Multi-chain synthetic receptors for simultaneous ligand-induced transcriptional regulation and membrane proximal signaling

Abstract

The present disclosure generally relates to multi-chain chimeric polypeptides or receptors having different polypeptide chains that associate post-translational to enable simultaneous activation of a signaling domain and release of a transcriptional regulatory factor following binding of a ligand. The disclosure also provides nucleic acid constructs, recombinant cells, vectors, pharmaceutical compositions and methods of treatment comprising the multi-chain chimeric polypeptides of the disclosure. The present disclosure also provides methods for simultaneous induction of T cell signaling and gene modulation in T cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 398,185, filed on August 15, 2022, the disclosure of which is incorporated herein by reference in its entirety (including any drawings). Statement Regarding Federally Funded Research

[0002] This invention was made with U.S. Government support under Grant No. OD025751-01 awarded by the National Institutes of Health. The Government has certain rights in this invention. Incorporation of Sequence Listing

[0003] This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The attached Sequence Listing file, named "2023-08-14Sequence_Listing_ST26 048536-727001WO.xml", was created on August 14, 2023 and is 345,882 bytes. Technical Field

[0004] The present disclosure relates generally 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 therapies comprising such receptors. Background Art

[0005] Many important advances have been made in the development of immunotherapies based on adoptive transfer of lymphocytes (e.g., T cells), the most important of which are T cells expressing chimeric immune receptors including chimeric antigen receptors (CAR-T cells). However, an important issue limiting these therapies is the inability to regulate or shut down CAR-T activity when needed. Regulation of CAR-T activity is particularly important for reducing or eliminating interactions that cause significant side effects when administering chimeric antigen receptor T cells.

[0006] Unlike T cell receptors (TCR), chimeric immune receptors such as CAR include molecules that combine tumor antigen recognition and intracellular activation. The structure and design of such immune receptors include, at a minimum, an extracellular antigen recognition domain connected to one or more intracellular activation domains by TMD. Examples of such immune receptors include synNotch receptors, which can bind to user-defined cell surface display ligands, thereby triggering the release of proteolytic cleavage and transcriptional regulators of receptors, which induce customized transcriptional programs in cells. Other examples include such receptors, which incorporate signal transduction (e.g., costimulation, CD3ζ), which can initiate T cell activation with customized transcriptional regulation. Those examples of immune receptors are designed as single protein chains with continuous fusions of costimulation, CD3ζ and transcriptional regulatory domains.

[0007] Receptors capable of simultaneous signal transduction and transcriptional regulation are also needed to more efficiently and reliably induce proximal T cell activation signals and gene regulation in primary T cells. Summary of the invention

[0008] The present disclosure generally relates to multi-chain chimeric polypeptides (e.g., chimeric immunoreceptors) having an architecture that enables transcriptional regulation and signal transduction using different protein chains. In particular, the present disclosure relates to multi-chain chimeric polypeptides having different polypeptide chains that associate with each other after translation (via residues located within the transmembrane domain) to enable simultaneous activation of the signaling domain and release of the transcriptional regulator upon binding of a ligand.

[0009] In particular, multi-chain chimeric polypeptides are provided herein, comprising (a) a first polypeptide having (i) an extracellular ligand-binding domain with binding affinity for a selected ligand, (ii) a first transmembrane domain (TMD) containing a first modification interface, and (iii) a first intracellular domain (ICD) containing a transcriptional regulator, and (b) a second polypeptide having (i) a second TMD containing a second interface and (ii) a second ICD containing a signaling domain, wherein the first modification interface and the second modification interface each comprise amino acid residues having opposite charges, wherein the first polypeptide is coupled to the second polypeptide via the first modification interface and the second interface, and wherein the binding of the selected ligand to the extracellular ligand-binding domain induces the activity of the signaling domain and the release of the transcriptional regulator. In some embodiments, the binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces the activity of the signaling domain and the release of the transcriptional regulator.

[0010] In some embodiments, the first polypeptide comprises (i) the extracellular ligand binding domain, (ii) the first TMD and (iii) the first intracellular domain in order from the N-terminus to the C-terminus of the first polypeptide. In some embodiments, the first TMD comprises (i) 10 to 25 consecutive valine residues or (ii) Notch 1 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) the second TMD and (ii) the second intracellular domain.

[0012] In some embodiments of the multi-chain chimeric polypeptide, the first modified interface comprises positively charged residues, the second interface comprises negatively charged residues, and the first polypeptide and the second polypeptide are coupled via electrostatic forces between the first modified interface and the second modified interface. 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 located 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 located at a position selected from positions 8 to 11 of SEQ ID NO: 18. In some embodiments in which the first transmembrane comprises SEQ ID NO: 21, the lysine or arginine residue is located at position 12 of SEQ ID NO: 21. In some embodiments in which the first transmembrane comprises SEQ ID NO: 18, the lysine or arginine residue is located 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 portion that binds (or is capable of binding) to a ligand on the surface of a cell. In some embodiments, the antigen binding portion is selected from antibodies, nanobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, F(ab) fragments, single chain variable fragments (scFv), single domain antibodies (sdAbs) and functional fragments thereof.

[0014] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the ligand comprises a protein or a 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 ligands include 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, placental alkaline phosphatase-like protein 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. In some embodiments, the transcriptional regulator comprises Ga14-VP16, Ga14-VP64, tetR-VP64, ZFHD1-VP64, Ga14-KRAB or HAP1-VP16.

[0017] In some embodiments of the multi-chain chimeric polypeptides 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 CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, 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, CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, 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-induced proteolytic cleavage site, an autoproteolytic peptide sequence, a nuclear localization signal, and a membrane-proximal domain. In some embodiments, the membrane-proximal domain is a polydomain. In some embodiments, the polydomain includes a Notch-1 or Notch-2 membrane-proximal domain. In some embodiments, the autoproteolytic peptide sequence is from porcine Teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus (ERAV) 2A (E2A), beta tetrasomic virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), malarial disease virus 2A (BmIFV2A), or a combination thereof.

[0021] In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the first polypeptide further comprises a hinge domain from CD8, CD28, OX40 or IgG4. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the hinge domain comprises a truncated CD8 alpha 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] In one aspect, the present invention also provides a multi-chain chimeric polypeptide, which comprises (a) a first polypeptide having (i) an extracellular ligand binding domain comprising a CD19scFv, (ii) a first TMD comprising a continuous valine residue segment containing a lysine or arginine residue, and (iii) a Notch 2 juxtamembrane domain, and (iv) a first ICD having a Gal4VP64 transcriptional regulator; and (b) a second polypeptide having a DNAX activating protein 12 (DAP12); wherein the first polypeptide is coupled to the second polypeptide via a lysine or arginine residue within the continuous valine residue segment, and wherein the binding of CD19 to the extracellular ligand binding domain simultaneously induces the activity of the signaling domain and releases the transcriptional regulator. In some embodiments, wherein the continuous valine residue segment comprises 5 to 25 valine residues, and the lysine residues are flanked by a continuous 5 to 15 valine residue segment. In some embodiments, the continuous valine residue segment comprises 5 to 25 valine residues. In some embodiments, the first TMD comprises 15 to 20 valine residues.In some embodiments, the lysine residue is located at position 9 or 10 of the first TMD.

[0024] In one aspect, the present invention also provides a multi-chain chimeric polypeptide, which comprises (a) a first polypeptide, the first polypeptide having (i) the extracellular ligand binding domain comprising CD19scFv, the first TMD comprising a continuous valine residue stretch comprising lysine or arginine residues, the first polypeptide also comprising a Notch 2 membrane-proximal domain and the first intracellular domain comprising a Gal4VP64 transcriptional regulatory factor; a second polypeptide, the second polypeptide comprising a CD3z signaling domain, and wherein the first polypeptide and the second polypeptide are coupled via a lysine residue within the continuous valine residue stretch, and wherein binding of CD19 to the extracellular ligand binding domain simultaneously induces the activity of the signaling domain and releases the transcriptional regulatory factor.

[0025] In one aspect, the present invention also provides a multi-chain chimeric polypeptide, comprising (a) a first polypeptide, the first polypeptide having (i) an extracellular ligand binding domain comprising a CD19scFv, the first TMD comprising a continuous valine residue stretch comprising a lysine or arginine residue, the first polypeptide further comprising a Notch 2 juxtamembrane domain and the first intracellular domain comprising a human or humanized transcriptional regulator; a second polypeptide, the second polypeptide comprising DNAX activating protein 12 (DAP12) or CD3z, and wherein the first polypeptide and the second polypeptide are coupled via a lysine residue within the continuous valine residue stretch, and wherein binding of CD19 to the extracellular ligand binding domain simultaneously induces the activity of the signaling domain and releases 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 immune receptor. In some embodiments, the immune receptor is a chimeric antigen receptor.

[0028] 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 transmembrane domain (TMD) comprising a first modification interface, and (iii) a first intracellular domain comprising a transcriptional regulator or a 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 modification interface and the second modification interface each comprise amino acid residues having opposite charges, and wherein the first polypeptide is coupled to the second polypeptide via the first modification interface and the second interface, and wherein binding of the selected ligand to the extracellular ligand-binding domain induces the activity of the signaling domain and the release of the transcriptional regulator.

[0029] In some embodiments, the first intracellular domain comprises a transcriptional regulator. In some embodiments, the transcriptional regulator comprises Ga14-VP16, Ga14-VP64, tetR-VP64, ZFHD1-VP64, Ga14-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. In some embodiments, the signaling domain comprises one or more of the following: CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, CD66d, 4-1BB, or a common γ chain signaling domain.

[0031] In some embodiments, the signaling domain of the second polypeptide comprises a signaling domain comprising a CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, CD66d, or a 4-1BB signaling domain.

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

[0033] In some embodiments, binding of the selected ligand to the extracellular ligand binding domain simultaneously induces activity 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) the extracellular ligand-binding domain, (ii) the first TMD, and (iii) the first intracellular domain.

[0035] In some embodiments, the first TMD comprises: (i) 10 to 25 consecutive valine residues, or (ii) a Notch 1 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) the second TMD and (ii) the second intracellular domain.

[0037] In some embodiments, the first modification interface comprises positively charged residues, and wherein the second interface comprises negatively charged residues, and wherein the first polypeptide and the second polypeptide are coupled via electrostatic forces between the first modification interface and the second modification interface. In some embodiments, the positively charged residues are 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 located 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 located at a position selected from positions 8 to 11 of SEQ ID NO: 18. In some embodiments, the lysine or arginine residue is located at (i) position 12 of SEQ ID NO: 21, or (ii) 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 cell surface. In some embodiments, the antigen binding portion is selected from antibodies, nanobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, F(ab) fragments, single chain variable fragments (scFv), single domain antibodies (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 ligands include 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, placental alkaline phosphatase-like protein 2 (ALPPL2), B cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein alpha (SIRPα).

[0044] In some embodiments, the cell is a human cell.

[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-induced proteolytic cleavage site, an autoproteolytic peptide sequence, a nuclear localization signal, and a membrane-proximal domain. In some embodiments, the membrane-proximal domain is a polydomain. In some embodiments, the polydomain includes a Notch-1 or Notch-2 membrane-proximal domain. In some embodiments, the autoproteolytic peptide sequence is from porcine Teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), eriovenous rhinitis virus (ERAV) 2A (E2A), beta tetrasomic virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), softening virus 2A (BmIFV2A), or a combination thereof.

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

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

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

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

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

[0052] In some embodiments, the autoproteolytic peptide is a Tetrasomal Betavirus 2A (T2A) peptide.

[0053] In some embodiments, the nucleic acid construct has a nucleic acid sequence that has 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 other aspects, the disclosure provides a vector comprising a recombinant nucleic acid molecule of the 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 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, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, and other T cells. 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 the recombinant cell of the present disclosure and a pharmaceutically acceptable excipient.

[0057] Also provided herein is a method for simultaneously inducing T cell signaling and gene regulation in T cells, the method comprising (a) providing a T cell having a multi-chain chimeric polypeptide or 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 the transcriptional regulator.

[0058] In another aspect, the present disclosure provides a method for simultaneously inducing T cell signaling and gene regulation in T cells, 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 T cells with one or more vectors, wherein the binding of a selected ligand to the extracellular ligand binding domain simultaneously induces intracellular signaling and the release of the transcriptional regulator. In some embodiments, the induced intracellular signaling of the T cells regulates the expression of selected genes involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, molecule secretion, cell adhesion and / or cytolytic activity.

[0059] In another 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 enhances intracellular signaling.

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

[0061] In some embodiments, the released transcriptional regulator regulates the expression of the 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 immune receptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immune activator, an immunosuppressant, or an inhibitory immune receptor.

[0062] In another aspect, provided herein is a method for treating a health condition of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant cell or a pharmaceutical composition of the present disclosure, wherein the recombinant cell or the pharmaceutical composition treats the health condition of the subject. In some embodiments, the administered recombinant cell modulates the activity of a target cell in an individual. In some embodiments, the activity of the target cell includes expression, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, molecular secretion, cell adhesion, and cytolytic activity of a selected gene. 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 regulating T cell activity, the method comprising (a) providing an effective amount of any one 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 and releases the transcriptional regulator, and simultaneously (ii) activates T cell signaling, wherein the released transcriptional regulator regulates the activity of the recombinant cell.

[0064] The foregoing summary is illustrative only and is not intended to be limiting in any way. In addition to the illustrative embodiments and features described herein, other aspects, embodiments, objects, and features of the present disclosure will become fully apparent from the drawings, detailed description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 A schematic diagram of a multi-chain chimeric polypeptide according to the present disclosure is shown, wherein the first chain / first polypeptide (right side) delivers transcriptional regulation via a transcription factor, and the second chain / second polypeptide (left side) delivers a signal transduction function. The first polypeptide comprises an extracellular ligand binding domain having binding affinity to a selected ligand, a first TMD having a first modified interface (positively charged amino acid residues), and a first ICD having a transcriptional regulator. The second polypeptide comprises a second TMD having a second interface (negatively charged amino acid residues) and a second ICD having a signal transduction domain. The coupling of the first polypeptide and the second polypeptide is carried out via the interaction between positively charged amino acid residues and negatively charged amino acid residues. The multi-chain chimeric polypeptide is activated by ligand (e.g., CD19) binding, resulting in the dissociation of a transcriptional regulator (e.g., Gal4VP64), which then enters the nucleus and activates the transcription of a gene (BFP in this example). A schematic diagram of a transcriptional regulation response box with a BFP gene is also shown.

[0066] Figure 2A-2D Schematically summarized are the results of experiments performed to evaluate the functionality of various chimeric multi-chain polypeptides / receptors in order to optimize multi-chain chimeric receptors that induce the activity of the signaling domain and the release of transcriptional regulators upon ligand binding to the extracellular ligand-binding domain. Figure 2A An embodiment of a multi-chain chimeric polypeptide (e.g., an immune receptor) referred to as 056 is shown. The 056 embodiment comprises i) a first polypeptide containing a CD19scFv binding domain, a TREM2 first TMD with a positively charged first modified interface, and a Gal4VP64 translational regulatory factor, and (ii) a second polypeptide chain containing a DAP12 with a negatively charged modified interface. The nucleic acid construct encoding the 056 embodiment (SEQ ID NO: 2) comprises DAP12, a T2A linker, and a cassette encoding the CD19scFV-TREM2-Gal4VP64 first polypeptide in the 5' to 3' direction. Figure 2BAnother design of a multi-chain chimeric polypeptide, denoted as 056C, is shown. The 056C embodiment comprises i) a first polypeptide comprising a CD19 scFv binding domain, a truncated CD8 α-hinge domain (labeled as CD8 Hinge 2), a Notch1 first TMD (Uniprot P46531 numbering Uniprot P46531 amino acids 1736 to 1757 with L1747K mutation) having a positively charged first modified interface (comprising a L1747K substitution), a Notch2 juxtamembrane domain, a Gal4VP64 translational regulator; and (ii) a second polypeptide chain comprising DAP12. The nucleic acid construct encoding the 056C embodiment (SEQ ID NO:4) comprises DAP12, a T2A linker and a box encoding a first polypeptide in the 5' to 3' direction, wherein the first polypeptide contains CD19scFV, a truncated CD8α hinge domain (CD8 hinge 2), Notch 1 with L1747K substitution (Uniprot P46531 amino acids 1736 to 1757 with L1747K mutation), Notch 2 juxtamembrane domain and Gal4VP64 transcriptional regulator. Figure 2C Different designs of multi-chain chimeric polypeptides (056D) are shown, comprising i) a first polypeptide containing a CD19scFv binding domain, a TREM2 hinge, a Notch1 first TMD (Uniprot P46531 amino acids 1736 to 1757 with a positively charged first modified interface (containing a L1747K substitution), a Notch2 juxtamembrane domain, a Gal4VP64 translation regulator; and (ii) a second polypeptide chain containing DAP12. The nucleic acid construct encoding the 056D embodiment (SEQ ID NO: 5) comprises DAP12, a T2A autoproteolytic peptide sequence, and a cassette encoding a CD19scFV-TREM2-Notch1 (Uniprot P46531 amino acids 1736 to 1757 with a L1747K substitution)-Gal4VP64 first polypeptide in the 5' to 3' direction. Figure 2DA multi-chain chimeric polypeptide represented as 056B is shown, comprising i) a first polypeptide having a CD19scFv binding domain, a TREM2 hinge, a Notch1 first TMD (containing a γ-secretase active site) having a first modified interface with a positive charge, a Notch2 proximal membrane domain, and a Gal4VP64 translational regulatory factor; and (ii) a second polypeptide chain (056B) containing DAP12. The nucleic acid construct encoding the 056B embodiment (SEQ ID NO: 3) comprises DAP12, a T2A autoproteinase peptide sequence, and a box encoding the CD19scFV-TREM2-Notch1 insert with a γ-secretase active site-TREM2 intracellular domain sequence-Gal4VP64 first polypeptide in the 5' to 3' direction. A comparison of the expression levels of four multi-chain receptors 056, 056C, 056D, and 056B is also shown. Among the four designs, 056C has the strongest transcriptional activity. Figure 2A-2D The bottom graph in each of the Figures shows the relative expression of the four receptors measured by anti-myc tag staining (representing the second chain (y-axis)) 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 a lentiviral construct (two may also be used, 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). DAP12 / TREM immune receptor (056) and DAP12 / hinge Notch hybrid (056C) showed some expression, while DAP12 / TREM-Notch hybrid 1 (056D) and DAP12 / TREM-Notch hybrid 2 (056B) did not show expression.

[0067] Figure 3A-3C Depicted in Figure 2A The design between the 056 and 056C multi-chain chimeric polypeptides described in ( Figure 3A ), activation dynamics ( Figure 3B ) and killing activity ( Figure 3C ) comparison. Figure 3B Shown is receptor transcriptional activation of the inducible BFP reporter gene measured using Fortessa X-50 (BD Sciences) in T cells expressing the anti-CD19 receptor in the presence of (i) no additional cells (upper trace), (ii) K562 cells (middle trace) or (iii) CD19+K562 cells (lower trace). Figure 3C A comparison of the killing activity of the 056 and 056C receptors is also shown. The 056C receptor has higher transcriptional activity than the 056 receptor, but does not produce a strong killing response.

[0068] Figure 4A-4D Additional designs of multi-chain chimeric polypeptides (056, 056E, 056F and 056G) are described. Figure 4A Receptor 056 is shown, which is Figure 2A Same as described in, Figure 4B Describe the design of a multi-chain chimeric polypeptide (056E), wherein the second polypeptide comprises DAP12 and Gal4VP64 transcriptional regulators, and the first polypeptide comprises a CD19scFv ligand binding domain, a TREM2 transmembrane domain, and an intracellular domain. The 056E receptor is encoded by a nucleic acid construct having DAP12, a Gal4VP64 box connected (via a T2A linker) to a box having CD19scFV, a TREM2 transmembrane domain, and an intracellular domain. This design has limited relative expression. Figure 4C An embodiment (056F) of a multi-chain chimeric polypeptide according to the present disclosure is depicted, wherein the 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 the second polypeptide comprises a DAP12 signaling domain. The 056F embodiment is encoded by a nucleic acid construct having DAP12 in the 5' to 3' direction, 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 An embodiment (056G) similar to the 056F embodiment is shown, except that the latter contains a polyvaline TMD with a V10K substitution. The 056F and 056G embodiments with the polyvaline TMD exhibited increased expression compared to the other embodiments.

[0069] Figure 5A-5D Depicted Figure 4A-4D Comparison of the killing activity (target clearance) and activation kinetics of the embodiments 056, 056E, 056F and 056G of the multi-chain polypeptide described in . Embodiments 056 and 056E did not show strong transcriptional activation, but were able to show killing activity of target cells. Embodiments 056F and 056G showed strong transcriptional activation, but only 056F achieved target killing.

[0070] Figure 6 A schematic diagram of the multi-chain receptor module engineering strategy using pRay056F as a prototype is shown.

[0071] Figure 7 shows the assay for multi-chain receptor function with CD3z substitution.

[0072] Figure 8 shows an assay for multi-chain receptor function with human transcription factors.

[0073] Fig. 9 A schematic diagram of a multichain receptor employing a modular engineering strategy using dual vector transduction is shown.

[0074] Fig.10 The expression profile of each multichain receptor pair is shown.

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

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

[0077] Fig.13 Screening of multi-chain receptors that enhance killing and survival is shown. The graph shows T cell counts on days 5, 12, and 20 at an ET ratio of 1:4.

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

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

[0080] Fig.16 Screening of multi-chain receptors that enhance killing and survival is shown. The graph shows K562 cell counts at days 5, 12, and 20 at an ET ratio of 1:4. DETAILED DESCRIPTION

[0081] It is well known in the art of chimeric polypeptides (e.g., receptors) that proximity to the membrane is important when adding a signaling domain and has an impact on how well the domain signals. The present disclosure provides chimeric polypeptides / receptors that have an architecture that is different than that generally known in the art and that allows for more control over where the signaling motif is located. The chimeric polypeptides / receptors of the present disclosure have a more controllable relationship to the membrane than previously known.

[0082] Thus, the present disclosure provides, among other things, multi-chain chimeric polypeptides having two different polypeptides (chains) such that a transcription factor is located on a first polypeptide close to the membrane and a signaling domain is located on a second polypeptide. The first and second polypeptides can associate to allow for simultaneous regulation of signaling (e.g., T cell signaling) from one chain and transcriptional regulation from the other chain in a ligand-dependent manner upon binding of a selected ligand to one of the polypeptides.

[0083] In particular, the disclosure relates to multi-chain chimeric polypeptides with different polypeptide chains that can associate post-translationally via residues located within the TMD of a first polypeptide and a second polypeptide. Binding of a ligand to an extracellular ligand binding domain (ECD) of a first polypeptide can activate cell signaling and release of transcriptional regulators from cleavage of the first polypeptide. Thus, cleaved transcriptional regulators can function without any other fused signaling domains, and vice versa.

[0084] In some embodiments, the multi-chain chimeric polypeptides presented herein can have the following unique architecture, wherein the first polypeptide can comprise (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. The second polypeptide can comprise (i) a second TMD having a second interface and (ii) a second intracellular domain having a signaling domain. The first polypeptide and the second polypeptide can associate via charged residues within the first modified interface and the second interface. Binding of the selected ligand to the extracellular ligand binding domain can simultaneously induce the activity of the signaling domain and the release of the transcriptional regulator.

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

[0086] The multi-chain chimeric polypeptides of the present disclosure constitute a platform for engineering T cells, which can not only detect and activate responses to tumors, but also can start one or more transcriptional programs, such as producing various biological agents, cytokines, inflammatory factors, regulatory RNAs, etc. The multi-chain chimeric polypeptides of the present disclosure allow receptor capabilities to be coupled with defined auxiliary functions, which are generally not encoded or triggered by signal transduction domains constructed in synthetic receptors of the prior art. Therefore, the multi-chain chimeric polypeptides of the present disclosure can achieve a broadened method to positively modify T cell function in a manner different from that known to date. 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 guide T cell differentiation into 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 can promote and achieve more immediate signaling capabilities of receptors that previously only linked antigen recognition to transcriptional responses (i.e., without other signaling effects). By incorporating signaling domains in the multi-chain form of the present disclosure, those skilled in the art can effectively couple auxiliary functions to transcriptional responses and activate both simultaneously in response to the same antigen. Auxiliary functions can include, for example, cell destruction (by using immunoreceptor tyrosine-based activation motif (ITAM) domains on the signaling chain) or by using costimulatory domains (such as 41BB, CD28 costimulatory domains, etc.) to promote survival signaling.

[0088] In the following detailed description, reference is made to the accompanying drawings which form a part thereof. In the accompanying drawings, similar symbols generally identify similar components unless the context otherwise dictates. The illustrative alternatives described in the detailed description, drawings, and claims are not intended 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 accompanying drawings may be arranged, substituted, combined, and designed in a variety of different configurations, all of which are expressly contemplated and constitute a part of this application. I. Definitions

[0089] Unless otherwise defined, all technical terms, symbols and other scientific terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein is not necessarily to be construed as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referred to herein are well understood by those skilled in the art and are commonly employed by those skilled in the art using conventional methods.

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

[0091] In the case of providing a range of values, it should be understood that unless the context clearly stipulates otherwise, each intermediate value (to one tenth of the unit of the lower limit) between the upper and lower limits of the range and any other stated or intermediate values ​​in the stated range are all included in the present disclosure. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the present disclosure, belonging to any explicitly excluded limits in the stated range. When the stated range includes one or two limits, the scope excluding any one or two of those included limits is also included in the present disclosure. Certain ranges are presented in this article with a numerical value preceded by the term "about", as used herein, and the term "about" has its approximately common meaning. The term "about" is used to provide textual support for the exact number thereafter and the number close to or approximating the number after the term. When determining whether a number is close to or approximating a specifically listed number, the close or approximate unlisted number can be a number that provides a substantially equivalent form of the number specifically listed in the context in which it is presented. If the approximation is not originally clear from the context, "about" means within ±10% of the provided value, or rounded to the nearest significant figure, in each case including the provided value. In some embodiments, the term "about" indicates a specified value ± at most 10%, at most ±5%, or at most ±1%.

[0092] As used herein, the terms "administration" and "administering" refer to the delivery of a biologically active composition or formulation by an administration route including, but not limited to, oral, intranasal, transdermal, intravenous, intraarterial, intramuscular, intranodal, intraperitoneal, subcutaneous, and intramuscular administration or a combination thereof. The terms include, but are not limited to, administration by a medical professional and self-administration.

[0093] It should be understood that terms such as "cell", "cell culture" and "cell line" refer not only to specific subject cells, cell cultures or cell lines, but also to the offspring or potential offspring of such cells, cell cultures or cell lines, without considering the number of transfers or passages in culture. It should be understood that not all progeny are exactly the same as parental cells. This is because certain modifications may occur in offspring due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so that offspring may actually be different from parental cells, but are still included in the scope of the terms as used herein, as long as the offspring retains the same functions as the original cells, cell cultures or cell lines.

[0094] It should be understood that aspects and embodiments of the disclosure described herein include "comprising" aspects and embodiments, "consisting of" and "consisting essentially of aspects and embodiments". As used herein, "comprising" is synonymous with "including", "containing" or "characterized by", and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. As used herein, "consisting of" excludes any 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 expression of the term "comprising" herein, particularly in the description of the components of the composition or in the description of the steps of the method, should be understood to cover those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0095] The term "cancer" refers to the presence of cells with typical characteristics of cancer cells (such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological characteristics). Some types of cancer cells can aggregate into masses, such as tumors, but some cancer cells can exist alone in the subject. Tumors can be solid tumors, soft tissue tumors, or metastatic lesions. 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 leukemias, lymphomas, and myelomas. Cancer can include pre-malignant cancers as well as malignant cancers.

[0096] The term "nucleic acid" is used herein to refer to DNA or RNA, or molecules containing deoxynucleotides and / or ribonucleotides. Nucleic acids may be naturally occurring or prepared synthetically, and thus include analogs of naturally occurring polynucleotides in which one or more nucleotides are modified based on naturally occurring nucleotides.

[0097] As used herein, the term "operably linked" refers to a physical or functional connection between two or more elements (eg, polypeptide sequences or polynucleotide sequences) that permits them to operate in their intended manner.

[0098] In the context of two or more nucleic acid sequences or proteins, the term "percent identity" as used herein refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (e.g., about 50% sequence identity or higher, such as 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 higher identity, when compared and aligned over a comparison window or a specified region to obtain maximum correspondence), as measured using, for example, the National Center for Biotechnology (NCBI) BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below or by manual alignment and visual inspection. Such sequences are then referred to as "substantially identical". This definition also relates to or can be applied to the complement of a sequence. This definition also includes sequences with deletions and / or additions and those with substitutions. Sequence identity can be calculated using published techniques and publicly available computer programs such as the GCS package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990) (incorporated herein by reference in its entirety). Sequence identity can be measured using sequence analysis software using its default parameters, such as the sequence analysis software package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705). 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 (including all values ​​therebetween) to the reference amino acid sequence. For polypeptides, the length of the comparison sequence will generally be 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 consecutive amino acids (e.g., the full-length sequence) (including all values ​​therebetween).For nucleic acids, the length of comparison sequences will typically 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 (e.g., the full length of the nucleotide sequence), including all values ​​therebetween.

[0099] As used herein, "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 doctor. Therefore, the subject can be a human patient or individual suffering from, at risk of suffering from, or suspected of suffering from one or more symptoms of a disease of interest (e.g., cancer) and / or a disease. The subject can also be an individual diagnosed as having a risk of a disease of interest at or after diagnosis. The term "non-human animal" includes all vertebrates, such as mammals (e.g., rodents, such as mice) and non-mammals, such as non-human primates, such as sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0100] When used in relation to nucleic acids, the term "recombinant" means that the nucleic acid has been altered or produced by human intervention, such as, for example, has been modified by a laboratory method or is the result of a laboratory method. Thus, for example, a recombinant nucleic acid comprises a viral genome and a nucleic acid produced by a laboratory method. A recombinant protein or recombinant polypeptide produced by a recombinant construct may contain amino acid residues not found in the natural (non-recombinant or wild-type) form of the protein, or may contain amino acid residues that have been modified (e.g., labeled). The term may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical modification of a peptide, protein, or nucleic acid sequence, including chemical modification of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; production of fusion proteins (e.g., fusion proteins comprising antibody fragments); and 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 may be a polypeptide that: 1) has been synthesized or modified in vitro, e.g., using chemical or enzymatic techniques; 2) comprises linked polypeptide sequences that are not linked in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more amino acids relative to a naturally occurring polypeptide sequence; and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements relative to a naturally occurring polypeptide.

[0102] As will be understood by those of ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be identified as fully describing the same range and enabling the same range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those of ordinary skill in the art, all such as "up to", "at least", "greater than", "less than", etc., include the numbers and relate to the ranges that can subsequently be decomposed into sub-ranges as described above. Finally, as will be understood by those of ordinary skill in the art, the range includes each individual member. Therefore, for example, a group having 1-3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to a group having 1, 2, 3, 4, or 5 articles, etc.

[0103] It should be understood that certain features of the present disclosure described in the context of a separate embodiment for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to the present disclosure are specifically encompassed in the present disclosure and disclosed herein as if each and every combination were individually and clearly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed in the present disclosure and disclosed herein as if each and every such sub-combination were individually and clearly 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 for clarity, the present disclosure may be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment. II. Composition A. Multi-chain chimeric polypeptides

[0105] The present disclosure provides, among other things, multi-chain chimeric polypeptides comprising two polypeptide chains. A multi-chain polypeptide can be a functional polypeptide consisting of two or more discrete polypeptide elements (i.e., "chains") that are covalently or non-covalently linked together by molecular association rather than by peptide bonding. The chains of a multi-chain polypeptide can be structurally different. The chains of a multi-chain polypeptide can be functionally different. The present disclosure provides a multi-chain chimeric polypeptide, wherein one polypeptide (a first polypeptide) comprises (i) an extracellular ligand binding domain with binding affinity for a selected ligand, (ii) a first TMD, and (iii) a first intracellular domain having a transcriptional regulator. Another polypeptide (a second polypeptide) comprises (i) a TMD and (ii) an intracellular domain having a signaling domain. The first polypeptide is activated by the binding of a ligand to an extracellular ligand binding domain, which thereafter results in the release of a transcriptional regulator from the first polypeptide and cell signaling via a signaling domain on the second polypeptide. The two polypeptides are associated together, for example, by oppositely charged residues in the TMD.

[0106] The multi-chain chimeric polypeptides disclosed herein can act as receptors and promote the activation of cell signaling or amplification of cellular pathways in certain cell and environmental contexts. Such receptor activity can be used to enhance and regulate 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 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 TMD comprising a first modification interface, and (iii) a first intracellular domain comprising a transcriptional regulator; 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 modification interface and the second modification interface each comprise amino acid residues having opposite charges, such that the first polypeptide is coupled to the second polypeptide via the first modification interface and the second interface, and wherein binding of the selected ligand to the extracellular ligand binding domain induces the activity of the signaling domain and the release of the transcriptional regulator. In some embodiments of the multi-chain chimeric polypeptides of the present disclosure, the first modification interface comprises positively charged residues, the second interface comprises negatively charged residues, and the first polypeptide is coupled to the second polypeptide via electrostatic forces between the first modification interface and the second modification interface.

[0108] In another aspect, provided herein are multi-chain chimeric polypeptides capable of achieving simultaneous transcriptional regulation and cellular activation (e.g., T cell activation) upon binding of a selected ligand to the extracellular ligand binding domain of the 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 immune receptor. In some embodiments, the immune receptor is a chimeric antigen receptor (CAR). 1. The first polypeptide

[0110] The multi-chain polypeptide of the present disclosure comprises a first chimeric polypeptide containing an extracellular ligand binding domain (part) having a 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 described herein, an "interface" refers to outward-facing residues within the transmembrane domain of a first polypeptide that interact with outward-facing residues within the transmembrane domain of another polypeptide. In some embodiments, the outward-facing residues within the transmembrane domain of the first polypeptide form a charged surface for interfacial interaction with a partner from a separate protein that displays a surface with an opposite charge.

[0111] In some embodiments, the 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 first 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. a. Extracellular ligand binding domain (ECD)

[0113] In some embodiments, the extracellular domain (ECD) of the first polypeptide of the multi-chain chimeric polypeptide disclosed herein has binding affinity to one or more target ligands. The target ligand can be expressed on the cell surface, or otherwise anchored, fixed or restricted, so that it can apply mechanical force to the chimeric receptor. In this way, without being bound by any particular theory, the binding of the ECD of the first polypeptide of the multi-chain chimeric polypeptide provided herein to the cell surface ligand does not necessarily remove the target ligand from the target cell surface, but generates mechanical pulling force on the chimeric receptor. For example, if other soluble ligands are bound to molecules in the surface or extracellular matrix, the other soluble ligands can be targeted.

[0114] In some embodiments, the target 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 a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the ligand is a differentiation cluster ligand (CD) and includes, but is not limited to, 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, placental alkaline phosphatase-like protein 2 (ALPPL2), B cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein alpha (SIRPα).

[0115] In some embodiments, the extracellular domain of the first polypeptide of the multi-chain chimeric polypeptide of the present disclosure includes an antigen binding portion. In some embodiments, the antigen binding portion can bind to a ligand on the cell surface. In some embodiments, the antigen binding portion can bind to one or more targets. In some embodiments, the antigen binding portion includes one or more antigen binding determinants of an antibody or its functional antigen binding fragment. It will be readily understood by those skilled in the art after reading the present disclosure that the term "functional fragment thereof" or "functional variant thereof" refers to a molecule having a common 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 is a functional fragment or functional variant that retains substantially 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 the retention of its epitope binding activity can be assessed using assays known to those skilled 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), a single chain variable fragment (scFv), a single domain antibody (sdAb), and any functional fragment thereof.

[0116] Antigen binding portion can comprise naturally occurring amino acid sequence or can be engineered, designed or modified to provide desired and / or improved characteristics, such as binding affinity. Generally, the binding affinity of antigen binding portion (e.g., antibody) to 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, binding affinity is measured by antigen / antibody dissociation rate. In some embodiments, binding affinity is measured by competitive radioimmunoassay. In some embodiments, binding affinity is measured by ELISA. In some embodiments, antibody affinity is measured by flow cytometry.

[0117] An antibody that "selectively binds" an antigen (such as CD19) is an antigen-binding portion that does not substantially bind other antigens but binds the antigen with high affinity, for example, with an equilibrium constant (KD) of 100 nM or less, such as 60 nM or less, for example 30 nM or less, such as 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, 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.

[0118] The technician can select the ECD based on the desired positioning or function of the cell that is genetically modified to express the multi-chain chimeric polypeptide or the first polypeptide of the present disclosure. For example, a first polypeptide having an ECD that includes an antibody specific for the HER2 antigen can target cells to breast cancer cells that express HER2. In some embodiments, the ECD of the disclosed first polypeptide is capable of binding to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). It will be understood by the skilled artisan that TAAs include molecules, such as proteins, that are present on tumor cells and normal cells or on many normal cells but at concentrations much lower than those on tumor cells. In contrast, TSAs typically include molecules, such as proteins, that are present on tumor cells but not on normal cells.

[0119] In some cases, the antigen binding portion is specific for an epitope present in an antigen (i.e., a tumor-associated antigen) expressed by a tumor cell. Tumor-associated antigens can be antigens related to, for example, breast cancer cells, B cell lymphomas, 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, gliomas, glioblastomas, colorectal cancer cells, etc. It will also be understood that tumor-associated antigens can also 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), placental alkaline phosphatase-like protein 2 (ALPPL2), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein alpha (SIRPα), CD123, CD171, CD179α, 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, GPRC5D, HER2(ERBB2 / neu), IGLL1, IL-11Rα, KIT(CD117), MUC1, NCAM, PAP, PDGFR-β, 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 selected from CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179α, 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, GPRC5D, HER2(ERBB2 / neu), IGLL1, IL-11Ra, KIT(CD117), MUC1, NCAM, PAP, PDGFR-β, PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72, TEM1 / CD2 48, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, Axl, GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL-13Rα1, IL-13Rα2, alpha-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), macrocystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melanin-A (melanoma antigen recognized by T lymphocytes;MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptic vesicle protein, thyroglobulin, thyroid transcription factor-1, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basic immunoglobulin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (subunit of heterodimeric IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, 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 α4, integrin α4β7, LFA-1 (CD11α), myostatin, OX-40, scleroscin, SOST, TGFβ1, TNF-α, VEGF-A, pyruvate kinase isoenzyme M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBC1, TRB C2, BCMA, CD38, CD123, CD93, CD34, CD1a, SLAMF7 / CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, kappa light chain, lambda light chain, CD16 / FcγRIII, CD64, FITC, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and its isoforms, TEM-8, sperm protein 17 (Sp17), mesothelin. ;

[0122] Additional non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma variable reading frame protein), Trp-p8, STEAP1 (prostatic six transmembrane epithelial antigen 1), abnormal ras protein, abnormal p53 protein, integrin beta 3 (CD61), prolactin, K-Ras (V-Ki-ras2 Colston rat sarcoma viral oncogene), Ral-B, GPC2, CD276 (B7H3) or IL-13Rα. 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 reporter proteins such as GFP and eGFP. Non-limiting examples of such antigen binding portions include LaG17 anti-GFP nanobodies. In some embodiments, the antigen binding portion of the ECD includes an anti-BCMA fully humanized VH domain (FHVH). In some embodiments, the antigen is signal regulatory protein alpha (SIRPα).

[0123] Additional antigens suitable for targeting by the first chimeric polypeptide and multi-chain CAR disclosed herein include, but are not limited to, GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL-13Rα1, IL-13Rα2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), and 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), macrocystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melanin-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptic vesicle protein, thyroglobulin, and thyroid transcription factor-1.

[0124] Additional antigens suitable for targeting by the polypeptides disclosed herein include, but are not limited to, those associated with inflammatory diseases, such as AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basal immunoglobulin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a subunit of the heteromeric IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, 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 α4, integrin α4β7, LFA-1 (CD11α), myostatin, OX-40, sclerostin, SOST, TGFβ1, TNF-α, and VEGF-A.

[0125] Other antigens suitable for targeting by the first chimeric polypeptide and multi-chain CAR disclosed herein include, but are not limited to, pyruvate kinase isozyme M2 type (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 / FcγRIII, CD64, FITC, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and its isotype variants, TEM-8, sperm protein 17 (Sp17), and mesothelin. Additional 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 (prostatic six transmembrane epithelial antigen 1), abnormal ras protein, abnormal p53 protein, integrin β3 (CD61), prolactin, K-Ras (V-Ki-ras2 Colston rat sarcoma viral oncogene) and Ral-B. In some embodiments, the antigen is GPC2, CD19, Her2 / neu, CD276 (B7H3), IL-13Rα1 or IL-13Rα2. 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 reporter proteins such as GFP and eGFP. Non-limiting examples of such antigen binding portions include LaG17 anti-GFP nanobodies. In some embodiments, the antigen binding portion of the ECD comprises an anti-BCMA fully humanized VH domain (FHVH).

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

[0127] In some embodiments, the antigen binding portion of the ECD is specific for a cell surface target, wherein non-limiting examples of cell surface targets 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 with an antigen binding portion that binds CD19, CEA, HER2, MUC1, CD20, ALPPL2, BCMA, or EGFR. In some embodiments, the multi-chain CAR provided herein comprises an extracellular domain containing an antigen binding portion that binds CD19. In some embodiments, the chimeric first polypeptide provided herein comprises an extracellular domain containing an antigen binding portion that binds ALPPL2. In some embodiments, the chimeric first polypeptide provided herein comprises an extracellular domain containing an antigen binding portion that binds BCMA. In some embodiments, the chimeric first polypeptide comprises an extracellular domain containing an antigen binding portion that binds Her2.

[0128] In some embodiments, antigens suitable for targeting by a chimeric first polypeptide disclosed herein include ligands derived from pathogens. b. The linker sequence between the ECD and the first transmembrane domain (TMD)

[0129] The multi-chain chimeric polypeptides and receptors of the present disclosure may include a linker sequence disposed between the ECD and the TMD. The linker sequence may be a natural or synthetic polypeptide. The linker sequence may be a flexible linker that provides structural flexibility and spacing from the flanking polypeptide region. The linker sequence may be a hinge domain inserted into the TMD at the N-terminus. 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%, 99% sequence identity to SEQ ID NO: 17. In some embodiments, the truncated CD8α comprises an amino acid sequence containing SEQ ID NO: 17. In some embodiments, the truncated CD8α is encoded by SEQ ID NO: 17.

[0130] The hinge polypeptide sequence suitable for the compositions and methods of the present disclosure can be a naturally occurring hinge polypeptide sequence (e.g., from those of naturally occurring immunoglobulins). Alternatively, the hinge polypeptide sequence can be a synthetic sequence corresponding to a naturally occurring hinge polypeptide sequence, or can be a fully synthetic hinge sequence, or can be engineered, designed or modified to provide desired and / or improved properties, such as regulating transcription. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD and IgG subclasses, such as IgG1 hinge domains, IgG2 hinge domains, IgG3 hinge domains and IgG4 hinge domains or their functional variants. 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 on this point can be found in, for example, Vidarsson G et al., Frontiers Immunol. 2014 October 20th recent review, the document is hereby incorporated by reference in its entirety.

[0131] Therefore, 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 CD8α hinge domains, CD28 hinge domains, CD152 hinge domains, PD-1 hinge domains, CTLA4 hinge domains, OX40 hinge domains, FcγRIIIα hinge domains, and functional variants thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from a CD8α hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from a CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain comprises a hinge polypeptide sequence derived from an OX40 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.

[0133] In principle, except that the hinge domain should be given flexibility, there is no particular restriction on its length and / or amino acid composition. However, it will be readily understood by those skilled in the art that the length and amino acid composition of the hinge polypeptide sequence can be optimized to change the orientation and / or proximity of ECD and TMD relative to each other, and the orientation and / or proximity of the first polypeptide and the second polypeptide relative to each other, to achieve the desired activity of the chimeric multi-chain polypeptide of the present disclosure. In some embodiments, any arbitrary single-chain peptide comprising about one 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, etc.) can be used as the hinge domain. In some embodiments, the hinge domain comprises about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the hinge domain comprises about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the hinge domain comprises about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the hinge domain comprises about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the hinge domain comprises a sequence having at least 80% sequence identity (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) to SEQ ID NO:17. c. First transmembrane domain

[0134] The multi-chain chimeric polypeptide of the present disclosure comprises a transmembrane domain (e.g., a first TMD) located in 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 comprises a continuous segment of valine residues. In some embodiments, the continuous segment of valine residues comprises 5 to 25 valine residues. In some embodiments, the first TMD comprises a continuous segment of 5 to 25 valine residues. In some embodiments, the first TMD comprises a continuous segment 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 valine residues. An example of a first TMD with a continuous segment of valine residues (referred to as a poly-V TMD) is shown in Figure 4C and Figure 4D, which respectively depict embodiments 056F and 056G of the multi-chain chimeric polypeptides of the present disclosure.

[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 in a continuous valine residue segment. In some embodiments, the positively charged residue is located after the first valine residue from N-terminal. In some embodiments, the positively charged residue is located after the second, or third, or fourth, or fifth, or sixth, or seventh, or eighth, or ninth, or tenth, or eleventh, or twelfth, or thirteenth, or fourteenth, or fifteenth, or sixteenth, or seventeenth, or eighteenth, or nineteenth, or twentieth, or twenty-first, or twenty-second, or twenty-third valine residue from N-terminal. In some embodiments, lysine or arginine residues replace the sixth, or seventh, or eighth, or ninth, or tenth, or eleventh, or twelfth valine residue from N-terminal. 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 residue number 8 or residue number 9 or residue number 10 or residue number 11 of SEQ ID NO: 18, or at a position corresponding to residue number 8 or residue number 9 or residue number 10 or residue number 11 of SEQ ID NO: 18.

[0136] The first TMD of the multi-chain chimeric polypeptide of the present disclosure may include a TMD from a Notch receptor. In some embodiments, the TMD includes a Notch 1 receptor ( Figure 2B-Figure 2D Depicted are embodiments 056C, 056D, and 056B of the multi-chain chimeric polypeptides of the present disclosure). In some embodiments, the Notch 1 receptor is a human Notch 1 receptor.

[0137] The first TMD may comprise one or more ligand-inducible proteolytic cleavage sites. Examples of ligand-inducible proteolytic cleavage sites in Notch receptors (e.g., S2 or S3) are described in U.S. Patent No. 11,202,801, which is incorporated herein by reference in its entirety. In some embodiments, the ligand-inducible proteolytic cleavage site may be cleaved by γ-secretase (a multiprotein enzyme complex), such as in, for example, Embodiment 056B ( Figure 2D ) as described in ).

[0138] The first polypeptide of the multi-chain chimeric polypeptide of the present disclosure may include a membrane-proximal domain (JMD). The JMD may be located at the C-terminus of the TMD. The JMD may include a highly charged domain. In some embodiments, the JMD is a Notch 2 JMD.

[0139] In principle, there is no particular limitation on the length and / or amino acid composition of the JMD. Any single-chain peptide comprising 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 the JMD. In some embodiments, the JMD comprises 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, such as 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 includes Notch-1 or Notch-2. In some embodiments, the polybasic domain comprises an amino acid sequence in which a majority (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%) of the residues are lysine and / or arginine and / or histidine and / or any combination thereof.

[0141] The JMD may comprise a sequence having at least 70% sequence identity, such as 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 sequence 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 having only Lys (K) or Arg (R) in the first 4 residues. In some embodiments, the JMD comprises one, two, three, four, five, or more basic residues. In some embodiments, the JMD comprises five, four, three, two, one, or zero aromatic residues or residues with hydrophobic and / or bulky side chains. d. First intracellular domain and transcriptional regulator

[0142] The intracellular domain of the first polypeptide of the multi-chain chimeric polypeptide of the present disclosure may include a transcriptional regulatory factor. The transcriptional regulatory factor of the present disclosure may be a polypeptide element that is used to activate or inhibit the transcription of a promoter-driven DNA sequence. The transcriptional regulatory factor suitable for the composition and method of the present disclosure may be a naturally occurring transcriptional regulatory factor or may be engineered, designed or modified to provide desired and / or improved properties, for example, to regulate transcription. As discussed above, the advantage of the engineered receptors of the present disclosure is that they can provide the ability to trigger a customized transcriptional program in an engineered cell. In some embodiments, the transcriptional regulatory factor of the present disclosure is a customized transcriptional regulatory factor that drives the transcription of a specific sequence that only occurs once in an engineered cell. In some embodiments, the transcriptional regulatory factor of the present disclosure includes a human or humanized sequence.

[0143] In some embodiments, the transcriptional regulatory factor directly regulates the differentiation of cells. In some embodiments, the transcriptional regulatory factor indirectly regulates (e.g., regulates) the differentiation of cells by regulating the expression of a second transcription factor. It will be understood by those of ordinary skill in the art that the transcriptional regulatory factor can be a transcriptional activator or a transcriptional repressor. In some embodiments, the transcriptional regulatory factor is a transcriptional repressor. In some embodiments, the transcriptional regulatory factor is a transcriptional activator. In some exemplary embodiments, the transcriptional regulatory factor of the chimeric receptor of the present disclosure includes a transcription factor DNA binding domain (DBD). Exemplary DBDs may include Gal4, tetR, ZFHD1, Zif268, and HAP1. In some exemplary embodiments, at least one transcriptional regulatory factor of the chimeric receptor of the present disclosure also includes a transactivation domain (TAD). In some embodiments, the transcriptional regulatory factor may also include a nuclear localization signal.

[0144] In some embodiments, the transcriptional regulator of the chimeric receptor of the present disclosure includes a zinc finger-containing transcriptional effector (ZTE) comprising one or more zinc finger proteins or zinc finger motifs (ZFs). For example, the transcriptional regulator of the chimeric receptor of the present disclosure may include a DBD containing one or more ZFs. ZFs are finger-like folds in proteins that allow them to interact with nucleic acid sequences such as DNA and RNA. Such finger-like folds are well known in the art. The fold is usually produced by the binding of specific amino acids in the protein to zinc atoms and is stabilized by the coordination of zinc ions between four essentially unchanged (depending on the type of zinc finger framework) Cys and / or His residues. Exemplary zinc finger proteins may include ZF3, ZF6, ZF10, etc.

[0145] As used herein, the term "motif" refers to a structural motif. The ZF motif is a relatively small polypeptide domain with a super secondary structure, and comprises about 30 amino acids and folds to form an alpha-helix adjacent to an antiparallel beta-sheet (called a beta beta alpha fold), and is stabilized by zinc ions. The ZF domain recognizes and binds to nucleic acid triplets or overlapping quadruplets (as described below) in a double-stranded DNA target sequence. Naturally occurring zinc finger domains (also referred to 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 viral assembly. Additional information in this regard can be found in, for example, U.S. Patent No. 10,138,493.

[0146] C2H2 zinc fingers (C2H2-ZFs) are the most common class of vertebrate DNA-binding domains and are usually found in tandem arrays (ZFAs), where consecutive C2H2-ZFs each contact three (or more) consecutive bases. C2H2-ZFs can be assembled in a modular manner. Given that a set of modules has a defined three-base specificity, modular assembly also provides a method for constructing artificial proteins with specific DNA binding preferences.

[0147] ZF-containing proteins typically contain strings or chains of ZF motifs, forming a ZF array (ZFA). Thus, a ZF protein may comprise two or more ZFs, such as a ZFA consisting of 2 or more ZF motifs, which may be directly adjacent to each other (e.g., separated by a short linker sequence), or may be separated by a longer, flexible or structured polypeptide sequence. For example, a ZFA may have six ZF motifs (6-finger ZFA), seven ZF motifs (7-finger ZFA), or eight ZF motifs (8-finger ZFA) arranged in series. It is generally expected that directly adjacent ZF domains bind to continuous nucleic acid sequences, such as adjacent trinucleotides / triplets. In some cases, cross-linking may also occur between adjacent ZFs and their respective target triplets, which may help to strengthen or enhance recognition of the target sequence and result in binding of overlapping quadruplet sequences. By comparison, long-range ZF domains within the same protein may recognize and / or bind to non-continuous nucleic acid sequences or even different molecules (e.g., proteins instead of nucleic acids).

[0148] In some embodiments, the multi-chain receptors of the present disclosure comprise a zinc finger-containing transcriptional effector (ZTE) 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 further detail below, exemplary effector domains suitable for the multi-chain receptors of the present disclosure include, but are not limited to, transcriptional activation domains (e.g., TADs), epigenetic effector domains, and DNA modification enzymes.

[0149] In some embodiments, the multi-chain receptor of the present disclosure comprises a transcriptional effector having a non-zinc finger DNA binding domain (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 domains derived from PAX6. As described in further detail below, 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 modification enzymes. In some embodiments, the multi-chain receptor of the present disclosure comprises 1 to about 10 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 acceptor of the present disclosure comprises a ZTE having two or more, such as 3 or more, such as 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more (e.g., up to about 30 or 32) ZF motifs, which are arranged adjacent to each other in series to form an array or ZFA of ZF motifs. 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 continuous. In some embodiments, in a eukaryotic genome, the ZTE of the engineered Notch receptor disclosed herein binds to a target orthogonal specific DNA sequence, and, for example, has a reduced or minimal functional binding potential.

[0151] In some embodiments of the present disclosure, the ZTE comprises: (a) a first domain including a DNA-binding zinc finger protein domain (ZF 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 are each independently an integer from 0 to 5, and at least one of a and b is not 0; wherein the ZF protein domain comprises 1 to about 10 zinc finger arrays (ZFA); wherein the ZFA comprises about 1 to 3, about 3 to 6, or about 6 to about 8 zinc finger motifs according to Formula II (from N-terminus to C-terminus): XcCXdCXe-(helix)-HXfH-L2 (Formula II) wherein L2 is a linker peptide having about 4-6 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 forming an alpha-helix, wherein ZFA is capable of binding to a specific nucleic acid sequence.

[0152] In some embodiments, the ZF protein domain of the multi-chain receptor disclosed herein comprises 1 to about 10 ZFAs, each ZFA independently comprising a sequence having at least about 90% identity to a sequence selected from 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 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 / SV40 NLS). 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 / Notch1 NLS). 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 / SV40 NLS). 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 / Notch1 NLS). 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 receptor disclosed herein comprises 1 to about 10 ZFAs, each ZFA independently comprising a sequence having about 100% sequence identity with a sequence selected from SEQ ID NOs: 62-67. In some embodiments, the ZFA sequence has about 100% sequence identity with the sequence of SEQ ID NO: 62 (ZF3). In some embodiments, the ZFA sequence has about 100% sequence identity with the sequence of SEQ ID NO: 63 (ZF6). In some embodiments, the ZFA sequence has about 100% sequence identity with the sequence of SEQ ID NO: 64 (ZF6 / SV40NLS). In some embodiments, the ZFA sequence has about 100% sequence identity with the sequence of SEQ ID NO: 65 (ZF6 / Notch1NLS). In some embodiments, the ZFA sequence has about 100% sequence identity with 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 / Notch1 NLS). In some embodiments, the ZFA sequence has about 100% sequence identity to the sequence of SEQ ID NO: 67 (ZF10 / Notch1 NLS). 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 having the same amino acid sequence. In some embodiments, the ZF protein domain comprises multiple ZFAs having amino acid sequences that are different from each other.

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

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

[0157] In some embodiments, the transcriptional regulator is 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 VP64, p65, KRAB transactivation variants and VP16. In some exemplary embodiments, the transcriptional regulator has a sequence derived from the TAD of human or humanized p65. In some exemplary embodiments, the transcriptional regulator has a sequence derived from human or humanized p65. In other exemplary embodiments, the transcriptional regulator has a sequence derived from the 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 together, with or without a linker sequence.

[0159] In some embodiments, the transcriptional regulator comprises a sequence having at least 80% sequence identity (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) with one or more of SEQ ID NOs: 68-75. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 90% sequence identity with 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 with one or more of SEQ ID NOs: 68-75. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 10 ...

[0160] In some embodiments, the transcriptional regulator comprises a sequence having at least 80% sequence identity (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) with one or more of SEQ ID NOs: 76-90. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 90% sequence identity with 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 with one or more of SEQ ID NOs: 76-90. In some embodiments, the transcriptional regulator comprises an amino acid sequence having at least 10 ...

[0161] In some embodiments, the transcriptional regulators of the multi-chain receptors of the present disclosure include ZF3, TADs derived from the TADs of human or humanized p65, and TADs derived from the TADs of human or humanized HSF1. In some embodiments, the transcriptional regulators of the multi-chain receptors of the present disclosure include ZF3, human or humanized p65, and human or humanized HSF1. In other exemplary embodiments, the transcriptional regulators of the multi-chain receptors of the present disclosure include ZF3, human or humanized p65, and human or humanized GRTau1. In some exemplary embodiments, the transcriptional regulators of the multi-chain receptors of the present disclosure include ZF3, human or humanized p65, and human or humanized p53. In other exemplary embodiments, the transcriptional regulators of the multi-chain receptors of the present disclosure include ZF3, human or humanized p65, and human or humanized ATF6.

[0162] In some embodiments, the multi-chain receptor transcriptional regulator of the present disclosure includes a TAD derived from the TAD of human or humanized p65 and a TAD derived from the TAD of human or humanized HNF1α. In some embodiments, the multi-chain receptor transcriptional regulator of the present disclosure includes 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 may be responsible for the propagation of downstream signaling after binding of the first polypeptide chain to a ligand. The intracellular signaling domain may have at least two different domains: at least one co-stimulatory domain and one 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 selected from 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, TWEAKR, EDAR, M1, TREM2, ILT7, ILT3, ILT4, TLT-1, CD200R, CD300a, CD300f, DC-SIGN, B7-2, allergen-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 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 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 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 act as substrates for phosphorylation (e.g., such as, for example, immunoreceptor tyrosine activation motifs (ITAMs)). In some embodiments, the activation domain comprises at least 1, at least 2, at least 3, at least 4, or at least 5 specific tyrosine-based motifs selected from the following that act as substrates for phosphorylation: ITAM motifs, ITIM motifs, or related intracellular motifs. In some embodiments of the present disclosure, the activation domain of the intracellular signaling domain comprises at least 1, at least 2, at least 3, at least 4, or at least 5 ITAMs. In general, any activation domain comprising ITAMs can be appropriately used for the construction of multi-chain receptors as described herein. ITAMs typically include conserved protein motifs that are often present in the tails of signaling molecules expressed in many immune cells. The motif may include two repeated amino acid sequences YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, resulting in a conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in ITAMs following activation of signaling molecules. ITAMs can also function as docking sites for other proteins involved in signaling pathways.

[0166] In some embodiments, the activation domain comprises one or more immunoreceptor tyrosine activation motifs (ITAMs). In some embodiments, the activation domain is derived from CD3ζ, CD3σ, CD3 / and CD3ε. For example, in some embodiments, ITAM is derived from CD3ζ, CD3σ, CD3 / and CD3ε. In an exemplary embodiment, ITAM is derived from CD3ζ. In certain embodiments, ITAM comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence with CD3ζITAM. In some embodiments, the activation domain comprises at least 1, at least 2, at least 3, at least 4 or at least 5 ITAMs, which are independently selected from the following ITAMs: CD3ζ, FcRγ and combinations thereof. In some embodiments, the activation domain comprises CD3ζITAM.

[0167] In some embodiments, the intracellular domain of the first polypeptide of the present disclosure comprises a signaling chain derived from the common γ chain of the IL-2 receptor. In some embodiments, the signaling chain derived from the common γ chain of the IL-2 receptor comprises the following amino acid sequence: 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 from known receptors, such as, for example, triggering receptor on myeloid cells 2 (TREM 2; Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 3A , Figure 4A and Figure 4B 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 the soluble TREM2 extracellular domain (sTREM2) into the extracellular fluid and generates a membrane-tethered C-terminal fragment (CTF). TREM2 CTF represents a substrate for intramembrane proteolysis by γ-secretase ( Figure 2A In some embodiments of the present disclosure, the first polypeptide may comprise a TREM2 TMD without its cytoplasmic domain. In some embodiments, the first polypeptide may comprise a TREM2 extracellular domain and a TMD having a Notch 1 gamma secretase cleavage site and / or a Notch-2 JMD ( Figure 2D ).

[0169] The first polypeptide of the multi-chain chimeric polypeptide of the present disclosure may comprise a first modified interface, and the first polypeptide associates with the second polypeptide via the first modified interface. The first modified interface may be from Notch 1TMD. The first modified interface may have positively charged residues, and the first polypeptide associates with the second polypeptide of the multi-chimeric polypeptide of the present disclosure via the positively charged residues. The positively charged residues may be lysine or arginine. In some embodiments, a lysine or arginine residue may replace the leucine at position 12 of Notch 2JMD (refer to SEQ ID NO:21, which corresponds to residue 1747 of the entire construct comprising SEQ ID NO:8). In some embodiments, the first polypeptide comprises a truncated CD8α hinge domain ECD, a Notch 1TMD having an L12K mutation, a Notch 2JMD, and a Gal4VP64 transcriptional regulator ( Figure 2B In some embodiments, the first polypeptide comprises a TREM2 ECD, a Notch 1 TMD with a L1747K mutation (Uniprot P46531 amino acids 1736 to 1757 with a L1747K mutation), a Notch 2 JMD, and a Gal4 VP64 transcriptional regulator ( Figure 2C In some embodiments, the first polypeptide comprises a TREM2 ECD, a Notch 1 TMD having a γ-secretase site, a Notch 2 JMD, and a Gal4 VP64 transcriptional regulator ( Figure 2D ).

[0170] In other aspects, the first polypeptide may comprise a truncated CD8α hinge domain (CD8-hinge 2) ECD, a polyvaline TMD having a lysine substitution at position 9 or 10, a Notch 2 JMD, and a Gal4VP64 transcriptional regulator ( Figure 4C and Figure 4D ).

[0171] In other aspects, the first polypeptide may comprise a truncated CD8α hinge domain (CD8-hinge 2) ECD, a polyvaline TMD having a lysine substitution at position 9 or 10, a Notch 2 JMD, and a human HNF1a transcriptional regulator ( Fig. 8A and Figure 8B ).

[0172] In other aspects, the first polypeptide may comprise a truncated CD8α hinge domain (CD8-hinge 2) ECD, a polyvaline TMD having a lysine substitution at position 9 or 10, a Notch 2 JMD, and a 4-1BB / CD3 ζ domain ( Fig.10 ).

[0173] In other aspects, the first polypeptide may comprise a truncated CD8 alpha hinge domain (CD8-hinge 2) ECD, a polyvaline TMD having a lysine substitution at position 9 or 10, a Notch 2 JMD, and a common gamma chain domain ( Fig.10 ).

[0174] In other aspects, the first polypeptide can comprise a truncated CD8 alpha hinge domain (CD8-hinge 2) ECD, a polyvaline TMD with a lysine substitution at position 9 or 10, a Notch 2 JMD, and a common gamma chain / CD3 zeta domain ( Fig.10 ). As will be appreciated by those skilled in the art, the first polypeptide may also include various other combinations of the components described herein. e. Other components

[0175] In some embodiments of the present disclosure, the first polypeptide may further comprise one or more of the following: a self-proteolytic peptide sequence or a nuclear localization signal.

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

[0177] In some embodiments, the autoproteolytic peptide sequence is from porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus (ERAV) 2A (E2A), beta-tetrasomal virus of the thrush moth 2A (T2A), BmCPV2A, malarial disease virus 2A (BmIFV2A), or a combination thereof.

[0178] The first polypeptide may also include a proteolytic cleavage site. In some embodiments, the proteolytic cleavage site may be cleaved by a gamma secretase. In some embodiments, the proteolytic cleavage site is a ligand-inducible 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-inducible, wherein the binding of the selected ligand to the extracellular ligand binding domain induces cleavage at the ligand-inducible proteolytic cleavage site. 2. Second polypeptide

[0179] The multi-chain chimeric polypeptide of the present disclosure comprises a second polypeptide comprising, 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 intracellular domain.

[0180] 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: 9 or SEQ ID NO: 11 or a functional variant thereof. a. Second transmembrane domain

[0181] The second polypeptide of the multi-chain chimeric polypeptide of the present disclosure may include a second TMD having a second interface. The second TMD may include any domain that is capable of associating with the first polypeptide of the multi-chain chimeric polypeptide of the present disclosure via a charged residue in the TMD, and having a signaling domain that is activated after the target ligand binds to the extracellular ligand binding domain of the first polypeptide. The second interface is a region in the second polypeptide that may include negatively charged residues, which associate with positively charged residues in the TMD of the first polypeptide of the present disclosure via the negatively charged residues. In some embodiments, the negatively charged residues may 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 and a DAP12 intracellular signaling domain (ie, a cytoplasmic domain), as explained in more detail below. b. Second intracellular domain

[0183] The second polypeptide of the multi-chain chimeric polypeptide of the present disclosure comprises an intracellular domain. The intracellular domain may be responsible for the propagation of downstream signal transduction after the first polypeptide chain binds to the ligand. The intracellular signaling domain may have at least two different domains: at least one costimulatory domain and one 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 selected from 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, TWEAKR, EDAR, M1, TREM2, ILT7, ILT3, ILT4, TLT-1, CD200R, CD300a, CD300f, DC-SIGN, B7-2, allergen-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 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 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 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 act as substrates for phosphorylation (e.g., such as, for example, immunoreceptor tyrosine activation motifs (ITAMs)). In some embodiments, the activation domain comprises at least 1, at least 2, at least 3, at least 4, or at least 5 specific tyrosine-based motifs selected from the following that act as substrates for phosphorylation: ITAM motifs, ITIM motifs, or related intracellular motifs. In some embodiments of the present disclosure, the activation domain of the intracellular signaling domain comprises at least 1, at least 2, at least 3, at least 4, or at least 5 ITAMs. In general, any activation domain comprising ITAMs can be appropriately used for the construction of multi-chain receptors as described herein. ITAMs typically include conserved protein motifs that are often present in the tails of signaling molecules expressed in many immune cells. The motif may include two repeated amino acid sequences YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, resulting in a conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in ITAMs following activation of signaling molecules. ITAMs can also function as docking sites for other proteins involved in signaling pathways.

[0186] In some embodiments, the activation domain comprises one or more immunoreceptor tyrosine activation motifs (ITAMs). In some embodiments, the activation domain is derived from CD3ζ, CD3σ, CD3 / and CD3ε. For example, in some embodiments, ITAM is derived from CD3ζ, CD3σ, CD3 / and CD3ε. In an exemplary embodiment, ITAM is derived from CD3ζ. In certain embodiments, ITAM comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence with CD3ζITAM. In some embodiments, the activation domain comprises at least 1, at least 2, at least 3, at least 4 or at least 5 ITAMs, which are independently selected from the following ITAMs: CD3ζ, FcRγ and combinations thereof. In some embodiments, the activation domain comprises CD3ζITAM.

[0187] In some embodiments, the intracellular domain is from DAP12 (12kDa DNAZ activating protein, TYROBP). The DAP12 intracellular domain (also referred to as the cytoplasmic domain of DAP12) contains ITAMs, which are phosphorylated after ligand binding to TREM2 and then regulate 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 CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d signaling domain. In some embodiments, the signaling domain is a CD3ζ signaling domain.

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

[0189] In some embodiments, the intracellular domain is 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, IL13, 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 include different chains, some of which are involved in ligand / cytokine interactions, while others are involved in signal transduction. For example, the IL-2 receptor includes an alpha chain, a beta chain, and a gamma chain.

[0190] The IL-2 receptor common gamma chain (also known as CD132) is shared between 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 intracellular domain is from an IL-2 receptor beta chain. In some embodiments, the amino acid sequence of the intracellular domain of an IL-2 receptor beta chain comprises RNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 92. In some embodiments, the IL-2 receptor β chain intracellular domain 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 intracellular domain is from an IL-4 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-4 receptor alpha chain intracellular domain comprises SEQ ID NO: 93. In some embodiments, the IL-4 receptor alpha chain intracellular domain 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 intracellular domain is from an IL-7 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-7 receptor alpha chain intracellular domain comprises KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ SEQ ID NO: 94. In some embodiments, the IL-7 receptor alpha chain intracellular domain 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 intracellular domain is from the IL-9 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-9 receptor alpha chain intracellular domain comprises KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEPCVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPAPPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSALPGNTQSSGPIPALACGLSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLLPSVLSKARSWTFSEQ ID NO: 95. In some embodiments, the IL-9 receptor alpha chain intracellular domain 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 intracellular domain is from the IL-13 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-13 receptor alpha chain intracellular domain comprises KRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQTKEETDSVVLIENLKKASQ SEQ ID NO: 96. In some embodiments, the IL-13 receptor alpha chain intracellular domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQID NO: 96 or a functional variant thereof.

[0196] In some embodiments, the intracellular domain is from the IL-15 receptor alpha chain. In some embodiments, the amino acid sequence of the IL-15 receptor alpha chain intracellular domain comprises KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL SEQ ID NO: 97. In some embodiments, the IL-15 receptor alpha chain intracellular domain 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 intracellular domain is from an IL-21 receptor alpha chain. In some embodiments, the amino acid sequence of the intracellular domain of an IL-21 receptor alpha chain comprises KTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS SEQ ID NO:98. In some embodiments, the IL-21 receptor alpha chain intracellular domain 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 intracellular domain is from a type II cytokine receptor. Type II cytokine receptors include receptors that bind type I and type II interferons, as well as receptors that bind members of the interleukin-10 family (interleukin-10, interleukin-20, and interleukin-22). 3. Additional Embodiments of Chimeric Polypeptides

[0199] The multi-chain chimeric polypeptide of the present disclosure may include various combinations of the above-mentioned first polypeptide and the second polypeptide. In some embodiments, the multi-chain chimeric polypeptide comprises (a) a first polypeptide containing (i) a CD19scFV as an extracellular ligand binding domain, (ii) a first TMD with a continuous valine residue segment, and (iii) a Notch 2 proximal membrane domain, and (iv) a first intracellular domain with a Gal4VP64 transcriptional regulator; and (b) a second polypeptide with DNAX activating protein 12 (DAP12), wherein the first polypeptide is coupled to the second polypeptide via a lysine residue within a continuous valine residue segment, and wherein the binding of CD19 to the extracellular ligand binding domain induces the activity of the signal transduction domain and releases the transcriptional regulator. In some embodiments, the flank of the lysine residue is a continuous segment of 5 to 15 continuous valine residues. The lysine residue can be located at position 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 of the N-terminus of the first TMD.

[0200] In some embodiments, the multi-chain chimeric polypeptide comprises (a) a first polypeptide containing (i) a CD19scFV as an extracellular ligand binding domain, (ii) a first TMD having a continuous valine residue stretch, and (iii) a Notch 2 juxtamembrane domain, and (iv) a first intracellular domain having a Gal4VP64 transcriptional regulator; and (b) a second polypeptide having a CD3ζ signaling domain, wherein the first polypeptide is coupled to the second polypeptide via a lysine residue within the continuous valine residue stretch, and wherein binding of CD19 to the extracellular ligand binding domain induces the activity of the signaling domain and releases the transcriptional regulator. In some embodiments, the lysine residues are flanked by a continuous stretch of 5 to 15 continuous valine residues. The lysine residues may be located at position 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 of the N-terminus of the first TMD.

[0201] In some embodiments, the multi-chain chimeric polypeptide comprises (a) a first polypeptide containing (i) a CD19scFV as an extracellular ligand binding domain, (ii) a first TMD having a continuous valine residue segment, and (iii) a Notch 2 juxtamembrane domain, and (iv) a first intracellular domain having a human transcriptional regulator; and (b) a second polypeptide having DNAX activating protein 12 (DAP12), wherein the first polypeptide is coupled to the second polypeptide via a lysine residue within the continuous valine residue segment, and wherein the binding of CD19 to the extracellular ligand binding domain induces the activity of the signaling domain and releases the transcriptional regulator. In some embodiments, the lysine residues are flanked by a continuous segment of 5 to 15 continuous valine residues. The lysine residues may be located at position 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 of 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 containing (i) a CD19 scFV as an extracellular ligand binding domain, (ii) a first TMD having a stretch of consecutive valine residues, and (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 ζ signaling domain, wherein the first polypeptide is coupled to the second polypeptide via a lysine residue within the stretch of consecutive valine residues, and wherein binding of CD19 to the extracellular ligand binding domain induces the activity of the signaling domain and releases the transcriptional regulator. In some embodiments, the lysine residues are flanked by a continuous stretch of 5 to 15 consecutive valine residues. The lysine residues may be located at position 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 of 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 containing (i) a CD19 scFV as an extracellular ligand binding domain, (ii) a first TMD having a stretch of consecutive valine residues, and (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 ζ signaling domain, wherein the first polypeptide is coupled to the second polypeptide via a lysine residue within the stretch of consecutive valine residues, and wherein binding of CD19 to the extracellular ligand binding domain induces the activity of the signaling domain and releases the transcriptional regulator. In some embodiments, the lysine residues are flanked by a continuous stretch of 5 to 15 consecutive valine residues. The lysine residues may be located at position 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 of the N-terminus of the first TMD. In some embodiments, the human transcriptional regulator is HNF1a. 4. Nucleic acid constructs encoding multi-chain chimeric polypeptides

[0204] The present disclosure also provides a recombinant nucleic acid construct comprising a nucleotide sequence encoding a multi-chain chimeric polypeptide of the present disclosure. The present disclosure also provides a recombinant nucleic acid construct having a nucleotide sequence encoding only a first polypeptide or only a second polypeptide. In the case where the recombinant nucleic acid construct encodes the first polypeptide or the second polypeptide, both types of constructs can be used to transduce a host cell 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 a multi-chain chimeric polypeptide of the present disclosure and a second cassette encoding a second polypeptide, wherein the two cassettes are on the same nucleic acid molecule. In some embodiments, the first cassette is located on the 5' side of the second cassette. In some embodiments, the first cassette is located on the 3' side of the second cassette. In some embodiments, the first cassette and the second cassette are connected by an autoproteolytic peptide. In some embodiments, the autoproteolytic peptide is a Tetrasomal Virus 2A (T2A) peptide of the Lymantria glaucosa.

[0206] In some embodiments, the recombinant nucleic acid construct of the present disclosure comprises a nucleotide sequence comprising the following sequence: 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. The 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, such as between about 15 Kb to 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 that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence encoded by the following sequence: 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. It will be appreciated that an expression cassette typically includes a construct of genetic material containing a coding sequence and sufficient regulatory information to direct the coding sequence to be correctly transcribed and / or translated in a recipient cell in vivo and / or ex vivo. Typically, the expression cassette can be inserted into a vector for targeting a desired host cell and / or in an individual. Thus, in some embodiments, the expression cassette of the present disclosure comprises: a coding sequence of a chimeric polypeptide as disclosed herein, the coding sequence being operably linked to an expression control element such as a promoter, and any one or combination of other nucleic acid sequences that optionally affect transcription or translation of the coding sequence.

[0210] The recombinant nucleic acid constructs provided can contain naturally occurring sequences, or sequences that are different 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., DNA produced by phosphoramidite-based synthesis)) or combinations or modifications of nucleotides within these types of nucleic acids. In addition, the recombinant nucleic acid molecules of the present disclosure can be double-stranded or single-stranded (e.g., sense strand or antisense strand).

[0211] Nucleic acid molecules are not limited to sequences encoding polypeptides; they may also include some or all non-coding sequences located upstream or downstream of a coding sequence (e.g., a coding sequence for a multi-chain chimeric polypeptide or a receptor). One of ordinary skill in the art of molecular biology is familiar with conventional procedures for isolating nucleic acid molecules. They can be produced, for example, by treating genomic DNA with restriction endonucleases or by performing a polymerase chain reaction (PCR). In the case where the nucleic acid molecule is a ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription. B. Carrier

[0212] The recombinant nucleic acid construct encoding the multi-chain polypeptide of the present disclosure may be contained in one or more vectors. Therefore, the present disclosure also provides vectors encoding or expressing the multi-chain chimeric polypeptide of the present disclosure.

[0213] One or more vectors of the present disclosure can express the first polypeptide chain or the second polypeptide chain or both of the multi-chain chimeric polypeptide, that is, the multi-chain polypeptide of the present disclosure can be expressed by one vector or by different vectors that are co-transduced into the cell.

[0214] In some embodiments, the multi-chain chimeric polypeptide is incorporated into an expression vector that is designed for transfer between host cells and can be used for transformation purposes, such as 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 cause 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 polypeptides or receptors of the present disclosure, one or more vectors may also contain, 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 that facilitate stable integration of the construct into the genome of the cell.

[0216] In some embodiments, expression vector can be a viral vector. As will be appreciated by those skilled in the art, the term "viral vector" is widely used to refer to nucleic acid molecules (e.g., transfer plasmids) comprising nucleic acid elements of viral origin that usually promote the transfer of nucleic acid molecules or are integrated into the genome of cells, or to refer to viral particles that mediate nucleic acid transfer. Viral particles will usually include various viral components, and sometimes also include host cell components in addition to one or more nucleic acids. The term viral vector can refer to viruses or viral particles that can transfer nucleic acid into cells or to refer to the nucleic acid itself transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are mainly derived from viruses.

[0217] The multi-chain chimeric polypeptide or chimeric antigen receptor of the present disclosure can 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 that are primarily derived from a retrovirus. In some embodiments, the multi-chain chimeric antigen receptor of the present disclosure can be incorporated into a lentiviral vector. The lentiviral vector can contain structural and functional genetic elements or portions thereof (including LTRs) that are primarily derived from a lentivirus (which is a genus of retroviruses).

[0218] Viral vectors that can be used in the present disclosure include, for example, adenoviral vectors and adeno-associated viral vectors, herpes viruses, simian virus 40 (SV40) and bovine papilloma virus vectors, retroviral vectors, lentiviral vectors (see, for example, Gluzman (ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY). For example, constructs as 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, Virginia). When selecting an expression system, care should be taken to ensure that the components are compatible with each other. A technician or ordinary technician can make such a decision. In addition, if guidance is needed when selecting an expression system, a skilled technician can consult P. Jones, "Vectors: Cloning Applications", John Wiley and Sons, New York, NY, 2009.

[0219] The multi-chain chimeric polypeptides of the present disclosure may be contained within one or more vectors that are capable of directing expression of the nucleic acid molecule in, for example, cells that have been transformed / transduced with one / more vectors. Suitable vectors for eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. In some embodiments, the vector is a lentiviral transfer vector comprising SEQ ID NO: 1 or any functional variant thereof.

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

[0221] The multi-chain chimeric polypeptide of the present disclosure can be introduced or transduced into a host cell or a recombinant cell, such as a human T lymphocyte, to produce a recombinant cell containing a nucleic acid molecule. Therefore, some embodiments of the present disclosure relate to a recombinant cell comprising a multi-chain chimeric polypeptide or CAR or a recombinant nucleic acid construct or vector of the present disclosure.

[0222] Introducing the constructs or vectors of the present disclosure into cells can be performed by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, liposome transfection, 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, and the like.

[0223] Therefore, in some embodiments, constructs or vectors can be delivered by viral or non-viral delivery vehicles known in the art.For example, constructs can be stably integrated in the host genome, or can be replicated as episomes, or present in recombinant host cells as mini-ring expression vectors for transient expression.Therefore, in some embodiments, constructs are maintained and replicated as episomal units in the recombinant host cells.In some embodiments, constructs are stably integrated into the genome of the recombinant cells.Stable integration can be achieved using classical random genome recombination techniques or with more precise techniques, such as CRISPR / Cas9 genome editing guided by guide RNA, or DNA-guided nuclease genome editing or TALEN genome editing (transcription activator-like effector nuclease) using NgAgo (Natronobacterium gregoryi Argonaute).In some embodiments, the recombinant nucleic acid molecules of the present disclosure are present in recombinant host cells as mini-ring expression vectors for transient expression.

[0224] The recombinant nucleic acid construct of the present disclosure can be encapsulated in a viral capsid or lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods (such as electroporation) known in the art. For example, the introduction of nucleic acid into a cell can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver the construct to a target cell via viral transduction. Several AAV serotypes have been described, and all known serotypes can infect cells from a variety of different tissue types. AAV can transduce a wide range of species and tissues in vivo without signs of toxicity, and it produces a relatively mild innate and adaptive immune response.

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

[0226] In some embodiments, the recombinant host cell can be genetically engineered (e.g., transduced or transformed or transfected) with a vector construct such as the present application, which can be, for example, a viral vector or a vector for homologous recombination (which comprises a nucleic acid sequence homologous to a portion of the host cell genome), or an expression vector for expressing a polypeptide of interest. The host cell can be an untransformed cell or a cell transfected with at least one nucleic acid molecule.

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

[0228] In some embodiments, the cell is a stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments of the cell, the cell is a lymphocyte. In some embodiments, the cell is a precursor T cell or a T regulatory (Treg) cell. In some embodiments, the cell is a CD34+ cell, a CD8+ cell or a CD4+ cell. In some embodiments, the cell is a CD8+T cytotoxic lymphocyte selected from the following: immature CD8+T cells, central memory CD8+T cells, effector memory CD8+T cells and large CD8+T cells. In some embodiments of the cell, the cell is a CD4+T helper lymphocyte selected from the following: immature CD4+T cells, central memory CD4+T cells, effector memory CD4+T cells and large CD4+T cells. In some embodiments, the cell can be obtained by leukocytosis performed on a sample obtained from a subject. In some embodiments, the subject is a human patient.

[0229] In another aspect, provided herein is a cell culture comprising at least one recombinant cell as disclosed herein and a culture medium. Typically, the culture medium can be any suitable culture medium for culturing cells as described herein. Techniques for transforming various host cells and species mentioned above are known in the art and are described in the technical and scientific literature. Therefore, a cell culture comprising at least one recombinant cell as 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. D. Pharmaceutical Composition

[0231] The present disclosure also provides a pharmaceutical composition comprising the recombinant cell of the present disclosure and a pharmaceutically acceptable excipient, such as a carrier.

[0232] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of 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.TM. (BASF in Parsippany, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be a fluid to the extent that it is easy to inject. It should be stable under manufacturing and storage conditions and must be preserved against the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, appropriate fluidity can be maintained by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant (e.g., sodium lauryl sulfate). Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol) and sodium chloride are typically included in the composition. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) in the composition.

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

[0234] In some embodiments, the multi-chain CAR 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 the methods described in the following literature: 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, errata: Am. J. Health Syst. Pharm. 53:325, 1996). III. Methods of the Disclosure A. Treatment methods

[0235] The present disclosure provides, among other things, a method for treating a health condition of a subject by administering to the subject a therapeutically effective amount of a recombinant cell, a pharmaceutical composition, a multi-chain chimeric polypeptide or a CAR of the present disclosure. The present disclosure also provides, among other things, a method for treating a health condition of a subject by administering to the subject a vector of the present disclosure.

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

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

[0238] Administration of any of the vectors or recombinant cells or pharmaceutical compositions described herein can be used to treat a patient's associated health condition or disease, such as cancer or an autoimmune disease or infection (e.g., chronic infection). In some embodiments, the vector or cell of the present disclosure can be incorporated into a composition (e.g., a drug or therapeutic composition) for use in treating an individual who suffers from, is suspected of suffering from, or is at high risk of suffering from one or more autoimmune disorders or diseases associated with checkpoint inhibition. Exemplary autoimmune disorders and diseases may include, but are not limited to, celiac disease, type I 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 amount of recombinant cells disclosed herein to the individual, wherein the recombinant cells inhibit the activity of the target cells in the individual. Generally, the target cells of the disclosed method can be any cell type in the individual, and can be, for example, cells from hematological malignancies, 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, hepatoma cells, lung cancer cells, mantle cell lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells, oligodendroglioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T cell lymphoma cells, renal cancer cells, sarcoma cells, gastric cancer cells, cancer cells, mesothelioma cells or sarcoma cells. In some embodiments, the target cell is a pathogenic cell.

[0240] In some embodiments, the methods of the present disclosure involve administering an effective amount of the recombinant cells of the present disclosure to an individual in need of such treatment. This administration step can be accomplished using any implantation delivery method in the art. For example, the recombinant cells of the present disclosure can be directly infused into the bloodstream of an individual or administered to the individual in other ways.

[0241] In some embodiments, the methods disclosed herein include administering (the term is used interchangeably with the terms "introducing", "implanting" and "transplanting") recombinant cells to an individual by a method or approach that results in at least a portion of the introduced cells being positioned at a desired site to produce one or more desired effects. The recombinant cells or their differentiated progeny can be administered by any appropriate approach that results in delivery to a desired location of an individual, where at least a portion of the administered cells or cell components remain viable. The viability period of the cells after administration to an individual can be as short as a few hours, such as twenty-four hours, to several days, to as long as several years, or even the life span of the individual, i.e., long-term transplantation.

[0242] When provided therapeutically, in some embodiments, the recombinant cells are provided at the onset of (or after) symptoms or indications of a disease or disorder, such as at the onset of the disease or disorder.

[0243] A therapeutically effective amount includes an amount of the recombinant cell sufficient to promote a specific beneficial effect when administered to an individual, such as an individual suffering from, suspected of suffering from, or at risk of suffering from a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of disease symptoms, to alter the course of disease symptoms (e.g., but not limited to, to slow the progression of disease symptoms), or to reverse disease symptoms. It should be understood that for any given case, one of ordinary skill in the art can determine an appropriate effective amount using routine experiments.

[0244] For use in the various embodiments described herein, an effective amount of a recombinant cell as disclosed herein may be at least 10 2 cells, at least 5x 10 2 cells, at least 10 3 cells, at least 5x 10 3 cells, at least 10 4 cells, at least 5x 10 4 cells, at least 10 5 cells, at least 2x 10 5 cells, at least 3x 10 5 cells, at least 4 x 10 5 cells, at least 5x 10 5 cells, at least 6 x 10 5 cells, at least 7x 10 5cells, at least 8 x 10 5 cells, at least 9x10 5 cells, at least 1x 10 6 cells, at least 2x 10 6 cells, at least 3x 10 6 cells, at least 4 x 10 6 cells, at least 5x 10 6 cells, at least 6 x 10 6 cells, at least 7x 10 6 cells, at least 8 x 10 6 cells, at least 9 x 10 6 Cells or multiples thereof. The recombinant cells may be derived from one or more donors, or may be obtained from autologous sources. In some embodiments, the recombinant cells are expanded in culture before being administered to an individual in need thereof.

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

[0246] In some embodiments, the recombinant cells are administered systemically, for example, via infusion or injection. For example, rather than administering the recombinant cell population directly to a target site, tissue, or organ, it enters the circulatory system of an individual to carry out metabolism and other similar biological processes.

[0247] The efficacy of a treatment for a disease or condition including any composition provided herein can be determined by a skilled clinician. However, it will be appreciated by those skilled in the art that if any or all of the signs or symptoms or markers of the disease are improved or ameliorated, the treatment is considered to be effective. The efficacy can also be measured by the failure of individual deterioration as assessed by a reduction in hospitalization or the need for medical intervention (e.g., disease progression stops or at least slows down). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a 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 symptoms to subside; and (3) preventing or reducing the possibility of symptom development.

[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 infection. 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 methods of the present disclosure, the administered recombinant cells modulate the activity of target cells in the individual. In some embodiments, the activity of the target cells includes expression of selected genes, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, molecular secretion, cell adhesion, and cytolytic activity.

[0251] As discussed 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 anticancer agents or anticancer therapies). "Combination" administration with one or more additional therapeutic agents includes simultaneous (concurrent) and continuous administration in any order. In some embodiments, the one or more additional therapeutic agents, chemotherapeutic agents, anticancer agents or anticancer therapies are selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy and surgery. "Chemistry" and "anticancer agents" are used interchangeably herein. Various types of anticancer agents can be used. Non-limiting examples include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec.RTM. or Glivec.RTM.)), hormone therapy, soluble receptors and other antitumor drugs. B. Methods for Simultaneous Induction of T Cell Signaling and Gene Regulation or Induction of Enhanced T Cell Signaling

[0252] The present disclosure also provides a method for simultaneously inducing T cell signaling and gene regulation in T cells, the method comprising (a) providing a T cell having 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 the release of transcriptional regulators.

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

[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 enhances intracellular signaling.

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

[0256] In some embodiments, the induced intracellular signaling of the T cell regulates the expression of selected genes involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, molecular secretion, cell adhesion and / or cytolytic activity. In some embodiments, the released transcriptional regulator regulates the expression of the payload in the T cell. In some embodiments, the payload includes chemokines, chemokine receptors, chimeric antigen receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, pathogen-derived proteins, proliferation inducers, receptors, RNA-guided nucleases, site-specific nucleases, T cell receptors, toxins, toxin-derived proteins, transcriptional regulators, transcriptional activators, transcriptional repressors, translational regulators, translational activators, translational repressors, activating immune receptors, antibodies, apoptosis inhibitors, apoptosis inducers, engineered T cell receptors, immune activators, immunosuppressants or inhibitory immune receptors.

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

[0258] In another aspect, various methods for regulating the activity of a cell are provided herein, comprising (a) providing an effective amount of any recombinant cell of the present disclosure; and (b) contacting the cell with a selected ligand, wherein the binding of the selected ligand to the extracellular ligand binding domain (i) induces cleavage of the ligand-inducible proteolytic cleavage site and releases the transcriptional regulator, and simultaneously (ii) activates T cell signaling, wherein the released transcriptional regulator regulates the activity of the recombinant cell. It will be understood by those skilled in the art after reading the present disclosure that the disclosed methods 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 regulatory factor regulates the expression of a gene product of the cell. In some embodiments, the released transcriptional regulatory factor regulates the expression of a heterologous gene product in the cell. A heterologous gene product is a gene product that is not usually found in natural cells, such as a gene product that is not usually produced by the cell. For example, a cell can 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 regulatory factor simultaneously regulates the expression of two or more heterologous gene products in the cell.

[0262] In some embodiments, the heterologous gene product in the cell is selected from 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 immune receptor, an antibody, an inhibitor of apoptosis, an inducer of apoptosis, an engineered T cell receptor, an immune activator, an immune suppressor, and an inhibitory immune receptor.

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

[0264] Also provided herein are systems and kits, which include multi-chain chimeric polypeptides, CARs, recombinant nucleic acids, recombinant cells or pharmaceutical compositions provided and described herein, and written instructions for their preparation and use. For example, in some embodiments, provided herein are systems and / or kits comprising one or more of the following: multi-chain chimeric polypeptides of the present disclosure, recombinant nucleic acids as described herein, recombinant cells as described herein, or pharmaceutical compositions as described herein. In some embodiments, the system and / or kit of the present disclosure also includes one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) for administering any of the provided recombinant nucleic acids, recombinant cells or pharmaceutical compositions to an individual. In some embodiments, the kit may have one or more additional therapeutic agents, which may be administered simultaneously or sequentially with other kit components for a desired purpose, such as for regulating the activity of cells, inhibiting target cancer cells or treating health conditions (e.g., diseases) in individuals in need.

[0265] In some embodiments, system or test kit may further include the instructions for practicing the method using the components of the test kit. The instructions for practicing the method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic, etc. The instructions can be present in the test kit as a drug instruction, in the label of the container of the test kit or its components (that is, associated with packaging or sub-packaging), etc. The instructions can exist as an electronic storage data file present in a suitable computer-readable storage medium (for example, CD-ROM, floppy disk, flash drive, etc.). In some cases, the actual instructions are not present in the test kit, but can provide the means for obtaining the instructions from a remote source (for example, via the Internet). The example of this embodiment is a test kit comprising a website, in which the instructions can be viewed and / or instructions can be downloaded therefrom. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0266] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be clear to those skilled in the art after reviewing this disclosure and are to be included within the spirit and scope of this application.

[0267] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. 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 the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and relevance of the cited documents. It will be clearly understood that although a number of information sources are referred to herein, including scientific journal articles, patent documents and textbooks; this reference does not constitute an admission that any of these documents form part of the common general knowledge in the field. Example

[0269] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art and fully explained in the literature (e.g., Sambrook, J. and Russell, DW (2012)). Molecular Cloning: A Laboratory Manual (4th edition). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J. and Russel, DW (2001). Molecular Cloning: A Laboratory Manual (3rd edition). 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 (including supplements through 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene 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 ImmunologyMethods 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, Ferré, F. and Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher;Greenfield,E.A. (2014). Antibodies: A Laboratory Manual (2nd Edition). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, The Netherlands: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference. .

[0270] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of the disclosure or the claims in any way. Example 1: Receptor Expression

[0271] This example describes flow cytometry data for receptor expression.

[0272] Primary human CD3+T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Gibco) and transduced with a lentiviral construct expressing a multi-chain receptor construct and another lentiviral construct containing a transcriptional reporter construct. Receptor expression was measured using an AlexaFluor647-labeled anti-myc antibody (Cell Signaling) for the myc tag on the chain containing the binder (CD19scFv). Reporter expression was measured by the constitutive mCitrine gene found on the reporter plasmid. Double positive cells were sorted on the 5th day after initial T cell stimulation and further amplified for activation testing. Here, the receptor design 056 ( Figure 2A ) and 056C( Figure 2B ) shows the highest expression for that cohort. Example 2: Receptor Activation

[0273] This example describes receptor activation and target killing for two receptor embodiments 056 and 056C ( Figure 3A ).

[0274] 1E5 double positive T cells expressing anti-CD19 receptor were co-cultured for 48 hours with: no addition (red, "T cells alone"), 1E5 K562 cells (blue, (+K562)), or 1E5 CD19+K562 cells (yellow, "+K562-CD19"). Transcriptional activation of the inducible BFP reporter gene was then measured using a Fortessa X-50 (BD). Figure 3B ). Target killing was measured by DRAQ7 staining and flow cytometry ( Figure 3C ). Receptor 056 showed better killing than receptor 056C, and receptor 056C showed better transcriptional activation. Example 3: Receptor Expression of Receptors 056F and 056G

[0275] This example describes flow cytometry data for receptor expression of embodiments of receptors 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 multi-chain receptor construct and another lentiviral construct containing a transcriptional reporter construct. Receptor expression was measured using an AlexaFluor647-labeled anti-myc antibody (Cell Signaling) for the myc tag on the chain containing the binder (CD19scFV). Reporter expression was measured by the constitutive mCitrine gene found on the reporter plasmid. Double positive cells were sorted on the 5th day after initial T cell stimulation and further amplified for activation testing. Receptor design 056F ( Figure 4C ) and 056G( Figure 4D ) shows the highest expression for that cohort. Example 4: Receptor Activation and Performance of Implementation Schemes 056, 056E-G

[0277] This example describes receptor activation and target killing assays for receptors 056, 056E, 056F, and 056G.

[0278] 1E5 double positive T cells expressing anti-CD19 receptor were co-cultured for 5 days with the following substances: no addition (red, "T cells alone"), 1E5 K562 cells (blue, "+K562") or 1E5 CD19+K562 cells (yellow, "+K562-CD19"). After 24 h, transcriptional activation of the inducible BFP reporter gene was measured using Fortessa X-50 (BD) (top row). Target killing was measured at 24 and 120 h by DRAQ7 staining and flow cytometry (bottom panel). Here, the receptor 056F ( Figure 5C ) showed both transcriptional and cytotoxic activity, receptor 056 ( Figure 5A ) and 056E( Figure 5B ) showed killing, and receptor 056G ( Figure 5D ) shows transcriptional activation only. Example 5: Multi-chain receptor module engineering strategy

[0279] This example describes a multi-chain receptor module engineering strategy using pRay056F as a prototype. Figure 6 As shown, one chain (DAP12 chain) with the DAP12 signaling domain is replaced by ITAM and signaling domains from TCR, co-stimulatory proteins and cytokine receptors. The transcription factor (TF) of the other chain (main chain, containing the transcription factor Gal4-VP64) is replaced by a human transcription factor or by ITAM and signaling domains from TCR, co-stimulatory proteins and cytokine receptors. Example 6: Multi-chain receptor function with CD3z substitution

[0280] This example describes the testing of multi-chain receptors with CD3z substitutions. Here, the DAP12 multi-chain receptor (056F, Fig. 7A ) was replaced by the ITAM signaling domain from CD3z, which contains three ITAMs (056I, Figure 7B ). 1E5 double positive T cells expressing anti-CD19 receptor were co-cultured for 5 days with the following substances: no addition (red, "T cells alone"), 1E5 K562 cells (blue, "+K562") or 1E5 CD19+K562 cells (yellow, "+K562-CD19"). After 24h, transcriptional activation of the inducible BFP reporter gene was measured using Fortessa X-50 (BD) (top row). Target killing was measured at 24 and 120 hours by DRAQ7 staining and flow cytometry (bottom graph). This substitution showed increased killing of target cells ( Figure 7B , bottom row). Transcriptional activation was slightly reduced but still strong ( Figure 7B , top row). From SNIPR receptors ( Figure 7C ) and standard BBz CAR( Fig.7D ) were included as positive controls for transcriptional activation and killing, respectively. Example 7: Multi-chain receptor function with human transcription factors

[0281] This example describes the testing of multi-chain receptors with human transcription factors. Fig. 8A ) and CD3z( Figure 8B), the Gal4-VP64 containing the non-human portion was replaced by a transcription factor containing the human portion. These receptors were designed to have the transcription and signaling domains constructed linearly on a single-chain receptor ( Figure 8C ) were compared. 1E5 double-positive T cells expressing the anti-CD19 receptor were co-cultured for 5 days with the following substances: no addition (red, "T cells alone"), 1E5 K562 cells (blue, "+K562"), or 1E5 CD19+K562 cells (yellow, "+K562-CD19"). After 24 h, transcriptional activation of the inducible BFP reporter gene was measured using Fortessa X-50 (BD) (top row). Target killing was measured at 24 and 120 h by DRAQ7 staining and flow cytometry (bottom panel). Both multi-chain receptors were able to mediate transcriptional activation and killing of target cells ( Fig. 8A and Figure 8B , top and bottom rows), whereas a single-chain design cannot ( Figure 8C , top and bottom rows). Transcriptional activation and killing from standard BBz CAR ( Fig.8D ) was included as a positive control for killing. Example 8: Multi-chain receptors using a modular engineering strategy using dual vector transduction

[0282] This example describes a modular engineering strategy for multi-chain receptors using dual vector transduction. Here, each chain is composed of DAP12 chains and backbone constructs as listed ( Fig. 9 ) were expressed from a separate promoter introduced by transduction of . Each side chain was combined in pairs with each backbone to evaluate the effect of pairing two signaling chains together. Three of the backbone variants contained signaling components in place of transcription factors, which were designed to deliver additional signaling capabilities rather than transcriptional activation. Example 9: Expression profiles of multi-chain receptor pairs

[0283] This example describes the expression profile of each multi-chain receptor pair. Cells containing the DAP-12 chain were detected by T2A-mCherry signaling, while cells containing the main chain receptors were detected by Myc tag staining. Myc tag staining was used to assess receptor expression levels. All receptors showed some level of expression, with some pairs showing better expression than others, as indicated by a higher double positive population ( Fig.10 ). Example 10: Screening for multi-chain receptors that enhance killing and survival

[0284] This example describes the screening of multi-chain receptors that enhance killing and survival. Primary human CD3+ T cells expressing multi-chain receptors were co-cultured with 100k K562 target cells at T cell to target ratios of 1:4, 1:2, and 1:1 and tracked over time. The co-cultures were maintained in human T cell culture medium without exogenous cytokines and fed every 5 days. On days 5, 12, and 20, half of the co-cultures were removed (replacement of culture medium), stained for T cell markers, and analyzed by flow cytometry. T cells showing ( Figure 11-13 ) and target cells ( Figure 14-16 ) survival counts were quantified. Standard BBzCAR T cells were included as controls (last column of each figure). As of the last time point (day 20), several multi-chain receptors appeared to mediate better T cell survival and target cell killing than BBz CAR. Example 11: Receptor and response element construct design

[0285] This example describes the design and construction of a family of multi-chain chimeric polypeptides (e.g., receptors) constructed by fusing CD19 scFv {Porter: 2011gr} to the corresponding receptor scaffold and Gal4DBD VP64. The receptor contains an n-terminal CD8α signal peptide (MALPVTALLLPLALLLHAARP) for membrane targeting and a myc tag (EQKLISEEDL) for easy determination of surface expression with α-myc A647 (cell-signaling #2233). For all primary T cell experiments, the receptor was cloned into a modified pHR'SIN:CSW vector containing a PGK promoter (SEQ ID NO: 1). Example 12: Isolation and culture of primary human T cells

[0286] This example describes the separation and culture of primary human T cells, which are subsequently used in various cell transduction experiments described in Example 3 below. In these experiments, primary CD4+ and CD8+ T cells were isolated from anonymous donor blood after apheresis (STEMCELL Technologies #15062 and 15063) by negative selection. Blood was obtained from the Blood Centers of the Pacific (San Francisco, California), as approved by the University Institutional Review Board. T cells were cryopreserved in RPMI-1640 (UCSF Cell Culture Center) with 20% human AB serum (Valley Biomedical Inc., #HP1022) and 10% DMSO. 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 IL-2 (NCI BRB preclinical repository) for all experiments. Example 13: Lentiviral transduction of human T cells

[0287] This embodiment describes a general scheme for the lentiviral transduction of human T cells. Generate pantropic VSV-G pseudotyped lentivirus via the following method: Lenti-X 293T cells (Clontech #11131D) are transfected with pHR'SIN:CSW transgenic expression vector and viral packaging plasmids pCMVdR8.91 and pMD2.G using Mirus TransIT-Lenti (Mirus #MIR 6606). Primary T cells are thawed on the same day, and after 24 hours of culture, stimulated with a 1:3 cell: bead ratio using human T activating factor CD3 / CD28Dynabeads (Life Technologies #11131D). At 48 hours, viral supernatants are harvested, and primary T cells are exposed to viruses for 24 hours. On the 5th day after T cell stimulation, Dynabeads are removed, and T cells are amplified until the 14th day, when they are left to rest and can be used in determination. T cells were sorted using Beckton Dickinson (BD) FACs ARIAII targeting assay. Example 14: Cancer cell lines

[0288] This example describes the generation of myeloid leukemia cells expressing CD19 at levels equivalent to Daudi tumors. The cancer cell line used is K562 myeloid leukemia cells (ATCC#CCL-243). Lentiviral transduction of K562 stably expresses human CD19 at levels equivalent to Daudi tumors, or expresses HER2 via a doxycycline inducible system. CD19 levels are determined by staining cells with α-CD19APC (Biolegend#302212), and HER2 levels are determined by staining cells with α-HER2 AF647 (Biolegend#324412). All cell lines were sorted for transgenic expression. Example 15: In vitro stimulation of primary T cells

[0289] For all in vitro T cell stimulations, 1 × 10 5 T cells were co-cultured with target cells in a 1:1 ratio in U-bottom 96-well tissue culture plates. Cultures were analyzed for reporter gene activation and / or target cell killing with a BD Fortessa X-50 at 24 hours or as indicated. All flow cytometric analyses were performed in FlowJo software (TreeStar). Example 16: Recombinant Constructs

[0290] The following are examples of recombinant nucleic acid constructs or boxes that encode examples of multi-chain chimeric polypeptides of the present disclosure. As described above, the construct comprises two boxes. In this embodiment, the box encoding the second polypeptide is located at the 5' side of the box encoding the first polypeptide. The T2A autoproteolytic peptide sequence is located between the second polypeptide and the first polypeptide. A construct having a first polypeptide located at the 5' side of the second polypeptide can be constructed in a similar manner.

Claims

1. 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 transmembrane domain (TMD) comprising a first modification interface, and (iii) a first intracellular domain comprising a transcriptional regulator; 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 modification interface and the second modification interface each comprise amino acid residues having opposite charges, And wherein the first polypeptide is coupled to the second polypeptide via the first modification interface and the second interface, and wherein binding of the selected ligand to the extracellular ligand binding domain induces the activity of the signaling domain and the release of the transcriptional regulator.

2. The multi-chain chimeric polypeptide of claim 1, wherein binding of the selected ligand to the extracellular ligand binding domain simultaneously induces the activity of the signaling domain and the 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) a Notch 1 transmembrane domain.

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

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

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 located 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 located 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 located at (i) position 12 of SEQ ID NO: 21, or (ii) position 9 of SEQ ID NO:

18.

10. The multi-chain chimeric polypeptide according to any one of claims 1 to 9, wherein the extracellular domain comprises an antigen binding portion capable of binding to a ligand on the surface of a cell.

11. The multi-chain chimeric polypeptide according to claim 10, wherein the antigen binding portion is selected from antibodies, nanobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, F(ab) fragments, single-chain variable fragments (scFv), single domain antibodies (sdAb) and functional fragments thereof.

12. The multi-chain chimeric polypeptide of any one of claims 1 to 11, wherein the ligand comprises a protein or a carbohydrate.

13. The multi-chain chimeric polypeptide according to any one of claims 1 to 12, 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 multi-chain chimeric polypeptide of claim 14, wherein the ligand comprises 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, placental alkaline phosphatase-like protein 2 (ALPPL2), B cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein alpha (SIRPα).

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

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

18. The multi-chain chimeric polypeptide according to any one of claims 1 to 17, wherein the transcriptional regulator 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 regulator comprises Ga14-VP16, Ga14-VP64, tetR-VP64, ZFHD1-VP64, Ga14-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 according to any one of claims 1 to 18, wherein the transcriptional regulator is HNF1a.

22. The multi-chain chimeric polypeptide of any one of claims 1 to 21, wherein the second polypeptide comprises a signaling domain comprising a CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d signaling domain.

23. The multi-chain chimeric polypeptide according to 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 membrane-proximal domain.

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

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

26. The multi-chain chimeric polypeptide according to any one of claims 23 to 25, wherein the autoproteolytic peptide sequence is from porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus (ERAV) 2A (E2A), beta-tetrasomal virus of the thrush moth 2A (T2A), BmCPV2A, malarial disease virus 2A (BmIFV2A), or a combination thereof.

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

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

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

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

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

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

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

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

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

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

37. 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 transmembrane domain (TMD) comprising a first modified 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 modification interface and the second modification interface each comprise amino acid residues having opposite charges, And wherein the first polypeptide is coupled to the second polypeptide via the first modification interface and the second interface, and wherein binding of the selected ligand to the extracellular ligand binding domain induces the activity of the signaling domain and the release of the transcriptional regulator.

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

39. The multi-chain chimeric polypeptide of claim 38, wherein the transcriptional regulator comprises Ga14-VP16, Ga14-VP64, tetR-VP64, ZFHD1-VP64, Ga14-KRAB or HAP1-VP16.

40. The multi-chain chimeric polypeptide of any one of claims 37 to 38, wherein the transcriptional regulator is a human or humanized transcriptional regulator. The multi-chain chimeric polypeptide of claim 40 , wherein the transcriptional regulator is HNF1a.

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

43. The multi-chain chimeric polypeptide of claim 42, wherein the signaling domain comprises one or more of: CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, CD66d, 4-1BB, or a common γ chain signaling domain.

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 CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, CD66d, or 4-1BB signaling domain.

45. 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. ​​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 intracellular domain.

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 simultaneously induces activity of the signaling domain and release of the transcriptional regulator.

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. 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) a Notch 1 transmembrane domain.

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

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

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

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 located 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 located at a position selected from positions 8 to 11 of SEQ ID NO:

18.

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

18.

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 multi-chain chimeric polypeptide of claim 55, wherein the antigen binding portion is selected from antibodies, nanobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, F(ab) fragments, single-chain variable fragments (scFv), single domain antibodies (sdAbs) and functional fragments thereof.

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

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. 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 multi-chain chimeric polypeptide of claim 59, wherein the ligand comprises 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, placental alkaline phosphatase-like protein 2 (ALPPL2), B cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein alpha (SIRPα).

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

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

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 membrane-proximal domain.

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

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

66. The multi-chain chimeric polypeptide of any one of claims 63 to 65, wherein the autoproteolytic peptide sequence is from porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus (ERAV) 2A (E2A), beta-tetrasomal virus of the thrush moth 2A (T2A), BmCPV2A, malarial disease virus 2A (BmIFV2A), or a combination thereof.

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

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

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

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

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

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

73. according to the nucleic acid construct described in any one in claim 69 to 72, wherein said nucleic acid construct comprises such nucleotide sequence, described nucleotide sequence and 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 its any functional variant have 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

74. The nucleic acid construct of claim 69 or claim 70, wherein the autoproteolytic peptide is a Tetrasomal Betavirus 2A (T2A) peptide.

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

76. The vector of claim 75, wherein the vector is an expression vector.

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

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

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

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

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

82. The recombinant cell of claim 80, wherein the recombinant cell is an immune cell.

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

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

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

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

88. A method of inducing enhanced T cell signaling in a T cell, the method comprising: (a) providing a T cell comprising the multi-chain chimeric polypeptide according to 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 enhances intracellular signaling.

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

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

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

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 immune receptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immune activator, an immune suppressor, or an inhibitory immune receptor.

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

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

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

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

97. The method of claim 94, wherein the cancer cell is a solid tumor cell or a hematological malignancy cell.

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

99. A method for regulating T cell activity, the method comprising: (a) providing an effective amount of any one of the recombinant cells according to claims 79 to 84; as well as (b) contacting the cell with a selected ligand, wherein binding of the selected ligand to the extracellular ligand binding domain induces cleavage of the ligand-inducible proteolytic cleavage site and (i) releases the transcriptional regulator, wherein the released transcriptional regulator regulates the activity of the recombinant cell; and simultaneously (ii) activates T cell signaling.

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