamma secretase site, a Notch2 JMD, and a Gal4VP64 transcriptional regulator (Figure 2D).

[0170] In other embodiments, the first polypeptide can include a truncated CD8α hinge domain (CD8-hinge2) ECD, a polyvaline TMD with a lysine substitution at position 9 or 10, a Notch2 JMD, and a Gal4VP64 transcriptional regulator (Figures 4C and 4D).

[0171] In other embodiments, the first polypeptide can include a truncated CD8α hinge domain (CD8-hinge2) ECD, a polyvaline TMD with a lysine substitution at position 9 or 10, a Notch2 JMD, and a human HNF1a transcriptional regulator (Figures 8A and 8B).

[0172] In other embodiments, the first polypeptide can include a truncated CD8α hinge domain (CD8-hinge2) ECD, a polyvaline TMD with a lysine substitution at position 9 or 10, a Notch2 JMD, and a 4-1BB / CD3 zeta domain (Figure 10).

[0173] In other embodiments, the first polypeptide can include a truncated CD8 alpha hinge domain (CD8-hinge2) ECD, a polyvaline TMD with a lysine substitution at position 9 or 10, a Notch2 JMD, and a common gamma chain domain (Figure 10).

[0174] In other embodiments, the first polypeptide can include a truncated CD8 alpha hinge domain (CD8-hinge2) ECD, a polyvaline TMD with a lysine substitution at position 9 or 10, a Notch2 JMD, and a common gamma chain / CD3 zeta domain (Figure 10). As will be appreciated by those of skill in the art, the first polypeptide can also include various other combinations of the components described herein.

[0175] e. Other ingredients In some embodiments of the present disclosure, the first polypeptide may further comprise one or more of the following: an autoproteolytic peptide sequence or a nuclear localization signal.

[0176] In some embodiments, the intracellular domain comprises a nuclear localization sequence and a transcriptional regulator, for example, Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, or HAP1-VP16.

[0177] In some embodiments, the autoproteolytic peptide sequence is derived from Porcine Teschovirus-1 2A (P2A), Foot-and-Mouth Disease Virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna Virus 2A (T2A), Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A), Flacheria Infection Virus 2A (BmIFV2A), or a combination thereof.

[0178] The first polypeptide may further comprise a proteolytic cleavage site. In some embodiments, the proteolytic cleavage site is cleavable by gamma secretase. In some embodiments, the proteolytic cleavage site is a ligand-induced proteolytic cleavage site. The proteolytic cleavage site may be located between the transcriptional regulator and the hinge domain. The proteolytic cleavage site may be ligand-induced, where cleavage at the ligand-induced proteolytic cleavage site is induced when a selected ligand binds to the extracellular ligand-binding domain.

[0179] 2. Second Polypeptide The multi-chain chimeric polypeptides of the present disclosure comprise, in order from the N-terminus to the C-terminus of the second polypeptide, (i) a second TMD having a second interface, and (ii) a second polypeptide having a second intracellular domain.

[0180] In some embodiments, the second polypeptide of the present disclosure comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9 or SEQ ID NO:11 or a functional variant thereof.

[0181] a. second transmembrane domain The second polypeptide of the multi-chain chimeric polypeptide of the present disclosure can include a second TMD having a second interface. The second TMD can associate with the first polypeptide of the multi-chain chimeric polypeptide of the present disclosure through charged residues in the TMD and can include any domain having a signaling domain that can be activated when a target ligand binds to the extracellular ligand-binding domain of the first polypeptide. The second interface is a region in the second polypeptide that can include negatively charged residues that associate with positively charged residues in the TMD of the first polypeptide of the present disclosure. In some embodiments, the negatively charged residue can be arginine or lysine.

[0182] In some embodiments, the second polypeptide comprises a DAP12 TMD. In some embodiments, the second polypeptide comprises a DAP12 domain as well as a DAP12 intracellular signaling domain (i.e., a cytoplasmic domain), as described in more detail below.

[0183] b. second intracellular domain The second polypeptide of the multi-chain chimeric polypeptide of the present disclosure comprises an intracellular domain. The intracellular domain may be involved in propagating downstream signaling upon binding of the first polypeptide chain to a ligand. The intracellular signaling domain may have at least two distinct domains: at least one costimulatory domain and an activation domain.

[0184] In some embodiments, the costimulatory domain comprises a sequence derived from a signaling molecule. The signaling molecule can be a protein selected from class 1 or class 3 human membrane proteins. In some embodiments, the signaling molecule is CD28, 4-1BB, OX40, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, CRTAM, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Ly108, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIR1, LAG3, CD160, CD27, GITR, CD30, TNFR1, TNFR2, HVEM, LT_R, DR3, DCR3, FAS, CD40, RANK, OPG, TRAILR1, TACI, BAFFR, BCMA, or TWEAK. Selected from R, EDAR, XEDAR, RELT, DR6, TROY, NGFR, CD22, SIGLEC-3, SIGLEC-5, SIGLEC-7, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR3DL1, CD94-NKG2A, CD300b, CD300e, TREM1, TREM2, ILT7, ILT3, ILT4, TLT-1, CD200R, CD300a, CD300f, DC-SIGN, B7-2, allergin-1, LAT, BLNK, LAYN, SLP76, EMB-LMP1, HIV-NEF, HVS-TIP, HVS-ORF5, and HVS-stpC. In some exemplary embodiments, the signaling molecule is selected from the list consisting of OX40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27, and CD226, and derivatives, mutants, variants, fragments, and combinations thereof. In other embodiments, the signaling molecule is selected from the list consisting of OX40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27, and CD226, and derivatives, mutants, variants, fragments, and combinations thereof.In some embodiments, the signaling molecule is selected from the group consisting of 4-1BB, BAFF-R, BCMA, BTLA, CD2, CD200R, CD244, CD28, CD300a, CD300f, CD40, CD7, CD72, CD96, CRACC, CRTAM, CTLA4, CXADR, DC-SIGN, GITR, HAVCR2, ICOS, ILT2, ILT3, ILT4, KIR2DL1, KIR3DL1, KLRG1, LAG3, LAIR1, NKG2D, NKR-P1A, NTB-A, PD1, Siglec-3, TACI, TIGIT, TLT-1, and TNR8 (CD30), and derivatives, mutants, variants, fragments, and combinations thereof. In other embodiments, the signaling molecule is CD28 or 4-1BB. In an exemplary embodiment, the costimulatory domain comprises a sequence derived from CD28. In another exemplary embodiment, the costimulatory domain comprises a sequence derived from 4-1BB.

[0185] In some embodiments, the activation domain comprises one or more conserved amino acid motifs that serve as substrates for phosphorylation, such as immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the activation domain comprises at least one, at least two, at least three, at least four, or at least five specific tyrosine-based motifs selected from ITAM motifs, ITIM motifs, or related intracellular motifs that serve as substrates for phosphorylation. In some embodiments of the present disclosure, the activation domain of the intracellular signaling domain comprises at least one, at least two, at least three, at least four, or at least five ITAMs. Generally, any activation domain containing an ITAM can be suitably used in constructing the multi-chain receptors described herein. ITAMs generally comprise a conserved protein motif often present in the tails of signaling molecules expressed in many immune cells. This motif can comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid that results in the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs in signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues within the ITAM after activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in signaling pathways.

[0186] In some embodiments, the activation domain comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the activation domain is derived from CD3ζ, CD3σ, CD3γ, and CD3ε. For example, in some embodiments, the ITAM is derived from CD3ζ, CD3σ, CD3γ, and CD3ε. In an exemplary embodiment, the ITAM is derived from CD3ζ. In certain embodiments, the ITAM comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a CD3ζ ITAM. In some embodiments, the activation domain comprises at least one, at least two, at least three, at least four, or at least five ITAMs independently selected from ITAMs derived from CD3ζ, FcRγ, and combinations thereof. In some embodiments, the activation domain comprises a CD3ζ ITAM.

[0187] In some embodiments, the intracellular domain is derived from DAP12 (12 kDa DNAZ-activating protein, TYROBP). The DAP12 intracellular domain, also known as the cytoplasmic domain of DAP12, contains ITAMs, which undergo phosphorylation upon ligand binding to TREM2, resulting in the regulation of several intracellular signaling pathways that control cell proliferation and differentiation, survival, phagocytosis, cytoskeletal remodeling, calcium mobilization, and / or cytokine production. In some embodiments, the signaling domain can be a CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fcε RI, DAP10, DAP12, or CD66d signaling domain. In some embodiments, the signaling domain is a CD3 zeta signaling domain.

[0188] In some embodiments, the signaling domain comprises an endodomain of a cytokine receptor.

[0189] In some embodiments, the endodomain is derived from a type I cytokine receptor. Type I cytokine receptors share a common amino acid motif (WSXWS) in the extracellular portion adjacent to the cell membrane. Type I cytokine receptors include (i) interleukin receptors, such as receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, and IL-27; (ii) colony-stimulating factor receptors, such as receptors for erythropoietin, GM-CSF, and G-CSF; and (iii) hormone receptors / neuropeptide receptors, such as hormone receptors and prolactin receptors. Members of the type I cytokine receptor family contain different chains, some of which are involved in ligand / cytokine interaction and others in signal transduction. For example, the IL-2 receptor contains an α chain, a β chain, and a γ chain.

[0190] The IL-2 receptor common gamma chain (also known as CD132) is shared among the IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, IL-15 receptor, and IL-21 receptor.

[0191] In some embodiments, the endodomain is derived from the IL-2 receptor beta chain. In some embodiments, the amino acid sequence of the IL-2 receptor beta chain endodomain is: [ka] In some embodiments, the IL-2 receptor beta chain endodomain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 92 or a functional variant thereof.

[0192] In some embodiments, the endodomain is derived from the IL-4 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-4 receptor alpha chain endodomain is: [ka] In some embodiments, the IL-4 receptor alpha chain endodomain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 93 or a functional variant thereof.

[0193] In some embodiments, the endodomain is derived from the IL-7 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-7 receptor alpha chain endodomain is: [ka] [ka] In some embodiments, the IL-7 receptor alpha chain endodomain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 94 or a functional variant thereof.

[0194] In some embodiments, the endodomain is derived from the IL-9 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-9 receptor alpha chain endodomain is: [ka] In some embodiments, the IL-9 receptor alpha chain endodomain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 95 or a functional variant thereof.

[0195] In some embodiments, the endodomain is derived from the IL-13 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-13 receptor alpha chain endodomain comprises KRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQTKEETDSVVLIENLKKASQ, SEQ ID NO: 96. In some embodiments, the IL-13 receptor alpha chain endodomain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 96 or a functional variant thereof.

[0196] In some embodiments, the endodomain is derived from the IL-15 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-15 receptor alpha chain endodomain comprises KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL, SEQ ID NO: 97. In some embodiments, the IL-15 receptor alpha chain endodomain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 97 or a functional variant thereof.

[0197] In some embodiments, the endodomain is derived from the IL-21 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-21 receptor alpha chain endodomain is: [ka] In some embodiments, the IL-21 receptor alpha chain endodomain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 98 or a functional variant thereof.

[0198] In some embodiments, the endodomain is derived from a type II cytokine receptor, including those that bind type I and type II interferons, as well as members of the interleukin-10 family (interleukin-10, interleukin-20, and interleukin-22).

[0199] 3. Additional Embodiments of Chimeric Polypeptides The multi-chain chimeric polypeptides of the present disclosure can include various combinations of the first and second polypeptides described above. In some embodiments, the multi-chain chimeric polypeptides include (a) a first polypeptide having CD19scFV as an extracellular ligand-binding domain, (ii) a first TMD having a contiguous stretch of valine residues, (iii) a Notch 2 juxtamembrane domain, and (iv) a first intracellular domain having a Gal4VP64 transcriptional regulator; and (b) a second polypeptide having DNAX-activation protein 12 (DAP12), wherein the first polypeptide is linked to the second polypeptide via a lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments, a contiguous stretch of 5 to 15 contiguous valine residues is located on either side of the lysine residues. The lysine residue can be at position 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 from the N-terminus of the first TMD.

[0200] In some embodiments, the multi-chain chimeric polypeptide comprises (a) a first polypeptide having a CD19scFV as an extracellular ligand-binding domain, (ii) a first TMD having a contiguous stretch of valine residues, (iii) a Notch 2 juxtamembrane domain, and (iv) a first intracellular domain having a Gal4VP64 transcriptional regulator; and (b) a second polypeptide having a CD3 zeta signaling domain, wherein the first polypeptide is linked to the second polypeptide through a lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments, a contiguous stretch of 5 to 15 contiguous valine residues is located on either side of the lysine residue. The lysine residue can be at position 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 from the N-terminus of the first TMD.

[0201] In some embodiments, the multi-chain chimeric polypeptide comprises (a) a first polypeptide having CD19scFV as an extracellular ligand-binding domain, (ii) a first TMD having a contiguous stretch of valine residues, (iii) a Notch 2 juxtamembrane domain, and (iv) a first intracellular domain having a human transcriptional regulator; and (b) a second polypeptide having DNAX-activation protein 12 (DAP12), wherein the first polypeptide is linked to the second polypeptide through a lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments, a contiguous stretch of 5 to 15 contiguous valine residues is located on either side of the lysine residue. The lysine residues can be at positions 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 from the N-terminus of the first TMD. In some embodiments, the human transcriptional regulator is HNF1a.

[0202] In some embodiments, the multi-chain chimeric polypeptide comprises (a) a first polypeptide having CD19scFV as an extracellular ligand-binding domain, (ii) a first TMD having a contiguous stretch of valine residues, (iii) a Notch 2 juxtamembrane domain, and (iv) a first intracellular domain having a human transcriptional regulator; and (b) a second polypeptide having a CD3 zeta signaling domain, wherein the first polypeptide is linked to the second polypeptide through a lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments, a contiguous stretch of 5 to 15 contiguous valine residues is located on either side of the lysine residue. The lysine residue can be at position 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 from the N-terminus of the first TMD. In some embodiments, the human transcriptional regulator is HNF1a.

[0203] In some embodiments, the multi-chain chimeric polypeptide comprises (a) a first polypeptide having CD19scFV as an extracellular ligand-binding domain, (ii) a first TMD having a contiguous stretch of valine residues, (iii) a Notch 2 juxtamembrane domain, and (iv) a first intracellular domain having a human transcriptional regulator; and (b) a second polypeptide having a CD3 zeta signaling domain, wherein the first polypeptide is linked to the second polypeptide through a lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. In some embodiments, a contiguous stretch of 5 to 15 contiguous valine residues is located on either side of the lysine residue. The lysine residue can be at position 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 from the N-terminus of the first TMD. In some embodiments, the human transcriptional regulator is HNF1a.

[0204] 4. Nucleic Acid Constructs Encoding Multi-Chain Chimeric Polypeptides The present disclosure also provides recombinant nucleic acid constructs comprising a nucleotide sequence encoding the multi-chain chimeric polypeptide of the present disclosure. The present disclosure also provides recombinant nucleic acid constructs having a nucleotide sequence encoding only the first polypeptide or only the second polypeptide. In cases where the recombinant nucleic acid construct encodes either the first polypeptide or the second polypeptide, both types of constructs can be used to transduce host cells to express the multi-chain chimeric polypeptide of the present disclosure.

[0205] The recombinant nucleic acid construct may comprise a first cassette encoding a first polypeptide of the multi-chain chimeric polypeptide of the present disclosure and a second cassette encoding a second polypeptide of the multi-chain chimeric polypeptide of the present disclosure, wherein both cassettes are on the same nucleic acid molecule. In some embodiments, the first cassette is 5' to the second cassette. In some embodiments, the first cassette is 3' to the second cassette. In some embodiments, the first cassette and the second cassette are joined by an autoproteolytic peptide. In some embodiments, the autoproteolytic peptide is Thosea asigna virus 2A (T2A) peptide.

[0206] In some embodiments, a recombinant nucleic acid construct of the present disclosure comprises a nucleotide sequence comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58 SEQ ID NO:99, or any functional variant thereof.

[0207] In some embodiments, the recombinant nucleic acid construct comprises a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 99, or any functional variant thereof. Recombinant nucleic acid constructs of the present disclosure can be of any length, including, for example, between about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, e.g., between about 15 Kb and 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, between about 5 Kb and about 25 Kb, or between about 30 Kb and about 50 Kb.

[0208] In some embodiments, the recombinant nucleic acid construct comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, or a functional variant thereof.

[0209] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or expression vector. An expression cassette is generally understood to include a genetic material construct containing a coding sequence and sufficient regulatory information to direct the appropriate transcription and / or translation of the coding sequence in recipient cells in vivo and / or ex vivo. Generally, the expression cassette can be inserted into a vector for targeting to a desired host cell and / or individual. Thus, in some embodiments, the expression cassette of the present disclosure includes the coding sequence of the chimeric polypeptide disclosed herein operably linked to an expression control element such as a promoter, and optionally any one or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0210] The provided recombinant nucleic acid constructs can contain naturally occurring sequences or sequences that differ from naturally occurring sequences but encode the same polypeptide, e.g., a first polypeptide or a second polypeptide, due to the degeneracy of the genetic code. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., produced by phosphoramidite-based synthesis), and can be composed of nucleotide combinations or modifications falling within these types of nucleic acids. Furthermore, the recombinant nucleic acid molecules of the present disclosure can be double-stranded or single-stranded (e.g., either the sense or antisense strand).

[0211] A nucleic acid molecule is not limited to a sequence encoding a polypeptide, but may also include some or all of the non-coding sequences present upstream or downstream of the coding sequence (e.g., the coding sequence for a multi-chain chimeric polypeptide or receptor). Those skilled in the art of molecular biology are familiar with conventional procedures for isolating nucleic acid molecules. Nucleic acid molecules can be generated, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). When the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0212] B. Vector Recombinant nucleic acid constructs encoding the multi-chain polypeptides of the disclosure can be contained in one or more vectors. Thus, the disclosure also provides vectors that encode or express the multi-chain chimeric polypeptides of the disclosure.

[0213] The vector(s) of the present disclosure may express either the first polypeptide chain or the second polypeptide chain of the multi-chain chimeric polypeptide, or may express both; i.e., the multi-chain polypeptides of the present disclosure may be expressed by one vector or by different vectors that are co-transduced into a cell.

[0214] In some embodiments, the multi-chain chimeric polypeptide can be incorporated into an expression vector designed for transfer between host cells and used for purposes of transformation, e.g., introducing heterologous DNA into host cells. Thus, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to effect replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0215] In addition to the components of the multi-chain chimeric polypeptide or receptor of the present disclosure, the vector or vectors may include, for example, one or more selectable markers, one or more origins of replication such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of a cell.

[0216] In some embodiments, the expression vector may be a viral vector. As will be understood by those skilled in the art, the term "viral vector" is broadly used to refer to either a nucleic acid molecule (e.g., a transfer plasmid) containing virus-derived nucleic acid elements that facilitate the transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. Viral particles generally contain various viral components, and sometimes also host cell components, in addition to nucleic acid(s). The term viral vector can refer to either a virus or viral particle that can transfer nucleic acid to a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from viruses.

[0217] The multi-chain chimeric polypeptide or chimeric antigen receptor of the present disclosure may be incorporated into a retroviral vector. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof derived primarily from retroviruses. In some embodiments, the multi-chain chimeric antigen receptor of the present disclosure may be incorporated into a lentiviral vector. The lentiviral vector may contain structural and functional genetic elements or portions thereof, including LTRs, derived primarily from lentiviruses, which are members of the retrovirus genus.

[0218] Viral vectors that can be used in the present disclosure include, for example, adenoviral vectors, adeno-associated viral vectors, herpesvirus, simian virus 40 (SV40), and bovine papillomavirus vectors, retroviral vectors, and lentiviral vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY). For example, the constructs disclosed herein can be produced in eukaryotic hosts such as mammalian cells (e.g., K562 cells, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, care should be taken to ensure that the components are compatible with each other. One of ordinary skill in the art can make such a determination. Furthermore, if guidance is needed in selecting an expression system, one of ordinary skill in the art can refer to P. Jones, "Vectors: Cloning Applications," John Wiley and Sons, New York, NY, 2009).

[0219] The multi-chain chimeric polypeptide of the present disclosure can be contained in one or more vectors, for example, can be directed to express in the cells transformed / transduced with one or more vectors.Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art, are commercially available, or can be easily prepared by those skilled in the art.In some embodiments, the vector is a lentivirus transfer vector comprising SEQ ID NO: 1 or any functional variant thereof.

[0220] DNA vectors can be introduced into eukaryotic cells by conventional transformation or transfection techniques.Suitable methods for transforming or transfecting host cells, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, biolistic introduction, nucleoporation, hydrodynamic shock and infection, can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals.

[0221] C. Recombinant Cells The multi-chain chimeric polypeptides of the present disclosure can be introduced or transduced into host cells or recombinant cells, such as human T lymphocytes, to produce recombinant cells containing the nucleic acid molecules. Thus, some embodiments of the present disclosure relate to recombinant cells comprising the multi-chain chimeric polypeptides or CARs or recombinant nucleic acid constructs or vectors of the present disclosure.

[0222] Introduction of the constructs or vectors of the present disclosure into cells can be achieved by methods known to those skilled in the art, such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0223] Thus, in some embodiments, the construct or vector can be delivered by a viral or non-viral delivery vehicle known in the art. For example, the construct can be stably integrated into the host genome, replicated as an episome, or present in the recombinant host cell as a minicircle expression vector for transient expression. Thus, in some embodiments, the construct is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the construct is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genome recombination techniques, or more sophisticated techniques such as guide RNA-guided CRISPR / Cas9 genome editing, or DNA-guided endonuclease genome editing by NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the recombinant nucleic acid molecule of the present disclosure is present in the recombinant host cell as a minicircle expression vector for transient expression.

[0224] The recombinant nucleic acid constructs of the present disclosure can be encapsulated in viral capsids or lipid nanoparticles, 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. In a non-limiting example, adeno-associated viruses (AAVs) are engineered to deliver constructs to target cells by viral transduction. Several AAV serotypes have been described, and all known serotypes are capable of infecting cells from multiple diverse tissue types. AAVs are capable of transducing a variety of species and tissues in vivo without evidence of toxicity, and induce relatively mild innate and adaptive immune responses.

[0225] Lentivirus-derived vector systems are also useful for construct delivery and gene therapy by viral transduction.Lentivirus vectors offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important gene and cell therapy target cell types; (iv) no viral protein expression after vector transduction; (v) ability to deliver complex genetic elements, such as polycistronic sequences or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector engineering and production.

[0226] In some embodiments, recombinant host cells can be genetically engineered (e.g., transduced or transformed or transfected) with a vector construct of the present application, which can be, for example, a viral vector or a vector for homologous recombination, or can be an expression vector for expression of a polypeptide of interest, that contains a nucleic acid sequence homologous to a portion of the host cell's genome. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0227] In some embodiments, the recombinant cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the mammalian cell is an immune cell, or a tumor cell, or a stem cell. In some embodiments, the recombinant cell is an immune system cell, for example, a lymphocyte (e.g., a T cell or NK cell), or a dendritic cell. In some embodiments, the immune cell is a B cell, monocyte, natural killer (NK) cell, basophil, eosinophil, neutrophil, dendritic cell, macrophage, regulatory T cell, helper T cell (Tx), cytotoxic T cell (Tcm), or other T cell. In some embodiments, the immune system cell is a T lymphocyte. In some embodiments, the cells are CAR-expressing reporter T (CAR-T) cells.

[0228] In some embodiments, the cells are stem cells. In some embodiments, the cells are hematopoietic stem cells. In some embodiments of the cells, the cells are lymphocytes. In some embodiments, the cells are precursor T cells or T regulatory (Treg) cells. In some embodiments, the cells are CD34+, CD8+, or CD4+ cells. In some embodiments, the cells are CD8+ T cytotoxic lymphocyte cells selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments of the cells, the cells are CD4+ T helper lymphocyte cells selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the cells can be obtained by leukapheresis performed on a sample obtained from a subject. In some embodiments, the subject is a human patient.

[0229] In another aspect, the present invention provides a cell culture comprising at least one recombinant cell disclosed herein and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming the above-mentioned various host cells and species are known in the art and are described in technical and scientific literature. Therefore, a cell culture comprising at least one recombinant cell disclosed herein is also within the scope of the present application.

[0230] Suitable methods and systems for generating and maintaining cell cultures are known in the art.

[0231] D. Pharmaceutical Compositions The present disclosure also provides a pharmaceutical composition comprising a recombinant cell of the present disclosure and a pharmaceutically acceptable excipient, e.g., a carrier.

[0232] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that easy syringability exists. The composition should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, such as sodium dodecyl sulfate. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, are commonly included in the composition.Absorptive agents, such as aluminum monostearate and gelatin, can be included in the composition to achieve sustained absorption of the injectable composition.

[0233] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0234] In some embodiments, the multi-chain CARs and chimeric polypeptides of the present disclosure can also be administered by transfection or infection using methods known in the art, including, but not limited to, those described in McCaffrey et al. (Nature 418: 6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-10, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-60, 1996, erratum at Am. J. Health Syst. Pharm. 53: 325, 1996).

[0235] III. Methods of the Disclosure A. Methods for Treating The present disclosure provides, inter alia, methods for treating a condition in a subject by administering to the subject a therapeutically effective amount of a recombinant cell, pharmaceutical composition, multi-chain chimeric polypeptide, or CAR of the present disclosure. The present disclosure also provides, inter alia, methods for treating a condition in a subject by administering to the subject a vector of the present disclosure.

[0236] The present disclosure also provides methods for inducing an immune response in a subject by administering to the subject a therapeutically effective amount of a recombinant cell, pharmaceutical composition, or CAR of the present disclosure.

[0237] Non-limiting examples of immune responses include a cytotoxic T lymphocyte (CTL) response, a B cell response (e.g., production of antibodies), an NK cell response, or any combination thereof, when administered to an immunocompetent subject.

[0238] Administration of any one of the vectors, recombinant cells, or pharmaceutical compositions described herein can be used to treat patients for related conditions or diseases, such as cancer or autoimmune diseases or infectious diseases (e.g., chronic infections). In some embodiments, the vectors or cells of the present disclosure can be incorporated into compositions, e.g., pharmaceutical or therapeutic compositions, for use in methods of treating individuals who have, are suspected of having, or may be at high risk of developing one or more autoimmune disorders or diseases associated with checkpoint inhibition. Exemplary autoimmune disorders and diseases can include, but are not limited to, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0239] In some embodiments, the method includes administering an effective number of recombinant cells disclosed herein to an individual, wherein the recombinant cells inhibit the activity of target cells in the individual. Generally, the target cells of the disclosed methods can be any cell type within an individual, such as cells from a hematological malignancy, multiple myeloma cells, solid tumor cells, acute myeloma leukemia cells, anaplastic lymphoma cells, astrocytoma cells, B-cell cancer cells, breast cancer cells, colon cancer cells, ependymoma cells, esophageal cancer cells, glioblastoma cells, glioma cells, leiomyosarcoma cells, liposarcoma cells, liver cancer cells, lung cancer cells, mantle cell lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells, oligodendrocyte glioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T-cell lymphoma cells, kidney cancer cells, sarcoma cells, gastric cancer cells, carcinoma cells, mesothelioma cells, or sarcoma cells. In some embodiments, the target cells are pathogenic cells.

[0240] In some embodiments, the methods of the present disclosure involve administering to an individual in need of such treatment an effective amount of recombinant cells of the present disclosure. This administering step can be accomplished using any method of transplant delivery known in the art. For example, the recombinant cells of the present disclosure can be injected directly into the individual's bloodstream or otherwise administered to the individual.

[0241] In some embodiments, the methods disclosed herein involve administering recombinant cells to an individual (this term is used interchangeably with the terms "introducing," "implanting," and "transplanting") by a method or route that results in at least partial localization of the introduced cells at a desired site so that a desired effect(s) occurs. The recombinant cells or their differentiated progeny can be administered by any suitable route that delivers them to a desired location in an individual where at least a portion of the administered cells or components of the cells remain viable. The survival period of the cells after administration to an individual can be as short as a few hours, e.g., 24 hours, to several days, years, or even the lifetime of the individual, i.e., long-term engraftment.

[0242] In some embodiments, when provided therapeutically, the recombinant cells are provided at (or after) the onset of a symptom or sign of a disease or condition, e.g., at the onset of the disease or condition.

[0243] A therapeutically effective amount includes a quantity of recombinant cells sufficient to promote a particular beneficial effect when administered to an individual, such as an individual with, suspected of having, or at risk for a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slow the progression of disease symptoms), or reverse disease symptoms. It will be understood that in any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0244] For use in the various embodiments described herein, an effective amount of the recombinant cells disclosed herein is at least 10 2 cells, at least 5 x 10 2 cells, at least 10 3 cells, at least 5 x 10 3 cells, at least 10 4 cells, at least 5 x 10 4 cells, at least 10 5cells, at least 2 x 10 5 cells, at least 3 x 10 5 cells, at least 4 x 10 5 cells, at least 5 x 10 5 cells, at least 6 x 10 5 cells, at least 7 x 10 5 cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 1 x 10 6 cells, at least 2 x 10 6 cells, at least 3 x 10 6 cells, at least 4 x 10 6 cells, at least 5 x 10 6 cells, at least 6 x 10 6 cells, at least 7 x 10 6 cells, at least 8 x 10 6 cells, at least 9 x 10 6 The recombinant cells may be derived from one or more donors or from an autologous source. In some embodiments, the recombinant cells are expanded in culture before being administered to an individual in need thereof.

[0245] In some embodiments, a recombinant cell composition or pharmaceutical composition (e.g., a composition comprising a plurality of recombinant cells according to any of the cells described herein) is delivered to an individual by a method or route that results in at least partial localization of the cell composition at a desired site. Compositions comprising recombinant cells can be administered by any suitable route that results in effective treatment in the individual, for example, administration of at least a portion of the delivered composition, e.g., at least 1 x 10 4The cells are delivered to the desired location of the individual for a certain period of time. Modes of administration include injection, infusion, and infusion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For cell delivery, delivery by injection or infusion is the preferred mode of administration.

[0246] In some embodiments, the recombinant cells are administered systemically, e.g., by infusion or injection, e.g., a population of recombinant cells is administered other than directly to a target site, tissue, or organ so that they enter the circulatory system of an individual and are therefore subject to metabolic and other similar biological processes.

[0247] The effectiveness of a treatment, including any of the compositions provided herein for treating a disease or condition, can be determined by a skilled clinician. However, those skilled in the art will understand that a treatment is considered effective if any one or all of the signs or symptoms or markers of the disease are improved or ameliorated. Efficacy can also be measured by an individual's not getting worse (e.g., the progression of the disease is stopped or at least slowed), as assessed by a reduced need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals), and includes (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the onset of symptoms.

[0248] Diseases suitable for treatment by the compositions and methods of the present disclosure include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. In some embodiments, the disease is cancer or a chronic infectious disease. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the disease is a solid tumor.

[0249] In some embodiments of the disclosed methods, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual has or is suspected of having a disease associated with inhibition of cell signaling mediated by a cell surface ligand or antigen.

[0250] In some embodiments of the disclosed methods, the administered recombinant cells modulate the activity of target cells in the individual, hi some embodiments, the target cell activity includes expression of a selected gene, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of molecules, cell adhesion, and cytolytic activity.

[0251] As noted above, the recombinant cells and pharmaceutical compositions described herein can be administered in combination with one or more additional therapeutic agents, such as, for example, chemotherapeutic agents or anti-cancer agents or anti-cancer therapies. Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential administration in any order. In some embodiments, the one or more additional therapeutic agents, chemotherapeutic agents, anti-cancer agents, or anti-cancer therapies are selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, and surgery. "Chemotherapy" and "anti-cancer agents" are used interchangeably herein. Various classes of anti-cancer agents can be used. Non-limiting examples include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxins, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone treatments, soluble receptors, and other antineoplastic agents.

[0252] Methods for simultaneously inducing T cell signaling and gene regulation or for inducing enhanced T cell signaling The present disclosure also provides a method for simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising: (a) providing a T cell comprising a multi-chain chimeric polypeptide or a chimeric antigen receptor of the present disclosure; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of a transcriptional regulator.

[0253] Also provided herein is a method for simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising: (a) providing a vector comprising a multi-chain chimeric polypeptide or CAR of the present disclosure; and (b) transducing a T cell with the vector, wherein binding of a selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of a transcriptional regulator.

[0254] Also provided herein is a method of inducing enhanced T cell signaling in a T cell, the method comprising: (a) providing a T cell comprising a multi-chain chimeric polypeptide of the present disclosure; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain results in enhanced intracellular signaling.

[0255] Also provided herein are methods for inducing enhanced T cell signaling in a T cell, the methods comprising: (a) providing a vector comprising a multi-chain chimeric polypeptide of the present disclosure, or a vector comprising a first polypeptide and a second vector comprising a second polypeptide of any one of the multi-chain polypeptides of the present disclosure; and (b) transducing a T cell with the one or more vectors, wherein binding of a selected ligand to the extracellular ligand-binding domain induces enhanced intracellular signaling.

[0256] In some embodiments, the induced intracellular signaling in the T cell modulates the expression of selected genes involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of molecules, cell adhesion, and / or cytolytic activity. In some embodiments, the released transcriptional regulator modulates the expression of a payload in the T cell. In some embodiments, the payload comprises a chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA-guided nuclease, a site-specific nuclease, a T cell receptor, a toxin, a toxin-derived protein, a transcriptional regulator, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immunoinhibitor, or an inhibitory immunoreceptor.

[0257] In some embodiments, enhanced T cell signaling can be a 1%, 5%, 10%, 25%, 50%, 75%, 100%, or greater than 100% improvement or enhancement in T cell signaling compared to a reference cell that does not express a chimeric receptor of the present disclosure.

[0258] C. Methods for Modulating Cellular Activity In another aspect, provided herein are various methods for modulating the activity of a cell, comprising: (a) providing an effective amount of any of the recombinant cells of the present disclosure; and (b) contacting the cell with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain (i) induces cleavage of the ligand-inducible proteolytic cleavage site, releasing a transcriptional regulator, and concomitantly (ii) activates T cell signaling, whereby the released transcriptional regulator modulates the activity of the recombinant cell. Upon reading this disclosure, one of skill in the art will understand that the methods of the present disclosure can be performed in vivo, ex vivo, or in vitro.

[0259] Non-limiting exemplary cellular activities that can be modulated using the methods provided herein include, but are not limited to, gene expression, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of gene products, cell adhesion, and cytolytic activity.

[0260] In some embodiments, the released transcriptional regulator modulates the expression of a gene product in the cell. In some embodiments, the released transcriptional regulator modulates the expression of a heterologous gene product in the cell. The heterologous gene product is one that is not normally found in, e.g., not normally produced by, the native cell. For example, the cell may be genetically modified with a nucleic acid comprising a nucleotide sequence encoding the heterologous gene product.

[0261] In some embodiments, the heterologous gene product is a secreted gene product. In some embodiments, the heterologous gene product is a cell surface gene product. In some cases, the heterologous gene product is an intracellular gene product. In some embodiments, the released transcriptional regulator simultaneously modulates the expression of two or more heterologous gene products in a cell.

[0262] In some embodiments, the heterologous gene product in the cell is selected from the group consisting of a chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA-guided nuclease, a site-specific nuclease, a T cell receptor (TCR), a chimeric antigen receptor (CAR), a toxin, a toxin-derived protein, a transcription regulator, a transcription activator, a transcription repressor, a translation regulator, a translation activator, a translation repressor, an activating immunoreceptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immunoinhibitor, and an inhibitory immunoreceptor.

[0263] In some embodiments, the released transcriptional regulator modulates differentiation of a cell, wherein the cell is an immune cell, a stem cell, a progenitor cell, or a precursor cell.

[0264] IV. Systems and Kits Also provided herein are systems and kits comprising the multi-chain chimeric polypeptides, CARs, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein, as well as written instructions for their production and use. For example, in some embodiments, provided herein are systems and / or kits comprising one or more of the multi-chain chimeric polypeptides of the present disclosure, the recombinant nucleic acids described herein, the recombinant cells described herein, or the pharmaceutical compositions described herein. In some embodiments, the systems and / or kits of the present disclosure further comprise one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, the kits may have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, for example, to modulate the activity of cells, inhibit target cancer cells, or treat a health condition (e.g., disease) in an individual in need thereof.

[0265] In some embodiments, the system or kit may further include instructions for practicing the method using the components of the kit. The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit as a package insert, on a label on the container of the kit or its components (i.e., associated with the package or subpackage), etc. The instructions may be present as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions may also be recorded on a suitable substrate.

[0266] The discussion of general methods provided herein is intended for illustrative purposes only. Other alternative methods and substitutions will become apparent to those skilled in the art upon review of this disclosure, and are intended to be within the spirit and scope of this application.

[0267] Throughout this specification, various patents, patent applications, and other types of publications are referenced (e.g., journal articles, electronic database entries, etc.). The disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0268] No admission is made that any reference cited herein constitutes prior art. The discussion of references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. Although several sources of information, including scientific journal articles, patent documents, and textbooks, are mentioned herein, it will be expressly understood that this reference is not an admission that any of these documents form part of the common general knowledge in the art. [Example]

[0269] The practice of the present invention will utilize, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those of skill in the art and which may be utilized in accordance with the teachings of Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (with addendums from 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Expression. Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology.New York, NY: Wiley; Mullis, KB, Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (with addendum from 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are hereby incorporated by reference.

[0270] Additional embodiments are disclosed in further detail in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the disclosure or claims in any way.

[0271] Example 1 Receptor expression In this example, flow cytometry data of receptor expression is described.

[0272] Primary human CD3+ T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Gibco) and transduced with a lentiviral construct expressing a multichain receptor construct and another lentiviral construct containing a transcriptional reporter construct. Receptor expression was measured using an AlexaFluor 647-tagged anti-myc antibody (Cell Signaling) directed against the myc tag on the binding agent (CD19scFv)-containing chain. Reporter expression was measured by a constitutive mCitrine gene found on the reporter plasmid. Double-positive cells were sorted 5 days after the initial T cell stimulation and further expanded for activation studies. Here, receptor designs 056 (Figure 2A) and 056C (Figure 2B) showed the highest expression among this cohort.

[0273] Example 2 Receptor activation This example describes receptor activation and target killing of two receptor embodiments, 056 and 056C (FIG. 3A).

[0274] 1 x 10 double-positive T cells expressing anti-CD19 receptor 5 cells were cultured for 48 hours without any additions (red, "T cells alone"), or 1 × 10 5 were co-cultured for 48 h with 1 × 10 K562 cells (blue, (+K562)) or 1 × 10 5 The receptors were co-cultured with CD19+K562 cells for 48 hours (yellow, "+K562-CD19"). Afterwards, transcriptional activation of the inducible BFP reporter gene (Figure 3B) was measured using a Fortessa X-50 (BD). Target killing (Figure 3C) was measured by DRAQ7 staining and flow cytometry. Receptor 056 showed better killing than receptor 056C, which also showed better transcriptional activation.

[0275] Example 3 Receptor expression of receptors 056F and 056G This example describes flow cytometry data for receptor expression for receptor embodiments 056F and 056G.

[0276] Primary human CD3+ T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Gibco) and transduced with a lentiviral construct expressing a multichain receptor construct and another lentiviral construct containing a transcriptional reporter construct. Receptor expression was measured using an AlexaFluor 647-tagged anti-myc antibody (Cell Signaling) directed against the myc tag on the binder (CD19scFV)-containing chain. Reporter expression was measured by a constitutive mCitrine gene found on the reporter plasmid. Double-positive cells were sorted 5 days after the initial T cell stimulation and further expanded for activation studies. Receptor designs 056F (Figure 4C) and 056G (Figure 4D) showed the highest expression in this cohort.

[0277] Example 4 Receptor activation and performance of embodiments 056, 056E-G This example describes receptor activation and target killing studies of receptors 056, 056E, 056F and 056G.

[0278] 1 x 10 double-positive T cells expressing anti-CD19 receptor 5 cells were cultured for 5 days without any additions (red, "T cells alone"), or 1 × 10 5 were co-cultured for 5 days with 1 × 10 K562 cells (blue, "+K562") or 1 × 10 5 These receptors were co-cultured with CD19+K562 cells for 5 days (yellow, "+K562-CD19"). After 24 h, transcriptional activation of the inducible BFP reporter gene (top panel) was measured using a Fortessa X-50 (BD). Target killing (bottom panel) was measured at 24 and 120 h by DRAQ7 staining and flow cytometry. Here, receptor 056F (Figure 5C) exhibits both transcriptional and killing activity, receptors 056 (Figure 5A) and 056E (Figure 5B) exhibit killing, and receptor 056G (Figure 5D) exhibits only transcriptional activation.

[0279] Example 5 Multichain receptor modular engineering strategies This example describes a multi-chain receptor modular engineering strategy using pRay056F as a prototype. As shown in Figure 6, one chain (DAP12-chain) containing the DAP12 signaling domain was replaced with ITAMs and signaling domains derived from TCR, costimulatory proteins, and cytokine receptors. For the other chain (main chain, containing the transcriptional factor Gal4-VP64), the transcription factor (TF) was replaced with a human transcription factor or ITAMs and signaling domains derived from TCR, costimulatory proteins, and cytokine receptors.

[0280] Example 6 Function of multichain receptors with CD3z substitutions This example describes the testing of a multi-chain receptor with a CD3z substitution, in which the DAP12 ITAM signaling domain of the DAP12 multi-chain receptor (056F, Figure 7A) is replaced with the ITAM signaling domain from CD3z, which contains three ITAMs (056I, Figure 7B). 1 x 10 double-positive T cells expressing anti-CD19 receptors were cultured. 5 cells were cultured for 5 days without any additions (red, "T cells alone"), or 1 × 10 5 were co-cultured for 5 days with 1 × 10 K562 cells (blue, "+K562") or 1 × 10 5 The BBz CAR-1000-CD19 cells were co-cultured with CD19+K562 cells for 5 days (yellow, "+K562-CD19"). After 24 h, transcriptional activation of the inducible BFP reporter gene (top panel) was measured using a Fortessa X-50 (BD). Target killing (bottom panel) was measured at 24 and 120 h by DRAQ7 staining and flow cytometry. This substitution appeared to increase target cell killing (Figure 7B, bottom panel). Transcriptional activation was slightly reduced but still potent (Figure 7B, top panel). Transcriptional activation and killing of the SNIPR receptor (Figure 7C) and standard BBz CAR (Figure 7D) were included as positive controls for transcriptional activation and killing, respectively.

[0281] Example 7 Function of multichain receptors with human transcription factors. This example describes the testing of multi-chain receptors with human transcription factors. Here, Gal4-VP64, which contains a non-human portion, is replaced with a transcription factor composed of a human portion, both in the context of DAP12 (056H, Figure 8A) and CD3z (Figure 8B). These receptor designs are compared with receptors designed with transcription and signaling domains linearly constructed on a single-chain receptor (Figure 8C). 1 x 10 double-positive T cells expressing anti-CD19 receptors were cultured. 5 cells were cultured for 5 days without any additions (red, "T cells alone"), or 1 × 10 5 were co-cultured for 5 days with 1 × 10 K562 cells (blue, "+K562") or 1 × 10 5 The BBz CAR was co-cultured with CD19+K562 cells for 5 days (yellow, "+K562-CD19"). After 24 h, transcriptional activation of the inducible BFP reporter gene (top panel) was measured using a Fortessa X-50 (BD). Target killing (bottom panel) was measured at 24 and 120 h by DRAQ7 staining and flow cytometry. While both multi-chain receptors are capable of mediating transcriptional activation and target cell killing (Figures 8A and 8B, top and bottom panels), the single-chain design is unable to mediate transcriptional activation and target cell killing (Figure 8C, top and bottom panels). Transcriptional activation and killing of the standard BBz CAR (Figure 8D) was included as a positive control for killing.

[0282] Example 8 Multichain receptors using a modular engineering strategy using dual vector transduction This example describes a modular engineering strategy for multichain receptors using dual vector transduction. Here, each chain is expressed from an individual promoter introduced by transduction of the listed DAP12 chain and backbone constructs (Figure 9). Each side chain is paired with each backbone to assess the impact of pairing the two signaling chains. Three of the backbone variants contain signaling components instead of transcription factors and are designed to deliver additional signaling capabilities rather than transcription activation.

[0283] Example 9 Expression profile of multichain receptor pairs In this example, we describe the expression profiles of each multi-chain receptor pair. Cells containing the DAP-12 chain are detected by T2A-mCherry signal, while cells containing the main chain receptor are detected by Myc-tag staining. Receptor expression levels were assessed using Myc-tag staining. All receptors showed some level of expression, with some pairs showing better expression than others, as highlighted by a larger double-positive population (Figure 10).

[0284] Example 10 Screening for multichain receptors that enhance killing and survival. This example describes a screen for multi-chain receptors that enhance killing and survival. Primary human CD3+ T cells expressing multi-chain receptors were co-cultured with 100,000 K562 target cells at T cell:target ratios of 1:4, 1:2, and 1:1 and followed over time. Co-cultures were maintained in human T cell medium without exogenous cytokines and fed every 5 days. On days 5, 12, and 20, half of the co-cultures were removed (with medium changes), stained for T cell markers, and analyzed by flow cytometry. Counts indicating T cell survival (Figures 11-13) and target cell survival (Figures 14-16) were quantified. Standard BBz CAR T cells were included as a control (last column in each plot). By the final time point (day 20), some multi-chain receptors appear to mediate superior T cell survival and target cell killing compared to BBz CAR.

[0285] Example 11 Design of receptor and response element constructs This example describes the design and construction of a family of multi-chain chimeric polypeptides (e.g., receptors) created by fusing CD19 scFv {Porter: 2011gr} with the corresponding receptor scaffold and Gal4 DBD VP64. The receptors contained an N-terminal CD8α signal peptide (MALPVTALLLPLALLLHAARP) for membrane targeting and a myc-tag (EQKLISEEDL) to facilitate determination of surface expression using α-myc A647 (cell-signaling #2233). For all primary T cell experiments, receptors were cloned into a modified pHR'SIN:CSW vector containing the PGK promoter (SEQ ID NO: 1).

[0286] Example 12 Isolation and culture of primary human T cells This example describes the isolation and culture of primary human T cells used in various cell transduction experiments, subsequently described in Example 3 below. For these experiments, primary CD4+ and CD8+ T cells were isolated by negative selection from anonymous donor blood after apheresis (STEMCELL Technologies #15062&15063). Blood was obtained from the Blood Centers of the Pacific (San Francisco, CA) with approval from the University Institutional Review Board. T cells were cryopreserved in RPMI-1640 (UCSF cell culture core) with 20% human AB serum (Valley Biomedical Inc., #HP1022) and 10% DMSO. For all experiments, after thawing, T cells were cultured in human T cell medium consisting of X-VIVO 15 (Lonza #04-418Q), 5% human AB serum, and 10 mM neutralized N-acetyl-L-cysteine ​​(Sigma-Aldrich #A9165), supplemented with 30 units / mL of IL-2 (NCI BRB Preclinical Repository).

[0287] Example 13 Lentiviral transduction of human T cells This example describes the general protocol used for lentiviral transduction of human T cells. Pantropic VSV-G pseudotyped lentivirus was generated using Mirus TransIT-Lenti (Mirus #MIR 6606) by transfection of Lenti-X 293T cells (Clontech #11131D) with the pHR'SIN:CSW transgene expression vector and viral packaging plasmids pCMVdR8.91 and pMD2.G. Primary T cells were thawed the same day and, after 24 hours of culture, stimulated with human T-activator CD3 / CD28 Dynabeads (Life Technologies #11131D) at a cell:bead ratio of 1:3. At 48 hours, viral supernatant was collected and primary T cells were exposed to virus for 24 hours. Five days after T cell stimulation, Dynabeads were removed, and T cells were expanded until day 14, at which point they were rested and ready for assay. T cells were sorted for assay using Beckton Dickinson (BD) FACs ARIA II.

[0288] Example 14 Cancer cell lines This example describes the generation of myeloid leukemia cells expressing CD19 at levels comparable to Daudi tumors. The cancer cell line used was K562 myeloid leukemia cells (ATCC #CCL-243). K562 cells were lentivirally transduced to stably express human CD19 at levels comparable to Daudi tumors or to express HER2 via a doxycycline-inducible system. CD19 levels were determined by staining cells with α-CD19 APC (Biolegend #302212), and HER2 levels were determined by staining cells with α-HER2 AF647 (Biolegend #324412). All cell lines were screened for transgene expression.

[0289] Example 15 In vitro stimulation of primary T cells For all in vitro T cell stimulations, 1 x 105 T cells were co-cultured with target cells at a 1:1 ratio in U-bottom 96-well tissue culture plates. Cultures were analyzed for reporter activation and / or target cell killing at 24 hours or at the indicated time points using a BD Fortessa X-50. All flow cytometry analyses were performed with FlowJo software (TreeStar).

[0290] Example 16 Recombinant constructs The following is an example of a recombinant nucleic acid construct or cassette encoding an example multi-chain chimeric polypeptide of the present disclosure. As described above, the construct contained two cassettes. In this example, the cassette encoding the second polypeptide is located 5' to the cassette encoding the first polypeptide. A T2A autoproteolytic peptide sequence is located between the second and first polypeptides. Constructs in which the first polypeptide is located 5' to the second polypeptide can also be constructed. [Table 1]

Claims

1. (a) a first polypeptide comprising: (i) an extracellular ligand-binding domain having binding affinity for a selected ligand; (ii) a first transmembrane domain (TMD) comprising a first engineered interface; and (iii) a first intracellular domain comprising a transcriptional regulator; (b) a second polypeptide comprising (i) a second TMD comprising a second interface and (ii) a second intracellular domain comprising a signaling domain; A multi-chain chimeric polypeptide comprising: the first and second modified interfaces each comprise oppositely charged amino acid residues; the first polypeptide is bound to the second polypeptide via the first modified interface and the second interface, and binding of the selected ligand to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. Multi-chain chimeric polypeptides.

2. The multi-chain chimeric polypeptide of claim 1, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces activation of the signaling domain and release of the transcriptional regulator.

3. The multi-chain chimeric polypeptide of claim 1 or 2, wherein the first polypeptide comprises, in order from the N-terminus to the C-terminus of the first polypeptide, (i) the extracellular ligand-binding domain, (ii) the first TMD, and (iii) the first intracellular domain.

4. The multi-chain chimeric polypeptide of any one of claims 1 to 3, wherein the first TMD comprises (i) 10 to 25 consecutive valine residues or (ii) the Notch1 transmembrane domain.

5. The multi-chain chimeric polypeptide of any one of claims 1 to 4, wherein the second polypeptide comprises, in order from the N-terminus to the C-terminus of the second polypeptide, (i) the second TMD and (ii) the second intracellular domain.

6. 6. The multi-chain chimeric polypeptide of any one of claims 1 to 5, wherein the first engineered interface comprises positively charged residues and the second interface comprises negatively charged residues, and the first polypeptide is bound to the second polypeptide via electrostatic forces between the first and second engineered interfaces.

7. The multi-chain chimeric polypeptide of claim 6 , wherein the positively charged residue is lysine or arginine.

8. The multi-chain chimeric polypeptide of claim 7, wherein (i) the first TMD comprises SEQ ID NO: 21 or a functional variant thereof, and the lysine or arginine residue is at a position selected from positions 10 to 14 of SEQ ID NO: 21, or (ii) the first TMD comprises SEQ ID NO: 18, and the lysine or arginine is at a position selected from positions 8 to 11 of SEQ ID NO:

18.

9. The multi-chain chimeric polypeptide of claim 8 , wherein the lysine or arginine residue is (i) at position 12 of SEQ ID NO:21, or (ii) at position 9 of SEQ ID NO:

18.

10. The multi-chain chimeric polypeptide of claim 1 , wherein the extracellular domain comprises an antigen-binding portion capable of binding to a ligand on the surface of a cell.

11. The antigen-binding moiety may be an antibody, nanobody, diabody, triabody, minibody, F(ab') ２ 11. The multi-chain chimeric polypeptide of claim 10, wherein the chimeric polypeptide is selected from the group consisting of a single-chain variable fragment, a F(ab) fragment, a single-chain variable fragment (scFv), a single-domain antibody (sdAb), and functional fragments thereof.

12. The multi-chain chimeric polypeptide of claim 1 , wherein the ligand comprises a protein or a carbohydrate.

13. The multi-chain chimeric polypeptide of claim 1 , wherein the ligand is a tumor-associated antigen or a tumor-specific antigen.

14. The multi-chain chimeric polypeptide of any one of claims 1 to 13, wherein the ligand comprises a cell surface receptor, an adhesion protein, an integrin, a mucin, a lectin, a tumor-associated antigen, or a tumor-specific antigen.

15. The ligand is CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8 a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD 94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (C 15. The multi-chain chimeric polypeptide of claim 14, comprising a nucleotide sequence selected from the group consisting of CD183 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein a (SIRPα).

16. The multi-chain chimeric polypeptide of any one of claims 10 to 15, wherein the cell is a human cell.

17. The multi-chain chimeric polypeptide of any one of claims 10 to 16, wherein the cell is a tumor cell.

18. The multi-chain chimeric polypeptide of any one of claims 1 to 17, wherein the transcriptional regulatory factor comprises a transcriptional activator or a transcriptional repressor.

19. The multi-chain chimeric polypeptide of any one of claims 1 to 18, wherein the transcriptional regulatory factor comprises Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, or HAP1-VP16.

20. The multi-chain chimeric polypeptide of any one of claims 1 to 18, wherein the transcriptional regulator is a human or humanized transcriptional regulator.

21. The multi-chain chimeric polypeptide of any one of claims 1 to 18, wherein the transcriptional regulatory factor is HNF1a.

22. 22. The multi-chain chimeric polypeptide of any one of claims 1 to 21, wherein the second polypeptide comprises a signaling domain comprising a CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, or CD66d signaling domain.

23. 23. The multi-chain chimeric polypeptide of any one of claims 1 to 22, wherein the first polypeptide further comprises one or more of the following: a hinge domain, a ligand-inducible proteolytic cleavage site, an autoproteolytic peptide sequence, a nuclear localization signal, a juxtamembrane domain.

24. 24. The multi-chain chimeric polypeptide of claim 23, wherein the juxtamembrane domain is a polybasic domain.

25. 25. The multi-chain chimeric polypeptide of claim 24, wherein the polybasic domain comprises a Notch-1 or Notch-2 juxtamembrane domain.

26. 26. The multi-chain chimeric polypeptide of any one of claims 23 to 25, wherein the autoproteolytic peptide sequence is derived from Porcine Teschovirus-1 2A (P2A), Foot-and-Mouth Disease Virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna Virus 2A (T2A), Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A), Flacheria Falcata Virus 2A (BmIFV2A), or a combination thereof.

27. 27. The multi-chain chimeric polypeptide of any one of claims 23 to 26, wherein the first polypeptide further comprises a hinge domain derived from CD8, CD28, OX40, or IgG4.

28. 28. The multi-chain chimeric polypeptide of claim 27, wherein the hinge domain comprises a truncated CD8α hinge domain.

29. 29. The multi-chain chimeric polypeptide of any one of claims 24 to 28, wherein the ligand-inducible proteolytic cleavage site is cleavable by gamma secretase.

30. 2. The multi-chain chimeric polypeptide of claim 1, wherein the extracellular ligand-binding domain comprises CD19 scFv, the first TMD comprises a contiguous stretch of valine residues containing a lysine or arginine residue, the first polypeptide further comprises a Notch 2 juxtamembrane domain, the first intracellular domain comprises a Gal4VP64 transcriptional regulator, and the second polypeptide comprises DNAX-activation protein 12 (DAP12), the first polypeptide is linked to the second polypeptide via the lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain simultaneously induces activation of the signaling domain and release of the transcriptional regulator.

31. The multi-chain chimeric polypeptide of claim 1, wherein the extracellular ligand-binding domain comprises CD19scFv, the first TMD comprises a contiguous stretch of valine residues containing a lysine or arginine residue, the first polypeptide further comprises a Notch 2 juxtamembrane domain, the first intracellular domain comprises a Gal4VP64 transcription regulator, the second polypeptide comprises a CD3z signaling domain, the first polypeptide is linked to the second polypeptide via the lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain simultaneously induces activity of the signaling domain and release of the transcription regulator.

32. 2. The multi-chain chimeric polypeptide of claim 1, wherein the extracellular ligand-binding domain comprises CD19 scFv, the first TMD comprises a contiguous stretch of valine residues containing a lysine or arginine residue, the first polypeptide further comprises a Notch 2 juxtamembrane domain, the first intracellular domain comprises a human or humanized transcriptional regulator, the second polypeptide comprises DNAX-activation protein 12 (DAP12) or CD3z, the first polypeptide is linked to the second polypeptide via the lysine residue within the contiguous stretch of valine residues, and binding of CD19 to the extracellular ligand-binding domain simultaneously induces activation of the signaling domain and release of the transcriptional regulator.

33. The multi-chain chimeric polypeptide of claim 32, wherein the transcriptional regulatory factor is HNF1a.

34. 31. The multi-chain chimeric polypeptide of claim 30, wherein the contiguous stretch of valine residues comprises 5 to 25 valine residues and the lysine or arginine residue is flanked on either side by a contiguous stretch of 5 to 15 valine residues.

35. 35. The multi-chain chimeric polypeptide of any one of claims 1 to 34, wherein the multi-chain chimeric polypeptide is an immunoreceptor.

36. 36. The multi-chain chimeric polypeptide of claim 35, wherein the immunoreceptor is a chimeric antigen receptor.

37. (a) a first polypeptide comprising: (i) an extracellular ligand-binding domain having binding affinity for a selected ligand; (ii) a first transmembrane domain (TMD) comprising a first modified interface; and (iii) a first intracellular domain comprising a transcriptional regulator or signaling domain; (b) a second polypeptide comprising (i) a second TMD comprising a second interface and (ii) a second intracellular domain comprising a signaling domain; A multi-chain chimeric polypeptide comprising: the first and second modified interfaces each comprise oppositely charged amino acid residues; the first polypeptide is bound to the second polypeptide via the first modified interface and the second interface, and binding of the selected ligand to the extracellular ligand-binding domain induces activation of the signaling domain and release of the transcriptional regulator. Multi-chain chimeric polypeptides.

38. 38. The multi-chain chimeric polypeptide of claim 37, wherein the first intracellular domain comprises a transcriptional regulator.

39. 39. The multi-chain chimeric polypeptide of claim 38, wherein the transcriptional regulator comprises Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, or HAP1-VP16.

40. 39. The multi-chain chimeric polypeptide of any one of claims 37 to 38, wherein the transcriptional regulator is a human or humanized transcriptional regulator.

41. 41. The multi-chain chimeric polypeptide of claim 40, wherein the transcriptional regulator is HNF1a.

42. 38. The multi-chain chimeric polypeptide of claim 37, wherein the first intracellular domain comprises a signaling domain.

43. 43. The multi-chain chimeric polypeptide of claim 42, wherein the signaling domain comprises one or more of the following: CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, CD66d, 4-1BB, or common gamma chain signaling domains.

44. 44. The multi-chain chimeric polypeptide of any one of claims 37 to 43, wherein the signaling domain of the second polypeptide comprises a signaling domain comprising a CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, CD66d, or 4-1BB signaling domain.

45. 44. The multi-chain chimeric polypeptide of any one of claims 37 to 43, wherein the signaling domain of the second polypeptide comprises a cytokine signaling domain.

46. 46. ​​The multi-chain chimeric polypeptide of claim 45, wherein the cytokine signaling domain comprises an IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-13R, IL-15R, or IL-21R endodomain.

47. 47. The multi-chain chimeric polypeptide of any one of claims 37 to 46, wherein binding of the selected ligand to the extracellular ligand-binding domain concomitantly induces activity of the signaling domain and release of the transcriptional regulator.

48. 48. The multi-chain chimeric polypeptide of any one of claims 37 to 47, wherein the first polypeptide comprises, in order from the N-terminus to the C-terminus of the first polypeptide, (i) the extracellular ligand-binding domain, (ii) the first TMD, and (iii) the first intracellular domain.

49. 49. The multi-chain chimeric polypeptide of any one of claims 37 to 48, wherein the first TMD comprises (i) 10 to 25 consecutive valine residues, or (ii) the Notch1 transmembrane domain.

50. 50. The multi-chain chimeric polypeptide of any one of claims 37 to 49, wherein the second polypeptide comprises, in order from the N-terminus to the C-terminus of the second polypeptide, (i) the second TMD, and (ii) the second intracellular domain.

51. 51. The multi-chain chimeric polypeptide of any one of claims 37-50, wherein the first engineered interface comprises positively charged residues and the second interface comprises negatively charged residues, and the first polypeptide is bound to the second polypeptide via electrostatic forces between the first and second engineered interfaces.

52. 52. The multi-chain chimeric polypeptide of claim 51, wherein the positively charged residue is lysine or arginine.

53. 53. The multi-chain chimeric polypeptide of claim 52, wherein (i) the first TMD comprises SEQ ID NO: 21 or a functional variant thereof, and the lysine or arginine residue is at a position selected from positions 10 to 14 of SEQ ID NO: 21, or (ii) the first TMD comprises SEQ ID NO: 18, and the lysine or arginine is at a position selected from positions 8 to 11 of SEQ ID NO:

18.

54. 54. The multi-chain chimeric polypeptide of claim 53, wherein the lysine or arginine residue is (i) at position 12 of SEQ ID NO:21, or (ii) at position 9 of SEQ ID NO:

18.

55. 55. The multi-chain chimeric polypeptide of any one of claims 37 to 54, wherein the extracellular domain comprises an antigen-binding portion capable of binding to a ligand on the surface of a cell.

56. The antigen-binding moiety may be an antibody, nanobody, diabody, triabody, minibody, F(ab') ２ 56. The multi-chain chimeric polypeptide of claim 55, wherein the chimeric polypeptide is selected from the group consisting of a single-chain variable fragment, a F(ab) fragment, a single-chain variable fragment (scFv), a single-domain antibody (sdAb), and functional fragments thereof.

57. 57. The multi-chain chimeric polypeptide of any one of claims 37 to 56, wherein the ligand comprises a protein or a carbohydrate.

58. 58. The multi-chain chimeric polypeptide of any one of claims 37 to 57, wherein the ligand is a tumor-associated antigen or a tumor-specific antigen.

59. 59. The multi-chain chimeric polypeptide of any one of claims 37 to 58, wherein the ligand comprises a cell surface receptor, an adhesion protein, an integrin, a mucin, a lectin, a tumor-associated antigen, or a tumor-specific antigen.

60. The ligand is CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8 a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD 94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (C 60. The multi-chain chimeric polypeptide of claim 59, comprising a nucleotide sequence selected from the group consisting of CD183 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein a (SIRPα).

61. 61. The multi-chain chimeric polypeptide of any one of claims 55 to 60, wherein the cell is a human cell.

62. 62. The multi-chain chimeric polypeptide of any one of claims 55 to 61, wherein the cell is a tumor cell.

63. 63. The multi-chain chimeric polypeptide of any one of claims 37 to 62, wherein the first polypeptide further comprises one or more of the following: a hinge domain, a ligand-inducible proteolytic cleavage site, an autoproteolytic peptide sequence, a nuclear localization signal, a juxtamembrane domain.

64. 64. The multi-chain chimeric polypeptide of claim 63, wherein the juxtamembrane domain is a polybasic domain.

65. 65. The multi-chain chimeric polypeptide of claim 64, wherein the polybasic domain comprises a Notch-1 or Notch-2 juxtamembrane domain.

66. 66. The multi-chain chimeric polypeptide of any one of claims 63 to 65, wherein the autoproteolytic peptide sequence is derived from Porcine Teschovirus-1 2A (P2A), Foot-and-Mouth Disease Virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna Virus 2A (T2A), Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A), Flacheria Falcata Virus 2A (BmIFV2A), or a combination thereof.

67. 67. The multi-chain chimeric polypeptide of any one of claims 63 to 66, wherein the first polypeptide further comprises a hinge domain derived from CD8, CD28, OX40, or IgG4.

68. 68. The multi-chain chimeric polypeptide of claim 67, wherein the hinge domain comprises a truncated CD8α hinge domain.

69. 69. A recombinant nucleic acid construct comprising, in a 5' to 3' direction, a first cassette and a second cassette, wherein the first cassette and the second cassette are joined by an autoproteolytic peptide, the first cassette encoding a first polypeptide of any one of the multi-chain chimeric polypeptides described in claims 1 to 68, and the second cassette encoding a second polypeptide of any one of the multi-chain chimeric polypeptides described in claims 1 to 68.

70. 69. A recombinant nucleic acid construct comprising, in a 5' to 3' direction, a first cassette and a second cassette, wherein the first cassette and the second cassette are joined by an autoproteolytic peptide, the first cassette encoding a second polypeptide of any one of the multi-chain chimeric polypeptides of claims 1 to 68, and the second cassette encoding a first polypeptide of any one of the multi-chain chimeric polypeptides of claims 1 to 68.

71. 69. A recombinant nucleic acid construct comprising a nucleic acid sequence encoding the first polypeptide of any one of the multi-chain chimeric polypeptides of claims 1 to 68.

72. 69. A recombinant nucleic acid construct comprising a nucleic acid sequence encoding the second polypeptide of any one of the multi-chain chimeric polypeptides of claims 1-68.

73. 73. The nucleic acid construct of any one of claims 69 to 72, comprising a nucleic acid sequence comprising 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or any functional variant thereof.

74. 71. The nucleic acid construct of claim 69 or claim 70, wherein the autoproteolytic peptide is a Thosea asigna virus 2A (T2A) peptide.

75. A vector comprising the nucleic acid construct of claims 69 to 74.

76. 76. The vector of claim 75, which is an expression vector.

77. 77. The vector of claim 75 or 76, which is a viral vector.

78. 78. The vector of any one of claims 75 to 77, wherein the viral vector comprises a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

79. 76. A recombinant cell comprising: a) a multi-chain chimeric polypeptide of any one of claims 1 to 68; b) a nucleic acid construct of any one of claims 69 to 74; and / or c) a vector of any one of claims 75 to 78.

80. 80. The recombinant cell of claim 79, which is a human cell.

81. 81. The recombinant cell of claim 79 or 80, which is a tumor cell.

82. 81. The recombinant cell of claim 80, which is an immune cell.

83. 83. The recombinant cell of claim 82, wherein the immune cell is a B cell, monocyte, natural killer cell, basophil, eosinophil, neutrophil, dendritic cell, macrophage, regulatory T cell, helper T cell, cytotoxic T cell, or other T cell.

84. 84. The recombinant cell of claim 83, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

85. 85. A pharmaceutical composition comprising the recombinant cell of any one of claims 79 to 84 and a pharmaceutically acceptable excipient.

86. 69. A method for simultaneously inducing T cell signaling and gene regulation in a T cell, comprising: (a) providing a T cell comprising the multi-chain chimeric polypeptide of any one of claims 1 to 68; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of the transcriptional regulator.

87. 1. A method for simultaneously inducing T cell signaling and gene regulation in a T cell, comprising: (a) providing a vector comprising the multi-chain chimeric polypeptide of any one of claims 1 to 68, or a vector comprising a first polypeptide and a second polypeptide of any one of the multi-chain polypeptides of claims 1 to 68; (b) transducing T cells with the one or more vectors; Including, The method wherein binding of the selected ligand to the extracellular ligand-binding domain induces a concomitant intracellular signal transduction and release of the transcriptional regulator.

88. 69. A method of inducing enhanced T cell signaling in a T cell, comprising: (a) providing a T cell comprising the multi-chain chimeric polypeptide of any one of claims 1 to 68; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain results in enhanced intracellular signaling.

89. 1. A method for inducing enhanced T cell signaling in a T cell, comprising: (a) providing a vector comprising the multi-chain chimeric polypeptide of any one of claims 1 to 68, or a vector comprising a first polypeptide and a second polypeptide of any one of the multi-chain polypeptides of claims 1 to 68; (b) transducing T cells with said one or more vectors; Including, The method wherein binding of the selected ligand to the extracellular ligand-binding domain induces enhanced intracellular signaling.

90. 90. The method of any one of claims 86 to 89, wherein the induced intracellular signaling of the T cells modulates the expression of selected genes involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of molecules, cell adhesion, and / or cytolytic activity.

91. 88. The method of claim 87, wherein the released transcriptional regulator modulates expression of a payload in the T cell.

92. 92. The method of claim 91 , wherein the payload comprises a chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA-guided nuclease, a site-specific nuclease, a T cell receptor, a toxin, a toxin-derived protein, a transcription regulator, a transcription activator, a transcription repressor, a translation regulator, a translation activator, a translation repressor, an activating immunoreceptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immunoinhibitor, or an inhibitory immunoreceptor.

93. 85. A method for treating a condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant cell of any one of claims 79 to 84 or a pharmaceutical composition of claim 85, wherein the recombinant cell or pharmaceutical composition treats the condition in the subject.

94. 92. The method of claim 91, wherein the administered recombinant cells modulate the activity of a target cell in the individual.

95. 93. The method of claim 92, wherein the target cell activity comprises expression of selected genes involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of molecules, cell adhesion, and cytolytic activity.

96. 94. The method of claim 93, wherein the target cell is a cancer cell.

97. 95. The method of claim 94, wherein the cancer cells are solid tumor cells or hematological malignancy cells.

98. 96. The method of claim 95, wherein the hematological malignancy cells are multiple myeloma cells.

99. 1. A method for modulating T cell activity, comprising: (a) providing an effective amount of any of the recombinant cells of claims 79 to 84; and (b) contacting the cell with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage of a ligand-inducible proteolytic cleavage site, thereby (i) releasing the transcriptional regulator, and the released transcriptional regulator modulates the activity of the recombinant cell, and concomitantly (ii) activating T cell signaling.

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