Chimeric receptors

Abstract

The present application relates to chimeric receptors, in particular chimeric receptors comprising homodimeric polypeptides. Uses of these receptors in tumor immunotherapy adoptive cell therapy) are also disclosed. The application further relates to methods of genetically modifying a therapeutic immune cell, eliciting an enhanced immune response against a target antigen. The application further relates to therapeutic cells expressing the chimeric receptors and methods of treating patients using the modified therapeutic cells.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 497,140, ​​filed April 19, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application includes a sequence list that has been electronically submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on April 19, 2024, named 243734_000199_SL.xml, and has a size of 235,430 bytes.

[0005] Statement regarding federally funded research or development

[0006] This invention was completed with the support of the U.S. government under license CA250401 granted by the National Institutes of Health. The U.S. government enjoys certain rights in this invention. Technical Field

[0007] This application relates to chimeric receptors, particularly chimeric receptors comprising homodimeric polypeptides containing at least one homodimerizing motif. This application also relates to the use of these chimeric receptors in tumor immunotherapy (e.g., adoptive cell therapy). This application further relates to methods for genetically modifying therapeutic immune cells to induce enhanced immune responses against target antigens. This application further relates to therapeutic cells expressing chimeric receptors and methods for treating patients using modified therapeutic cells. Background Technology

[0008] Immunotherapy utilizing immune cells, including T cells expressing chimeric antigen receptors (CARs), has revolutionized the treatment of patients with hematologic malignancies. However, immune cells, including CAR T cells, are less effective against solid tumors and brain tumors. While the lack of potency is likely multifactorial, limited persistence in the absence of stimulation and inability to function in the context of chronic antigen exposure have become major obstacles.

[0009] To improve the ability of CAR T cells to survive in the absence of antigen exposure and to function in the context of chronic antigen exposure, gamma cytokines (including IL-2, IL-7, IL-15, and IL-21) or members of other cytokine families (e.g., IL-18) have been overexpressed in CAR T cells. CAR T cells expressing cytokines have shown improved antitumor activity in preclinical models, but safety concerns exist because these secreted or membrane-bound cytokines have the potential to activate bystander immune cells. Therefore, there is a need to develop chimeric receptors that can provide signals to genetically modified immune cells. Summary of the Invention

[0010] This document discloses chimeric receptors, particularly chimeric receptors comprising homodimeric polypeptides containing at least one homodimerization motif. The use of chimeric receptors in tumor immunotherapy (e.g., adoptive cell therapy) is also disclosed. Furthermore, methods for genetically modifying therapeutic immune cells to induce enhanced immune responses against target antigens are disclosed. Methods for treating patients using therapeutic cells expressing chimeric receptors and methods for treating patients using modified therapeutic cells are also disclosed.

[0011] In one aspect, this paper provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer comprises:

[0012] i) An extracellular region containing at least one homodimerizing motif;

[0013] ii) Transmembrane region; and

[0014] iii) Intracellular region, which contains at least one signal transduction region derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof.

[0015] In some embodiments, the extracellular region comprises at least one homodimeric leucine zipper motif, at least one ligand-dependent homodimeric motif, or at least one single-chain variable fragment, or a combination thereof.

[0016] In some embodiments, the extracellular region comprises at least one homodimeric leucine zipper motif. In some embodiments, the homodimeric leucine zipper motif comprises at least five heptad repeat amino acids, with a leucine residue at every sixth position. In some embodiments, the homodimeric leucine zipper motif is derived from the transcription factor c-Jun. In some embodiments, the homodimeric leucine zipper motif comprises the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 80% identity with it. In some embodiments, the homodimeric leucine zipper motif is encoded by the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 80% identity with it.

[0017] In some embodiments, the ligand-dependent homodimerization motif undergoes homodimerization in the presence of a chemical inducer. In some embodiments, the ligand-dependent homodimerization motif comprises FKBP12. F36V Structural domain.

[0018] In some implementations, signal transduction domains derived from cell surface receptors or signal transduction adaptors, or portions or variants thereof, activate the Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway, the nuclear factor kappa light chain enhancer (NFκB) pathway for B cell activation, and / or additional signal transduction pathways activated by the MyD88 signal transduction complex (Myddosome).

[0019] In some implementations, additional signaling pathways activated by the MyD88 signaling complex (Myddosome) are one or more of the following: interleukin-1 receptor-associated kinase (IRAK), TNF receptor (TNFR)-associated factor (TRAF), TANK-binding kinase (TBK), mitogen-activated protein kinase (MAPK), protein kinase B or AKT (PKB / AKT) and / or phosphatidylinositol-3-kinase (PI3K) pathway.

[0020] In some implementations, the intracellular region includes a first signal transduction region derived from a cell surface receptor, or a portion thereof or a variant thereof, and a second signal transduction region derived from a signal transduction adaptor protein, or a portion thereof or a variant thereof.

[0021] In some embodiments, the signal transduction region is derived from a cytokine receptor, or a portion thereof or a variant thereof. In some embodiments, the cytokine receptor, or a portion thereof or a variant thereof, is an erythropoietin receptor (EpoR), a growth hormone receptor (GHR), a prolactin receptor (PRLR), a leptin receptor (LEPR), a granulocyte colony-stimulating factor (G-CSFR), a thrombopoietin receptor (TpoR), an interleukin-23 receptor (IL-23R), an interleukin-10 receptor beta subunit (IL-10R2), an IL-6 beta chain (gp130), IL-2Rβ, IL-18Rα, or IL-18Rβ.

[0022] In some embodiments, the signal transduction region is derived from EpoR, or a portion thereof or a variant thereof. In some embodiments, the EpoR-derived signal transduction region does not contain a phosphatase-1 (SHP1) binding site containing a Src homology 2 domain. In some embodiments, the EpoR-derived signal transduction region comprises the amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80% identity with it. In some embodiments, the EpoR-derived signal transduction region is encoded by the nucleotide sequence of SEQ ID NO: 45, or a sequence having at least 80% identity with it. In some embodiments, the EpoR-derived signal transduction region comprises the amino acid sequence of SEQ ID NO: 49, or a sequence having at least 80% identity with it. In some embodiments, the EpoR-derived signal transduction region is encoded by the nucleotide sequence of SEQ ID NO: 154, or a sequence having at least 80% identity with it.

[0023] In some embodiments, the signal transduction region is derived from GHR, or a portion thereof or a variant thereof. In some embodiments, the GHR-derived signal transduction region comprises the amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80% identity with it. In some embodiments, the GHR-derived signal transduction region is encoded by the nucleotide sequence of SEQ ID NO: 43, or a sequence having at least 80% identity with it.

[0024] In some embodiments, the signal transduction region is derived from a receptor tyrosine kinase, or a portion thereof or a variant thereof. In some embodiments, the receptor tyrosine kinase is the epidermal growth factor receptor (EGFR).

[0025] In some embodiments, the signal transduction region is derived from a Toll-like receptor, or a portion thereof or a variant thereof. In some embodiments, the Toll-like receptor is TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10.

[0026] In some embodiments, the signal transduction region is derived from a signal transduction adaptor protein, or a portion thereof or a variant thereof. In some embodiments, the signal transduction adaptor protein is MyD88. In some embodiments, the MyD88-derived signal transduction region comprises the amino acid sequence of SEQ ID NO: 38 or 220, or a sequence having at least 80% identity with it. In some embodiments, the MyD88-derived signal transduction region is encoded by the nucleotide sequence of SEQ ID NO: 37 or 219, or a sequence having at least 80% identity with it.

[0027] In some embodiments, the transmembrane region and the signal transduction region are derived from the same cell surface receptor. In some embodiments, the transmembrane region is derived from EpoR, or a portion thereof or a variant thereof. In some embodiments, the transmembrane region derived from EpoR comprises the amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from EpoR is encoded by the nucleotide sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109, or SEQ ID NO: 131, or a sequence having at least 80% identity with it.

[0028] In some embodiments, the transmembrane region is derived from GHR, or a portion thereof or a variant thereof. In some embodiments, the transmembrane region derived from GHR comprises the amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from GHR is encoded by the nucleotide sequence of SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, or SEQ ID NO: 145, or a sequence having at least 80% identity with it.

[0029] In some embodiments, the transmembrane region is derived from IL-18Rα, IL-18Rβ, CD28, IL-2Rβ, CD8, CD4, CD3ζ, CD40, CD134 (OX-40), CD19, or CD7. In some embodiments, the transmembrane region is derived from IL-18Rα, or a portion thereof or a variant thereof.

[0030] In some embodiments, the transmembrane region derived from IL-18Rα comprises the amino acid sequence of SEQ ID NO: 40, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from IL-18Rα is encoded by the nucleotide sequence of SEQ ID NO: 39, or a sequence having at least 80% identity with it.

[0031] In some embodiments, the transmembrane region is derived from IL-18Rβ, or a portion thereof or a variant thereof. In some embodiments, the transmembrane region derived from IL-18Rβ comprises the amino acid sequence of SEQ ID NO: 36, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from IL-18Rβ is encoded by the nucleotide sequence of SEQ ID NO: 35, or a sequence having at least 80% identity with it.

[0032] In some embodiments, the transmembrane region is derived from CD28, or a portion thereof or a variant thereof. In some embodiments, the transmembrane region derived from CD28 comprises the amino acid sequence of SEQ ID NO: 42, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from CD28 is encoded by the nucleotide sequence of SEQ ID NO: 41, or a sequence having at least 80% identity with it.

[0033] In some implementations, each polypeptide monomer of the chimeric receptor comprises:

[0034] i) Extracellular region, which contains a homodimeric leucine zipper motif;

[0035] ii) Transmembrane regions derived from EpoR, or portions thereof or variants thereof; and

[0036] iii) Intracellular regions, which contain signal transduction regions derived from EpoR, or portions thereof or variants thereof.

[0037] In some embodiments, the signal transduction region derived from EpoR does not contain a phosphatase-1 (SHP1) binding site containing a Src homology 2 domain. In some embodiments, the signal transduction region derived from EpoR contains the amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80% identity with it. In some embodiments, the signal transduction region derived from EpoR is encoded by the nucleotide sequence of SEQ ID NO: 45, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from EpoR contains the amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from EpoR is encoded by the nucleotide sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109, or SEQ ID NO: 131, or a sequence having at least 80% identity with it. In some embodiments, each polypeptide monomer comprises the amino acid sequence of SEQ ID NO: 112, 114, 116, or 118, or a sequence having at least 80% identity with it. In some embodiments, each polypeptide monomer is encoded by the nucleotide sequence of SEQ ID NO: 111, 113, 115, or 117, or a sequence having at least 80% identity with it.

[0038] In some implementations, each polypeptide monomer of the chimeric receptor comprises:

[0039] i) Extracellular region, which contains a homodimeric leucine zipper motif;

[0040] ii) Transmembrane regions derived from GHR, or portions thereof or variants thereof; and

[0041] iii) Intracellular regions, which contain signal transduction regions derived from GHR, or portions thereof or variants thereof.

[0042] In some embodiments, the signal transduction region derived from GHR comprises the amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80% identity with it. In some embodiments, the signal transduction region derived from GHR is encoded by the nucleotide sequence of SEQ ID NO: 43, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from GHR comprises the amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from GHR is encoded by the nucleotide sequence of SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, or SEQ ID NO: 145. In some embodiments, each polypeptide monomer comprises the amino acid sequence of SEQ ID NO: 120, or a sequence having at least 80% identity with it. In some embodiments, each polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 119, or a sequence having at least 80% identity with it.

[0043] In some implementations, each polypeptide monomer of the chimeric receptor comprises:

[0044] i) Extracellular region, which contains a homodimeric leucine zipper motif;

[0045] ii) Transmembrane regions derived from IL-18Rα, IL-18Rβ, or CD28, or portions thereof or variants thereof; and

[0046] iii) Intracellular regions, which contain signal transduction regions derived from MyD88, or a portion thereof or variant thereof.

[0047] In some embodiments, the signal transduction region derived from MyD88 comprises the amino acid sequence of SEQ ID NO: 38 or 220, or a sequence having at least 80% identity with it. In some embodiments, the signal transduction region derived from MyD88 is encoded by the nucleotide sequence of SEQ ID NO: 37 or 219, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region comprises the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 42, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region is encoded by the nucleotide sequence of SEQ ID NO: 39, SEQ ID NO: 35, or SEQ ID NO: 41, or a sequence having at least 80% identity with it. In some embodiments, each polypeptide monomer comprises the amino acid sequence of SEQ ID NO: 122, 124, or 126, or a sequence having at least 80% identity with it. In some embodiments, each polypeptide monomer is encoded by the nucleotide sequence of SEQ ID NO: 121, 123, or 125, or a sequence having at least 80% identity with it.

[0048] In some implementations, each polypeptide monomer of the chimeric receptor comprises:

[0049] i) Extracellular region, which contains a homodimeric leucine zipper motif;

[0050] ii) Transmembrane regions derived from GHR, or portions thereof or variants thereof; and

[0051] iii) Intracellular regions, which include signal transduction regions derived from GHR, or a portion thereof or variant thereof, and signal transduction regions derived from MyD88, or a portion thereof or variant thereof.

[0052] In some embodiments, the signal transduction region derived from GHR comprises the amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80% identity with it. In some embodiments, the signal transduction region derived from GHR is encoded by the nucleotide sequence of SEQ ID NO: 43, or a sequence having at least 80% identity with it. In some embodiments, the signal transduction region derived from MyD88 comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 220, or a sequence having at least 80% identity with it. In some embodiments, the signal transduction region derived from MyD88 is encoded by the nucleotide sequence of SEQ ID NO: 37 or SEQ ID NO: 219, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from GHR comprises the amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144, or a sequence having at least 80% identity with it. In some embodiments, the transmembrane region derived from GHR is encoded by the nucleotide sequence of SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, or SEQ ID NO: 145. In some embodiments, each polypeptide monomer comprises amino acids 28-611 of the amino acid sequence of SEQ ID NO: 221, or a sequence having at least 80% identity with it. In some embodiments, each polypeptide monomer is encoded by nucleotides 82-1833 of the nucleotide sequence of SEQ ID NO: 222, or a sequence having at least 80% identity with it.

[0053] In some embodiments, each polypeptide monomer of the chimeric receptor further comprises a leader sequence. In some embodiments, the leader sequence is derived from the immunoglobulin heavy chain variable region or the colony-stimulating factor 2 receptor alpha chain (CSF2RA). In some embodiments, the leader sequence derived from the immunoglobulin heavy chain variable region comprises the amino acid sequence SEQ ID NO: 7.

[0054] In some embodiments, each polypeptide monomer further comprises a hinge region. In some embodiments, the hinge region is derived from the same cell surface receptor as the signal transduction region. In some embodiments, the hinge region is derived from IgG1, IgG2, IgG3, IgG4, CD28, or CD8α.

[0055] In some embodiments, the polypeptide monomer further comprises one or more additional polypeptide sequences. In some embodiments, the one or more additional polypeptide sequences comprise a subset selected from one or more cell markers, epitope tags, cytokines, safety switches, dimerization moieties, and degradation moieties.

[0056] On the other hand, this document provides a polynucleotide encoding the chimeric receptor described herein. In some embodiments, one or more nucleotide sequences are expressed in an inducible manner, achieved by an inducible promoter, an inducible expression system, an artificial signal transduction circuit, and / or drug-induced splicing. In some embodiments, the nucleotide sequence is operatively linked to a promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a T cell-specific promoter or an NK cell-specific promoter.

[0057] In some embodiments, the polynucleotide further comprises one or more additional nucleotide sequences encoding one or more additional polypeptide sequences. In some embodiments, the one or more additional polypeptide sequences are selected from one or more cell markers, epitope tags, cytokines, safety switches, dimerization moieties, and degradation moieties. In some embodiments, the epitope tag is FLAG or Myc. In some embodiments, the cell marker is mClover3 or mRuby. In some embodiments, the nucleotide sequence encoding the chimeric receptor is operatively linked to one or more additional nucleotide sequences encoding one or more additional polypeptide sequences via a sequence encoding a self-cleaving peptide and / or an internal ribosome entry site (IRES). In some embodiments, the self-cleaving peptide is a 2A peptide. In some embodiments, the 2A peptide is a T2A, P2A, E2A, or F2A peptide. In some embodiments, the 2A peptide is a P2A peptide. In some embodiments, the P2A peptide comprises the amino acid sequence SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity with it.

[0058] In some implementations, the polynucleotides described herein are DNA molecules.

[0059] In some implementations, the polynucleotides described herein are RNA molecules.

[0060] On the other hand, this document provides a recombinant vector containing the polynucleotides described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, lentiviral vector, adenovirus vector, adeno-associated virus vector, alphavirus vector, herpesvirus vector, baculovirus vector, or vaccinia virus vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a minicircle plasmid, Sleeping Beauty transposon, piggyBac transposon, or a single-stranded or double-stranded DNA molecule used as a template for homology-directed repair (HDR) based gene editing.

[0061] In another aspect, this document provides an isolated host cell containing the polynucleotides or recombinant vectors described herein. In yet another aspect, this document provides an isolated host cell containing the chimeric receptors described herein.

[0062] In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell, natural killer (NK) cell, mesenchymal stem cell (MSC), or macrophage. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is an αβ T cell receptor (TCR) T cell, γδ T cell, CD8+ T cell, CD4+ T cell, cytotoxic T cell, invariant natural killer T (iNKT) cell, memory T cell, or memory stem T cell (T... SCM The host cells may be naive T cells, effector T cells, helper T cells, or regulatory T cells (Tregs). In some embodiments, the host cells are NK cells. In some embodiments, the host cells are NK cells and / or cell lines (e.g., NK92 cells) derived from peripheral blood, umbilical cord blood, induced pluripotent stem (iPS) cells (iPSCs).

[0063] In some embodiments, the host cell further expresses one or more antigen recognition molecules. In some embodiments, the one or more antigen recognition molecules are selected from chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TRuCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell engagers, natural or transgenic T-cell receptors, and antibodies, or combinations thereof.

[0064] In some embodiments, the host cell is further genetically modified using gene editing technologies (e.g., CRISPR-Cas9 or transcription activator-like effector nucleases (TALENs)) to enhance its function by expressing one or more additional genes or deleting one or more repressive genes. In some embodiments, the one or more additional genes are selected from one or more transcription factors. In some embodiments, the transcription factor is c-Jun. In some embodiments, the one or more repressive genes are REGNASE-1 or DNMT3A. In some embodiments, the gene editing technology is CRISPR-Cas9 or transcription activator-like effector nucleases (TALENs).

[0065] In some implementations, the host cells have been activated and / or expanded in vitro.

[0066] In some implementations, the host cell is an allogeneic cell. In other implementations, the host cell is an autologous cell.

[0067] In some implementations, immune cells are derived from induced pluripotent stem (iPS) cells (iPSCs).

[0068] On the other hand, this article provides a pharmaceutical composition comprising the host cells described herein and pharmaceutically acceptable carriers and / or excipients.

[0069] On the other hand, this document provides a method for enhancing the effector function of immune cells, wherein the immune cells express one or more antigen recognition molecules, the method comprising genetically modifying the cells with the polynucleotides or recombinant vectors described herein. In some embodiments, the one or more antigen recognition molecules are selected from chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TruCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell connectors, natural or transgenic T-cell receptors, and antibodies, or combinations thereof. In some embodiments, the effector function is one or more of amplification, persistence, and / or cytotoxicity (e.g., antitumor activity).

[0070] On the other hand, this document provides a method for generating isolated host cells as described herein, the method comprising genetically modifying the host cells with the polynucleotides or recombinant vectors described herein. In some embodiments, the method further comprises genetically modifying the host cells to express one or more antigen recognition molecules. In some embodiments, the one or more antigen recognition molecules are selected from chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TruCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell connectors, natural or transgenic T-cell receptors, and antibodies, or combinations thereof. In some embodiments, the genetic modification step is performed via viral gene delivery. In some embodiments, the genetic modification step is performed via non-viral gene delivery. In some embodiments, the genetic modification step is performed in vitro. In some embodiments, the method further comprises in vitro activation and / or amplification of the host cells before, after, and / or during the genetic modification.

[0071] On the other hand, this document provides a method for treating a disease, the method comprising administering to a subject an effective amount of the host cells or pharmaceutical compositions described herein. In some embodiments, the method includes:

[0072] a) Isolate T cells or NK cells from the subject or donor;

[0073] b) In vitro modification of T cells or NK cells with the polynucleotides or recombinant vectors described herein;

[0074] c) Optionally, T cells or NK cells are modified in vitro to express one or more antigen recognition molecules that bind to disease-associated antigens;

[0075] d) Optionally, before, after, and / or during step b) or c), expand and / or activate modified T cells or NK cells; and

[0076] e) Introduce a therapeutically effective amount of modified T cells or NK cells into the subject.

[0077] In some embodiments, one or more antigen recognition molecules are selected from chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TruCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell adapters, natural or transgenic T-cell receptors, and antibodies, or combinations thereof.

[0078] In some implementation schemes, the disease is cancer, infection, or autoimmune disease.

[0079] In some implementations, the subjects are humans.

[0080] On the other hand, this paper provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer comprises:

[0081] i) Extracellular region, which contains means for homodimerizing two polypeptide monomers;

[0082] ii) Transmembrane region, which includes means for connecting the intracellular region to the extracellular region; and

[0083] iii) Intracellular region, which contains means for enhancing the effector functions of immune cells.

[0084] In some embodiments, the means for homodimerizing two polypeptide monomers includes at least one homodimerizing leucine zipper motif, at least one ligand-dependent homodimerizing motif, or at least one single-chain variable fragment, or a combination thereof. In some embodiments, the means for homodimerizing two polypeptide monomers includes at least one homodimerizing leucine zipper motif.

[0085] In some embodiments, the homodimeric leucine zipper motif comprises at least five heptapeptide repeating amino acids, with a leucine residue every six positions. In some embodiments, the homodimeric leucine zipper motif is derived from the transcription factor c-Jun. In some embodiments, the homodimeric leucine zipper motif comprises the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 80% identity with it. In some embodiments, the homodimeric leucine zipper motif is encoded by the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 80% identity with it.

[0086] In some implementations, the ligand-dependent homodimerization motif undergoes homodimerization in the presence of a chemical inducer.

[0087] In some embodiments, means for enhancing effector function include at least one signal transduction region derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof. In some embodiments, means for enhancing effector function include a signal transduction region derived from EpoR, or a portion thereof or a variant thereof. In some embodiments, means for enhancing effector function include a signal transduction region derived from GHR, or a portion thereof or a variant thereof. In some embodiments, means for enhancing effector function include a signal transduction region derived from MyD88, or a portion thereof or a variant thereof. In some embodiments, means for enhancing effector function include a signal transduction region derived from GHR, or a portion thereof or a variant thereof, and a signal transduction region derived from MyD88, or a portion thereof or a variant thereof.

[0088] On the other hand, this paper provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer comprises:

[0089] i) Extracellular region, which contains means for homodimerizing two polypeptide monomers;

[0090] ii) Transmembrane region; and

[0091] iii) Intracellular region, which contains at least one signal transduction region derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof.

[0092] On the other hand, this paper provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer comprises:

[0093] i) An extracellular region containing at least one homodimerizing motif;

[0094] ii) Transmembrane region; and

[0095] iii) Intracellular region, which contains means for enhancing the effector functions of immune cells.

[0096] On the other hand, this document provides a chimeric receptor comprising i) an extracellular region; ii) a transmembrane region; and iii) an intracellular region, wherein the intracellular region comprises at least one signal transduction region derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof, and the improvement comprises the extracellular region comprising at least one homodimerization motif.

[0097] On the other hand, this article provides a chimeric receptor, which is improved to include two transmembrane polypeptide monomers each comprising: i) an extracellular region containing at least one homodimerizing motif and ii) an intracellular region containing at least one signal transduction region derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof.

[0098] In some embodiments, the improvement includes the extracellular region comprising at least one homodimeric leucine zipper motif, at least one ligand-dependent homodimeric motif, or at least one single-stranded variable fragment, or a combination thereof. In some embodiments, the improvement includes the extracellular region comprising at least one homodimeric leucine zipper motif. In some embodiments, the improvement includes the homodimeric leucine zipper motif comprising at least five heptapeptide repeating amino acids, wherein a leucine is present every six positions. In some embodiments, the improvement includes the homodimeric leucine zipper motif being derived from the transcription factor c-Jun. In some embodiments, the improvement includes the homodimeric leucine zipper motif comprising the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 80% identity with it. In some embodiments, the improvement includes the homodimeric leucine zipper motif being encoded by the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 80% identity with it. In some embodiments, the improvement includes the ligand-dependent homodimeric motif undergoing homodimerization in the presence of a chemical inducer. In some embodiments, the improvement further includes that the intracellular region comprises a signal transduction region derived from EpoR, or a portion thereof or a variant thereof. In some embodiments, the improvement further includes that the intracellular region comprises a signal transduction region derived from GHR, or a portion thereof or a variant thereof. In some embodiments, the improvement further includes that the intracellular region comprises a signal transduction region derived from GHR, or a portion thereof or a variant thereof, and a signal transduction region derived from MyD88, or a portion thereof or a variant thereof. In some embodiments, the improvement further includes that the intracellular region comprises a signal transduction region derived from GHR, or a portion thereof or a variant thereof, and a signal transduction region derived from MyD88, or a portion thereof or a variant thereof.

[0099] On the other hand, this article provides a polynucleotide that encodes the chimeric receptor described herein.

[0100] On the other hand, this article provides a recombinant vector containing the polynucleotides described herein.

[0101] On the other hand, this article provides an immune cell that expresses the chimeric receptor described herein.

[0102] On the other hand, this article provides an immune cell containing either the polynucleotides or recombinant vectors described herein.

[0103] On the other hand, this article provides a method for enhancing the effector function of immune cells, wherein the improvement includes the expression of the chimeric receptor described herein by the immune cells.

[0104] On the other hand, this article provides a method for improving the expansion, persistence, or cytotoxicity of antigen-activated immune cells, wherein the improvement includes the expression of the chimeric receptor described herein by the immune cells.

[0105] On the other hand, this article provides a method for treating a disease, the method comprising administering an effective amount of immune cells to a subject, wherein the modification includes the immune cells expressing the chimeric receptor described herein. Attached Figure Description

[0106] Figure 1A-1D This study demonstrated that Jun.MyD88 homodimer activates NFκB signaling and improves the antitumor activity of CAR T cells. Figure 1A NFκB activity in Ramos Blue NFκB reporter cells transduced with the indicator construct (N=3 technical replicates, paired ANOVA) p<0.05). Figure 1B NFκB activity in reporter cells transduced with indicator constructs of Ramos Blue NFκB (N=5 technical replicates, paired ANOVA) p<0.0001). Figure 1C Transduction efficiency in human T cells (N=1). Figure 1D Repeated stimulation assays using A673 tumor cells and EphA2-CAR T cells (N=1).

[0107] Figures 2A-2F The study showed that Jun leucine-based homodimers activate STAT5, leading to improved T cell viability, which is inhibited by ruxolitinib. Figure 2A and 2B Geometric mean fluorescence intensity of pSTAT5 in Jun ZipR+ cells Figure 2A ) or positive percentage ( Figure 2B (N=4 biological replicates, one-way ANOVA, p<0.01, p<0.001, p<0.0001). Figure 2C-2E The frequency of pSTAT5+ cells treated in vitro for 48 hours with an indicated concentration of ruxolitinib (N=3 biological replicates). Figure 2F The frequency of dead cells (annexin V+ dead+), apoptotic cells (annexin V+ dead-), necrotic cells (annexin V- dead+), and live cells (annexin V- dead-) after 7 days of cytokine starvation in vitro with or without 5 μM ruxolitinib treatment (N = 2 biological replicates, two-way ANOVA, live cell population) p<0.0001).

[0108] Figures 3A-3C This demonstrates that Jun homodimer ZipR does not alter the human T-cell immune phenotype. Figure 3A Transduction efficiency in human T cells (N=4, biological replicates). Figure 3B The frequency of CD4+ or CD8+ cells (N=4, biological replicates). Figure 3C T cell immunophenotype of CD4+ (left) or CD8+ (right) cells (N=4, biological replicates, T cells) EMRA : Re-expressing the effect memory of CD45RA [CD45+CCR7-]; T EM : Effect memory [CD45RA-CCR7-], T CM Central Memory [CD45RA-CCR7-]; T N -like: childlike [CD45RA+CCR7+]).

[0109] Figures 4A-4B The study demonstrated successful transduction of reporter cell lines and corresponding STAT5 phosphorylation using retroviral constructs encoding Jun.GHR and Jun.GHR.MyD88. Figure 4A The results show that the STAT5 reporter cell line was successfully transduced using retroviral constructs encoding Jun.GHR and Jun.GHR.MyD88. Figure 4B It is shown that Jun.GHR and Jun.GHR.MyD88 phosphorylate STAT5.

[0110] Figures 5A-5B This demonstrates an evaluation of the functionality of the MyD88 structure domain in Jun.GHR.MyD88 ZipR. Figure 5A The transduction of Ramos Blue MyD88 signaling reporter cell lines using Jun.GHR and Jun.GHR.MyD88 is shown. Figure 5B It is shown that, in contrast to Jun.GHR, Jun.GHR.MyD88 activates the MyD88 signaling. Detailed Implementation

[0111] Despite recent advances in cancer treatment, patients with relapsed or refractory disease continue to experience poor outcomes and require novel approaches. Genetically modified T cells that express chimeric antigen receptors (CARs) can kill chemotherapy-resistant tumor cells, thus having the potential to improve outcomes and reduce treatment-related toxicities from conventional therapies [1, 2].

[0112] CARs typically consist of four components: i) extracellular antigen recognition domains, most commonly single-chain variable fragments (scFv), ii) structural components, such as hinge and transmembrane domains, iii) co-stimulatory domains that provide signals to maintain CAR T cell effector function, and iv) CD3ζ activation domains [1-3].

[0113] First-generation CARs provide signaling only via CD3ζ1. Second-generation CARs also provide signaling2, most commonly through co-stimulation with CD28 or 4-1BB to maintain the expansion of activated CAR T cells. Although activated CAR T cells produce cytokines such as interleukin-2 (IL-2), production decreases after repeated exposure to tumor cells[9], and some cytokines important for T cell effector function (such as IL-12 and IL-15) are produced at low levels or not at all by T cells[23, 24]. Due to these limitations, engineered CAR T cells are needed to enhance cytokine-mediated signaling.

[0114] Among other things, this application provides a chimeric receptor capable of signaling genetically modified immune cells. The chimeric receptor described herein comprises a homodimeric polypeptide. Such chimeric receptors represent a significant advance in the field of immunotherapy. Because only one polypeptide chain is required, the chimeric receptor described herein will simplify the production of immune cells expressing the chimeric receptor, and thus streamline its clinical translation.

[0115] While the data disclosed herein (see the Examples section below) focuses on CAR expression in T cells, the methods and / or compositions disclosed herein are applicable to a variety of other cell therapy platforms that are being actively explored, including but not limited to NK cells, NK92 cells, NKT cells, and γδ T cells. Similarly, while the methods and / or compositions disclosed herein can be used to treat cancer, the invention is also broadly applicable to adoptive immunotherapy for non-malignant diseases, including but not limited to infectious diseases (e.g., viral infections) and autoimmune diseases.

[0116] definition

[0117] As used herein, the term "chimeric receptor" can refer to a cell surface receptor engineered to have at least a portion of at least one domain (e.g., extracellular region, transmembrane region, intracellular region, which may include, for example, a signal transduction region, i.e., an intracellular signal transduction region) derived from one or more different sources.

[0118] As used herein, the term "chimeric antigen receptor" or "CAR" is defined as a cell surface receptor comprising an extracellular target-binding region, a transmembrane region, and an intracellular region, wherein the intracellular region comprises a lymphocyte activation domain and optionally at least one co-stimulatory signaling domain, all in a combination not naturally occurring together on a single protein. This particularly includes receptors in which the extracellular and intracellular regions are not naturally occurring together on a single receptor protein. The chimeric antigen receptor (CAR) of the present invention can be used with lymphocytes such as T cells and natural killer (NK) cells.

[0119] A "homodimerizing motif" is any molecule that has the ability to associate (covalently or non-covalently) with molecules of the same structure.

[0120] The terms “T cell” and “T lymphocyte” are used interchangeably and synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be helper T (Th) cells, such as type 1 helper T (Th1) cells or type 2 helper T (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+CD8+ T cells, or any other subset of T cells. Other illustrative T cell populations suitable for a particular embodiment include naive T cells and memory T cells. Also included is “NKT cells,” which refers to a specialized population of T cells that expresses a semi-constant αβ T cell receptor but also expresses multiple molecular markers commonly associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8- cells. The TCR on NKT cells is unique because it recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. NKT cells can have protective or destructive effects due to their ability to produce cytokines that promote inflammation or immune tolerance. Also included are "gamma-delta T cells (γδ T cells)," a specialized subset of T cells possessing distinct TCRs on their surface. Unlike most T cells, where the TCR consists of two glycoprotein chains named α-TCR and β-TCR, the TCR in γδ T cells is composed of both γ and δ chains. γδ T cells play a role in immune surveillance and immune regulation and have been found to be an important source of IL-17 and induce robust CD8+ cytotoxic T cell responses. Also included are "regulatory T cells" or "Tregs," which are T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Treg cells are typically Foxp3-positive CD4+ T cells, and may also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0121] The terms “natural killer cell” and “NK cell” are used interchangeably and synonymously herein. As used herein, NK cells refer to differentiated lymphocytes with a CD16+CD56+ and / or CD57+ TCR- phenotype. NK cells are characterized by their ability to bind to and kill cells that do not express “self” MHC / HLA antigens by activating specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands of NK activation receptors, and to release protein molecules called cytokines that stimulate or suppress immune responses.

[0122] As used herein, the term "antigen" refers to any substance (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, part thereof, or combination thereof) that can be bound by T cell receptors. Antigens can also elicit an immune response. Examples of immune responses may involve, but are not limited to, antibody production, or activation of immunologically competitive cells, or both. Those skilled in the art will understand that antigens do not necessarily have to be encoded by "genes." It will be apparent that antigens can be synthesized, derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids containing other biological components, organisms, protein / antigen subunits, cytotoxic or inactivated whole cells or lysates.

[0123] The term “antigen recognition molecule” refers to any molecule capable of recognizing antigens as described herein. Non-limiting examples of antigen recognition molecules include T cell receptors (TCRs) (e.g., αβ TCRs), synthetic T cell receptor and antigen receptor (STARs), chimeric antigen receptors (CARs), T cell antigen couplers (TACs), T cell receptor fusion constructs (TruCs), HLA-independent T cell receptors (HITs), bispecific T cell connectors, and antibodies (e.g., bispecific antibodies) or antibody fragments.

[0124] The term "antibody" refers to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intracellular antibodies, minibody antibodies, diabody antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the above antibodies. The term "antibody" also refers to covalently coupled double-chain antibodies (such as those disclosed in U.S. Patent Application Publication 2007 / 0004909) and Ig-DARTS (such as those disclosed in U.S. Patent Application Publication 2009 / 0060910). Antibodies that can be used as TCR-binding molecules include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2), or subclass.

[0125] The term "host cell" refers to any cell containing heterologous nucleic acid. Heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism that has been selected, modified, transformed, grown, used, or manipulated in any way to produce substances through the cell, such as expressing genes, DNA or RNA sequences, proteins, or enzymes. A suitable host can be determined. For example, a host cell can be selected based on the vector backbone and the desired outcome. As an example, plasmids or cosmids can be introduced into prokaryotic host cells for the replication of several types of vectors. Bacterial cells such as, but not limited to, DH5α, JM109, and KCB, SURE® competent cells, and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. Additionally, bacterial cells such as E. coli LE392 can be used as host cells for bacteriophage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells used for vector replication and / or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. In some embodiments, the host cell is autologous. In some embodiments, the host cell is allogeneic.

[0126] The host cells disclosed herein include immune cells (e.g., T cells and natural killer cells, or macrophages) or stem cells (e.g., mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs) [iPSCs]) that contain DNA or RNA sequences encoding the chimeric receptors described herein and express the chimeric receptors on their cell surface. Host cells can be used to enhance immune cell activity (e.g., effector function), treat tumors, treat infectious diseases, and treat autoimmune diseases.

[0127] The terms “activation” or “stimulation” refer to the induction of a change in the biological state of cells (e.g., T cells and NK cells) that causes the cells to express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All of these changes can be generated by primary stimulus signals. Co-stimulatory signals can amplify the amplitude of the primary signal and inhibit cell death after initial stimulation, resulting in a more persistent state of activation and thus greater cytotoxic capacity. A “co-stimulatory signal” is a signal that, in combination with a primary signal (such as the TCR / CD3 linker), causes T cell and / or NK cell proliferation and / or upregulation or downregulation of key molecules.

[0128] The term "expression" refers to the permission or initiation of the production of information from a gene or DNA sequence, such as by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence to produce proteins. DNA sequences are expressed in or by cells to form "expression products" such as proteins. The expression product itself (e.g., the resulting protein) can also be described as being "expressed" by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0129] The term "tumor" refers to a benign or malignant abnormal growth of tissue. The term "tumor" includes cancer.

[0130] The term "effective function" refers to the specialized function of a cell. For example, the effector function of T cells can be cytolytic activity (e.g., tumor-killing activity) or auxiliary activity including the secretion of cytokines.

[0131] As used herein, the term “safety switch” refers to any mechanism that can remove or inhibit the effects of the chimeric receptor described herein from a system (e.g., a culture or a subject).

[0132] As used in this article, the term "site-specific nuclease" refers to a nuclease that can specifically recognize and cleave nucleic acid (DNA or RNA) sequences.

[0133] The terms “genetically modified” or “genetically engineered” refer to the addition of additional genetic material in the form of DNA or RNA to a cell.

[0134] As used herein, in the context of a protein or polypeptide (e.g., a chimeric receptor or its domain), the terms "derivative" or "derived from" refer to: (a) a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the polypeptide as a derivative; (b) a polypeptide encoded by a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the nucleotide sequence encoding the polypeptide as a derivative; and (c) containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, relative to the polypeptide as a derivative. 19. A polypeptide with 20 or more amino acid mutations (i.e., additions, deletions, and / or substitutions); (d) a polypeptide encoded by a nucleotide sequence capable of hybridizing with a nucleic acid encoding a polypeptide as a derivative under high, medium, or typical stringent hybridization conditions; (e) a polypeptide encoded by a nucleotide sequence capable of hybridizing with a nucleotide sequence encoding a fragment of a polypeptide as a derivative under high, medium, or typical stringent hybridization conditions, said fragment being at least 20 consecutive amino acids, at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 75 consecutive amino acids, at least 100 consecutive amino acids, at least 125 consecutive amino acids, or at least 150 consecutive amino acids; or (f) a fragment of a polypeptide as a derivative.

[0135] Percentage sequence identity can be determined using any method known to those skilled in the art. In a particular embodiment, percentage identity is determined using the “Best Fit” or “Gap” procedure of a sequence analysis software package (version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (e.g., high, medium, and typical stringent conditions) has been described, see, for example, U.S. Patent Application Publication No. 2005 / 0048549 (e.g., paragraphs 72-73), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0136] The term "portion," when used in relation to a polypeptide or protein, refers to any component of the polypeptide or protein, including but not limited to subunits, domains, or fragments. For example, a portion of a dimeric cell surface receptor can be a subunit (or chain) of that receptor.

[0137] As used herein, a "functional fragment" refers to a fragment of a polypeptide or protein, or a polynucleotide encoding said polypeptide or protein, that retains at least one function of the full-length polypeptide or protein. A functional fragment may include one, two, three, or more fragments of a full-length polypeptide or protein, or a polynucleotide encoding said polypeptide or protein. Each fragment may contain an amino acid sequence of at least 5 consecutive amino acid residues, at least 6 consecutive amino acid residues, at least 7 consecutive amino acid residues, at least 8 consecutive amino acid residues, at least 9 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 11 consecutive amino acid residues, at least 12 consecutive amino acid residues, at least 13 consecutive amino acid residues, at least 14 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the full-length polypeptide or protein amino acid sequence.

[0138] The terms "vector," "cloning vector," "recombinant vector," and "expression vector" refer to a vehicle that can introduce DNA or RNA sequences (e.g., foreign genes) into a host cell to genetically modify the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthetic RNA and DNA molecules, bacteriophages, viruses, etc. In some embodiments, the vector is a viral vector, such as, but not limited to, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors, herpesvirus vectors, baculovirus vectors, and vaccinia virus vectors.

[0139] As used herein, the terms “operably linked” and similar phrases, when used with respect to nucleic acids or amino acids, refer to the operational linking of nucleic acid or amino acid sequences placed together in a functional relationship. For example, operably linked promoter, enhancer elements, open reading frames (OPFs), 5' and 3' UTRs, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in transcription of the OPF and ultimately the production of a polypeptide (i.e., expression of the OPF). As another example, an operably linked peptide is a peptide in which functional domains are placed at appropriate distances from each other to confer the intended function of each domain.

[0140] As used herein, the term "promoter" refers to any sequence capable of driving transcription of a coding sequence in a cell. Therefore, promoters used in the vectors of this disclosure may comprise cis-acting transcriptional control elements and regulatory sequences relating to the timing and / or rate of gene transcription. As non-limiting examples, promoters may be cis-acting transcriptional control elements, including enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences involved in transcriptional regulation. Such cis-acting sequences typically interact with proteins or other biomolecules to perform (on / off, regulation, control, etc.) transcription. "Constitutive" promoters are those that continuously drive expression under most environmental conditions and in cell development and / or differentiation states. "Inducible" or "regulatory" promoters direct nucleic acid expression under developmental and / or environmental influences. Non-limiting examples of environmental conditions that can affect transcription via inducible promoters include increased temperature, drought, the presence of light, and anaerobic conditions.

[0141] The terms "enhance," "promote," "increase," "expand," or "improve" generally refer to the ability of the composition, as intended herein, to produce, initiate, or induce a greater physiological response (i.e., downstream effect) compared to the response induced by the vehicle or control molecule / composition. Measurable physiological responses may include immune cell expansion, activation, effector function, increased persistence, and / or increased tumor cell killing capacity, as well as other physiological responses that are apparent from the understanding in the art and from the description herein. In some embodiments, the “increased” or “enhanced” amount can be a “statistically significant” amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) in the response produced by the solvent or control composition (including all integers and decimals in the middle and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0142] "Decrease," "lower," "lessen," "reduce," or "abate" generally refers to the ability of the composition, as intended herein, to produce, initiate, or induce a smaller physiological response (i.e., downstream effect) compared to the response induced by the solvent or control molecule / composition. In some embodiments, the amount of "decrease" or "abate" can be a "statistically significant" amount and can include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimals in the middle and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) in the response induced by the solvent, control composition (reference response), or response in a particular cell lineage.

[0143] The term "treat" or "treatment" for a state, disease, symptom, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of the development of at least one clinical or subclinical symptom of a state, disease, symptom, or condition in a subject who may have or be susceptible to the state, disease, symptom, or condition but has not yet experienced or displayed clinical or subclinical symptoms of the state, disease, symptom, or condition; or (2) inhibiting the state, disease, symptom, or condition, i.e., preventing, reducing, or delaying the development of the state, disease, symptom, or condition or its recurrence or at least one clinical or subclinical symptom; or (3) alleviating the state, disease, symptom, or condition, i.e., causing the resolution of at least one of the state, disease, symptom, or condition or its clinical or subclinical symptoms. The benefit to the subject to be treated is statistically significant or at least perceptible to the patient or physician.

[0144] The term "effective" when applied to dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that is effective when administered alone. The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, the severity of the condition being treated, one or more specific medications being used, and the mode of administration, etc.

[0145] As used herein, the term "pharmaceutical composition" means a composition comprising the polynucleotides, carriers, peptides, compositions or host cells described herein, formulated for administration to a subject for the treatment, relief or prevention of disease.

[0146] The phrase “pharmaceutical acceptable” as used in conjunction with the compositions described herein refers to the molecular entity and other components of such compositions that are physiologically tolerable and generally do not produce adverse effects when administered to mammals (e.g., humans). Preferably, the term “pharmaceutical acceptable” means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in mammals, more specifically in humans.

[0147] As used herein, the term "protein" encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including but not limited to glycoproteins, as well as all other types of modified proteins (e.g., proteins produced by phosphorylation, acetylation, myristylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.).

[0148] Unless otherwise stated, the terms “nucleic acid,” “nucleotide,” and “polynucleotide” cover both DNA and RNA. “Nucleic acid sequence” or “nucleotide sequence” refers to a nucleic acid sequence that encodes an amino acid. The term can also refer to a nucleic acid sequence that includes portions encoding any amino acids added as an artifact, including any amino acids encoding a linker.

[0149] The terms “patient,” “individual,” “subject,” and “animal” are used interchangeably herein and refer to mammals, including but not limited to humans and veterinary animals (e.g., cats, dogs, cattle, horses, sheep, pigs, etc.) and laboratory animal models. In a preferred embodiment, the subject is a human.

[0150] The term "carrier" refers to a diluent, adjuvant, excipient, or solvent applied together with a compound. Such drug carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water or aqueous solutions, saline solutions, dextrose solutions, and glycerol solutions are preferred as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more binders (for compressed pellets), flow aids, encapsulating agents, flavorings, and colorings. Suitable drug carriers are described in EWMartin's "Remington's Pharmaceutical Sciences".

[0151] Unless the context clearly specifies otherwise, the singular forms “a / an / a kind of” and “the” include a plural indicator. Thus, for example, a reference to “method” includes one or more methods and / or steps of the type described herein and / or that will become apparent to a person skilled in the art upon reading this disclosure.

[0152] The terms "about" or "approximately" encompass a range that is statistically significant for a value. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The permissible deviation covered by the terms "about" or "approximately" depends on the specific system being studied and is readily understood by those skilled in the art.

[0153] If an aspect of this disclosure is described as “comprising” or a version thereof (e.g., containing a feature), then the implementation is also contemplated as “consisting of the feature” or “substantially consisting of the feature”.

[0154] Unless otherwise stated, the practice of this disclosure employs conventional techniques of statistical analysis, molecular biology (including recombinant technologies), microbiology, cell biology, and biochemistry, which are within the art. Such tools and techniques are described in detail in, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al., ed. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional technical explanations can be found, for example, in U.S. Patent No. 7,912,698 and U.S. Patent Application Publication Nos. 2011 / 0202322 and 2011 / 0307437.

[0155] The techniques described illustratively herein may be practiced appropriately in the absence of any element(s) not specifically disclosed herein.

[0156] The terms and expressions used are used as descriptive rather than restrictive terms, and the use of such terms and expressions does not exclude any equivalents of the features shown and described or any parts thereof, and various modifications may be made within the scope of the claimed technology.

[0157] Chimeric receptor

[0158] In some embodiments, this disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise:

[0159] i. Extracellular region containing at least one homodimerizing motif;

[0160] ii. Transmembrane region; and

[0161] iii. Intracellular region, which contains at least one signal transduction region.

[0162] In some implementations, at least one signal transduction region may be derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof.

[0163] extracellular region

[0164] In some embodiments, the extracellular region of the chimeric receptor may contain at least one homodimerizing motif. In some embodiments, the extracellular region of the chimeric receptor may contain more than one homodimerizing motif, such as two, three, four, five or more homodimerizing motifs.

[0165] In some implementations, the homodimerization motif may be a homodimerization leucine zipper motif, a ligand-dependent homodimerization motif, or a homodimerization motif that can otherwise bring the signal transduction region close together (e.g., a single-stranded variable fragment).

[0166] In some embodiments, the extracellular region may contain at least one leucine zipper motif. In some embodiments, the extracellular region may contain more than one leucine zipper motif, such as two, three, four, five or more leucine zipper motifs.

[0167] In some embodiments, the leucine zipper motif may contain at least two, three, four, or five heptapeptide repeating amino acids, with a leucine residue every six positions. In some embodiments, the leucine zipper motif may contain at least five heptapeptide repeating amino acids, with a leucine residue every six positions.

[0168] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif may comprise the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the leucine zipper motif is encoded by the following: the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3. In some embodiments, the leucine zipper motif comprises the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0169] In some embodiments, the leucine zipper motif may include any homodimeric leucine zipper motif as described, for example, in Moll et al., 2001 [6], Reike et al., 2013 [7] and Newman et al., 2003 [8], the contents of which are incorporated herein by reference in their entirety for all purposes.

[0170] In some embodiments, the zipper motifs (e.g., leucine zipper motifs) that may be used according to this disclosure may be derived from any number of human proteins within the knowledge of those skilled in the art. Non-limiting examples of human proteins containing zipper motifs that may be used according to this disclosure are described in Table 1. With respect to Table 1 (see also Newman JRS and Keating AE. Comprehensive identification of human bZIP interactions with coiled-coilarrays. Science. 2003 Jun 27;300(5628):2097-101, which are incorporated herein by reference in their entirety for all intended purposes), a: other names of the proteins specified on the left, or names of other proteins that share the same coiled-coil sequence used herein. b: sequences used in array experiments divided into five parts. From left to right: [cloning vector sequence] - [containing basic regions for cloning] - [coiled-coil domain] - [additional residues that facilitate cloning] - [cloning vector]. The N-terminus (left side) of the coiled-helical domain was determined by referencing the Fos / Jun crystal structure, and the C-terminus was determined by the point where the PAIRCOIL probability drops below 10%. The register of the coiled-helical domain is shown at the bottom of the column, starting from position f. Sequences were aligned using CLUSTALX based on sequence similarity of the coiled-helical domains. The additional protein sequences indicated by the numbers in square brackets are as follows: [1] to [4]-MSYYHHHHHHLESTSLYKKA (SEQ ID NO: 207), followed by GSGS (SEQ ID NO: 208) [1], GSEF (SEQ ID NO: 209) [2], GSGR (SEQ ID NO: 210) [3] and GFDD (SEQ ID NO: 211) [4]; [5]-LE; [6]-KVE; [7]-QLE; [8]-RLE; [9]-E;

[10] -FLE;

[11] -YLEII;

[12] -ALE;

[13] -NLE;

[14] -CSSNTQLSCTKWLIRGC (SEQ ID NO: 213);

[15] -SSNTQLSCTKWLIRGC (SEQ ID NO: 214);

[16] -HLE;

[17] -STCRDL (SEQ ID NO: 214) NO: 215);

[18] -FSTCRDL (SEQ ID NO: 216);

[19] -LDLQRSMNRRY (SEQ ID NO: 217);

[20] -PAEIYES (SEQ ID NO: 218). The initial methionine in sequences [1] to [4] is cleaved in vivo during expression.

[0171] Table 1. Non-limiting examples of human proteins containing zipper motifs

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] In some embodiments, the extracellular region of the chimeric receptor disclosed herein may contain at least one ligand-dependent homodimerization motif. For example, the ligand-dependent homodimerization motif may contain a dimerized chemically induced (CID) binding domain, such as the FKBP12 domain (see Mata et al., 2017 [9], Juillerat et al., 2019

[17] , Leung et al., 2019 [5], the contents of which are incorporated herein by reference in their entirety for all purposes). In one embodiment, the ligand-dependent homodimerization motif contains FKBP12. v36 Domain. In one implementation, the ligand-dependent homodimerization motif comprises a single-stranded variable segment.

[0178] In some embodiments, the extracellular region of the chimeric receptor disclosed herein may contain other homodimerization motifs that allow close proximity of the signal transduction domains, such as single-stranded variable fragments as a non-limiting example (see, for example, Katsarou et al., 2021

[10] , the contents of which are incorporated herein by reference in their entirety for all purposes). The single-stranded variable fragment may induce homodimerization by binding a single ligand, and may be another example of a “ligand-dependent homodimerization motif” or may induce homodimerization by hydrophobic interactions between two variable fragments.

[0179] In some implementations, when using two or more homodimerization motifs, the two or more homodimerization motifs can be operatively linked to each other via any of a variety of connectors. Example connector sequences may include (GGGGS)n (SEQ ID NO: 150), where n is any integer (e.g., 1, 2, 3, 4, 5).

[0180] In some embodiments, the adapter comprises the amino acid sequence of SEQ ID NO: 151, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 151. In some embodiments, the nucleotide sequence encoding the adapter comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 151, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 151. In some embodiments, the nucleotide sequence encoding the adapter comprises the nucleotide sequence shown in SEQ ID NO: 152, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 152. In some embodiments, the nucleotide sequence encoding the adapter comprises the nucleotide sequence shown in SEQ ID NO: 153, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 153. In some embodiments, the adapter comprises the amino acid sequence shown in SEQ ID NO: 151. In some embodiments, the nucleotide sequence encoding the adapter comprises the nucleotide sequence shown in SEQ ID NO: 152. In some embodiments, the nucleotide sequence encoding the adapter comprises the nucleotide sequence shown in SEQ ID NO: 153.

[0181] Hinge area

[0182] In some implementations, the extracellular region of the chimeric receptor disclosed herein may include a hinge region.

[0183] The hinge region may be derived from all or part of a naturally occurring molecule, such as from all or part of an extracellular region derived from CD8, CD4, or CD28, or from all or part of an antibody constant region. Optionally, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge region sequence, or it may be a fully synthetic hinge region sequence.

[0184] Non-limiting examples of hinge regions that can be used according to the invention include a portion of human CD8α, a portion of the extracellular region of CD28, the Fc receptor, IgG, IgM, IgA, IgD, IgE, the Ig hinge, or functional fragments thereof. The hinge may be mutated to prevent Fc receptor binding. The hinge region may be derived from the CD8α stalk, CD28, or IgG1. In some embodiments, the hinge region is derived from the CD8α stalk. In various embodiments, the hinge region is derived from CD28. The hinge region provides flexibility and accessibility between the extracellular region and the transmembrane region.

[0185] In some implementations, the hinge may be derived from, for example, IgG1, IgG2, IgG3, IgG4, CD28, or CD8α.

[0186] In some embodiments, the hinge region may be derived from the same cell surface receptor as the intracellular signaling region. In some embodiments, the hinge region may be derived from a different molecule than the cell surface receptor derived from the intracellular signaling region.

[0187] The hinge region may contain up to 300 amino acids, 10 to 100 amino acids, or 25 to 50 amino acids.

[0188] Leader sequence

[0189] In various embodiments, the extracellular region of the chimeric receptor disclosed herein may include a leader sequence. The leader sequence may be located at the N-terminus of the extracellular region. Optionally, the leader sequence may be cleaved from the extracellular region during cellular processing and localization of the chimeric receptor to the cell membrane. Any of a variety of leader sequences known to those skilled in the art may be used as a leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include FcεR, the variable region of human immunoglobulin heavy chain, CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In some embodiments, the leader sequence is derived from human immunoglobulin heavy chain.

[0190] In some embodiments, the leader sequence may be derived from the variable region of the immunoglobulin heavy chain or the colony-stimulating factor 2 receptor alpha chain (CSF2RA).

[0191] In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the leader sequence comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the leader sequence comprises the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the leader sequence comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the leader sequence comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0192] Transmembrane region

[0193] In some implementations, the chimeric receptor disclosed herein includes a transmembrane region.

[0194] In some implementations, the transmembrane region may be derived from the same cell surface receptor as the intracellular signal transduction region.

[0195] In some implementations, the transmembrane domain may be derived from EpoR, or a portion thereof or a variant thereof.

[0196] In some embodiments, the transmembrane region derived from EpoR may comprise an amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from EpoR comprises a nucleotide sequence encoding the following amino acid sequence: SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130 or SEQ ID NO: 108, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.In some implementations, the transmembrane region derived from EpoR may be encoded by the following nucleotide sequence: SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109 or SEQ ID NO: 131, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109 or SEQ ID NO: 131. In some embodiments, the transmembrane region derived from EpoR comprises the amino acid sequence shown in SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from EpoR comprises the nucleotide sequence shown in SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109, or SEQ ID NO: 131.

[0197] In some implementations, the transmembrane region may be derived from the GHR, or a portion thereof or a variant thereof.

[0198] In some embodiments, the transmembrane region derived from GHR may comprise an amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from GHR comprises a variant encoding the following nucleotide sequence: SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142 or SEQ ID NO: 144, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.In some embodiments, the transmembrane region derived from GHR may be encoded by the following nucleotide sequences: SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, or variants thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the transmembrane region derived from EpoR comprises the amino acid sequence shown in SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from EpoR comprises the nucleotide sequence shown in SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, or SEQ ID NO: 145.

[0199] In some implementations, the transmembrane domain may be derived from IL-18Rα, IL-18Rβ, CD28, IL-2Rβ, CD8, CD4, CD3ζ, CD40, CD134 (OX-40), CD19, or CD7.

[0200] In some implementations, the transmembrane domain may be derived from IL-18Rα, or a portion thereof or a variant thereof.

[0201] In some embodiments, the transmembrane region derived from IL-18Rα may contain the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 40. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from IL-18Rα comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 40. In some embodiments, the transmembrane region derived from IL-18Rα may be encoded by the nucleotide sequence of SEQ ID NO: 39, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 39. In some embodiments, the transmembrane region derived from IL-18Rα comprises the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from IL-18Rα comprises the nucleotide sequence shown in SEQ ID NO: 39.

[0202] In some implementations, the transmembrane region may be derived from IL-18Rβ, or a portion thereof or a variant thereof.

[0203] In some embodiments, the transmembrane region derived from IL-18Rβ may contain the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 36. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from IL-18Rβ comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 36. In some embodiments, the transmembrane region derived from IL-18Rβ may be encoded by the nucleotide sequence of SEQ ID NO: 35, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35. In some embodiments, the transmembrane region derived from IL-18Rβ comprises the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from IL-18Rβ comprises the nucleotide sequence shown in SEQ ID NO: 35.

[0204] In some implementations, the transmembrane region may be derived from CD28, or a portion thereof or a variant thereof.

[0205] In some embodiments, the transmembrane region derived from CD28 may contain the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 42. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from CD28 comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 42. In some embodiments, the transmembrane region derived from CD28 may be encoded by the nucleotide sequence of SEQ ID NO: 41, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 41. In some embodiments, the transmembrane region derived from CD28 comprises the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from CD28 comprises the nucleotide sequence shown in SEQ ID NO: 41.

[0206] In some embodiments, the transmembrane region is derived from CD8α, CD28, CD8, CD4, CD3ζ, CD40, CD134 (OX-40), CD19, or CD7. In a specific embodiment, the transmembrane region is derived from CD8α. In a specific embodiment, the transmembrane region is derived from CD28. The transmembrane region may be fused within the frame or operatively connected between the extracellular region and the intracellular signaling region.

[0207] In some embodiments, the transmembrane region can be modified by the deletion or addition of amino acids to optimize intracellular orientation. In some embodiments, the transmembrane region can be modified by the deletion or addition of 1, 2, 3, 4, 5, or 6 amino acids to optimize intracellular orientation. For example, the transmembrane region described herein can be modified by the deletion of 1, 2, 3, 4, 5, or 6 amino acids at the N-terminus or C-terminus of the transmembrane region. For example, the transmembrane region described herein can be modified by the addition of 1, 2, 3, 4, 5, or 6 amino acids (e.g., alanine addition) at the N-terminus or C-terminus of the transmembrane region.

[0208] In some cases, transmembrane regions can be modified by amino acid substitution, deletion, or insertion to avoid binding to proteins naturally associated with the transmembrane region. In some embodiments, the transmembrane region includes additional amino acids to allow for flexibility and / or optimal distance between regions attached to the transmembrane region.

[0209] The transmembrane region may be derived from a natural source or from a synthetic source. In the case of a natural source, the region may be derived from any membrane-binding protein or transmembrane protein. Non-limiting examples of transmembrane regions for specific uses in this disclosure may be derived from (i.e., transmembrane regions comprising at least the following): the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD7, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (OX-40), CD137, or CD154. Optionally, the transmembrane region may be synthetic, in which case the transmembrane region will primarily contain hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan, and / or valine may be found at each end of a synthetic transmembrane region.

[0210] In some embodiments, it is desirable to utilize transmembrane regions of the ζ, η, or FcεR1γ chain containing cysteine ​​residues capable of disulfide bonding, such that the resulting chimeric protein can form disulfide-linked dimers with itself or with unmodified versions of the ζ, η, or FcεR1γ chain or related proteins. In some cases, the transmembrane region will be selectively or modified by amino acid substitution to prevent such regions from binding to transmembrane regions of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In other cases, it is desirable to employ transmembrane regions of ζ, η, or FcεR1γ and -β, MB1 (Igα.), B29 or CD3-γ, ζ, or η to maintain physical association with other members of the receptor complex.

[0211] Intracellular signal transduction region

[0212] In some embodiments, the chimeric receptor disclosed herein may comprise an intracellular region including at least one signal transduction region (also referred to herein as an intracellular signal transduction region). The at least one intracellular signal transduction region may be derived from a cell surface receptor or a signal transduction adaptor protein, or a portion thereof or a variant thereof.

[0213] In some embodiments, the intracellular region may contain more than one signal transduction region, such as two, three, four or more signal transduction regions. In some embodiments, the intracellular region may contain a first signal transduction region derived from a cell surface receptor, or a portion thereof or a variant thereof, and a second signal transduction region derived from a signal transduction adaptor protein, or a portion thereof or a variant thereof.

[0214] In some embodiments, the signal transduction region is derived from a natural homodimeric cell surface receptor or a signal transduction adaptor protein. In some embodiments, the signal transduction region is derived from a non-natural homodimeric cell surface receptor or a signal transduction adaptor protein.

[0215] In some implementations, signal transduction domains derived from cell surface receptors or signal transduction adaptors, or portions or variants thereof, can activate the Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway, the nuclear factor kappa light chain enhancer (NFκB) pathway for B cell activation, and / or additional signal transduction pathways activated by the MyD88 signal transduction complex (Myddosome).

[0216] In some implementations, additional signaling pathways activated by the MyD88 signaling complex (Myddosome) are one or more of the following: interleukin-1 receptor-associated kinase (IRAK) (e.g., IRAK1, IRAK2, IRAK4), TNF receptor (TNFR)-associated factor (TRAF) (e.g., TRAF6), TANK-binding kinase (TBK) (e.g., TBK1), mitogen-activated protein kinase (MAPK), protein kinase B or AKT (PKB / AKT) and / or phosphatidylinositol-3-kinase (PI3K) pathway (see, for example, Fitzgerald and Kagan, Cell 180, March 19, 2020, the contents of which are incorporated herein by reference in their entirety for all purposes).

[0217] In some embodiments, the signal transduction region may be derived from a cytokine receptor, or a portion thereof or a variant thereof. In some embodiments, the signal transduction region may be derived from a natural homodimeric cytokine receptor. In some embodiments, the signal transduction region may be derived from a non-natural homodimeric cytokine receptor.

[0218] In some implementations, the cytokine receptor may be erythropoietin receptor (EpoR), growth hormone receptor (GHR), prolactin receptor (PRLR), leptin receptor (LEPR), granulocyte colony-stimulating factor (G-CSFR), thrombopoietin receptor (TpoR), interleukin-23 receptor (IL-23R), interleukin-10 receptor beta subunit (IL-10R2), IL-6 beta chain (gp130), IL-2Rβ, IL-18Rα, or IL-18Rβ.

[0219] In some implementations, the signal transduction region may be derived from a non-natural homodimer, such as the interleukin-23 receptor (IL-23R) (see, for example, Engelowski et al., 2018

[14] , the contents of which are incorporated herein by reference in their entirety for all purposes) or the interleukin-10 beta subunit (IL-10R2) (see, for example, Mossner et al., 2020

[15] , the contents of which are incorporated herein by reference in their entirety for all purposes).

[0220] In some implementations, the signal transduction region may be derived from the IL-6 beta chain (gp130) (see, for example, Stuhlmann-Laeisz et al., 2006

[16] , the contents of which are incorporated herein by reference in their entirety for all purposes).

[0221] In some implementations, the signal transduction region may be derived from the native Janus kinase (JAK) / signal transducer and transcription activator (STAT) homodimer, including but not limited to: thrombopoietin receptor (TpoR), leptin receptor (LEPR), prolactin receptor (PRLR) and granulocyte colony-stimulating factor receptor (GCSFR) (see, for example, Staerk et al., 2011

[11] , the contents of which are incorporated herein by reference in their entirety for all purposes).

[0222] In some embodiments, the signal transduction region may be derived from EpoR, or a portion thereof or a variant thereof. In a further embodiment, the signal transduction region derived from EpoR does not contain a phosphatase-1 (SHP1) binding site containing a Src homology 2 domain.

[0223] In some embodiments, the signal transduction region derived from EpoR may contain the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from EpoR comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. In some embodiments, the signal transduction region derived from EpoR may be encoded by the nucleotide sequence of SEQ ID NO: 45, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 45. In some embodiments, the signal transduction region derived from EpoR may comprise the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from EpoR comprises the nucleotide sequence shown in SEQ ID NO: 45.

[0224] In some embodiments, the signal transduction region derived from EpoR may contain the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 49. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from EpoR comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 49. In some embodiments, the signal transduction region derived from EpoR may be encoded by the nucleotide sequence of SEQ ID NO: 154, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 154. In some embodiments, the signal transduction region derived from EpoR may comprise the amino acid sequence of SEQ ID NO: 49. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from EpoR comprises the nucleotide sequence shown in SEQ ID NO: 154.

[0225] In some implementations, the signal conduction region may be derived from the GHR, or a portion thereof or a variant thereof.

[0226] In some embodiments, the signal transduction region derived from GHR may contain the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 44. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from the GHR comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 44. In some embodiments, the signal transduction region derived from GHR may be encoded by the nucleotide sequence of SEQ ID NO: 43, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 43. In some embodiments, the signal transduction region derived from GHR may comprise the amino acid sequence of SEQ ID NO: 44. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from EpoR comprises the nucleotide sequence shown in SEQ ID NO: 43.

[0227] In some implementations, the signal transduction region may be derived from a receptor tyrosine kinase, or a portion thereof or a variant thereof. As an example, the receptor tyrosine kinase may be the epidermal growth factor receptor (EGFR) (see, for example, Kourouniotis et al., 2016

[12] , the contents of which are incorporated herein by reference in their entirety for all purposes).

[0228] In some embodiments, the signal transduction region may be derived from a Toll-like receptor, or a portion thereof or a variant thereof. In some embodiments, the Toll-like receptor may be: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. In some embodiments, the Toll-like receptor may be the Toll-like receptor described in Zhang et al. 2002

[13] , the contents of which are incorporated herein by reference in their entirety for all purposes.

[0229] In some embodiments, the signal transduction region may be derived from a signal transduction adaptor protein, or a portion thereof or a variant thereof. In some embodiments, the signal transduction adaptor protein may be MyD88.

[0230] In some embodiments, the signal transduction region derived from MyD88 may contain the amino acid sequence of SEQ ID NO: 38 or 220, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 38 or 220. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from MyD88 comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 38 or 220, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 38 or 220. In some embodiments, the signal transduction region derived from MyD88 may be encoded by the following: a nucleotide sequence of SEQ ID NO: 37 or 219, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 37 or 219. In some embodiments, the signal transduction region derived from MyD88 may comprise the amino acid sequence of SEQ ID NO: 38 or 220. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from MyD88 comprises the nucleotide sequence shown in SEQ ID NO: 37 or 219.

[0231] Non-limiting examples of chimeric receptors

[0232] In some embodiments, this disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise:

[0233] 1) Extracellular region, which contains a homodimeric leucine zipper motif;

[0234] 2) Transmembrane regions, derived from EpoR, or portions thereof or variants thereof; and

[0235] 3) Intracellular regions, which include signal transduction regions derived from EpoR, or a portion thereof or variant thereof, and optionally signal transduction regions derived from MyD88, or a portion thereof or variant thereof.

[0236] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif may comprise the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the leucine zipper motif is encoded by the following: the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3. In some embodiments, the leucine zipper motif comprises the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0237] In some embodiments, the transmembrane region derived from EpoR may comprise an amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from EpoR comprises a nucleotide sequence encoding the following amino acid sequence: SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130 or SEQ ID NO: 108, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.In some implementations, the transmembrane region derived from EpoR may be encoded by the following nucleotide sequence: SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109 or SEQ ID NO: 131, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109 or SEQ ID NO: 131. In some embodiments, the transmembrane region derived from EpoR comprises the amino acid sequence shown in SEQ ID NO: 48, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 106, SEQ ID NO: 130, SEQ ID NO: 108, or SEQ ID NO: 132. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from EpoR comprises the nucleotide sequence shown in SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 127, SEQ ID NO: 107, SEQ ID NO: 129, SEQ ID NO: 109, or SEQ ID NO: 131.

[0238] In some implementations, the signal transduction region derived from EpoR does not contain a phosphatase-1 (SHP1) binding site containing the Src homology region 2 domain.

[0239] In some embodiments, the signal transduction region derived from EpoR may contain the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from EpoR comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. In some embodiments, the signal transduction region derived from EpoR may be encoded by the nucleotide sequence of SEQ ID NO: 45, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 45. In some embodiments, the signal transduction region derived from EpoR may comprise the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from EpoR comprises the nucleotide sequence shown in SEQ ID NO: 45.

[0240] In some embodiments, each polypeptide monomer of the chimeric receptor may comprise an amino acid sequence of SEQ ID NO: 112, 114, 116 or 118, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116 or SEQ ID NO: 118. In some embodiments, the nucleotide sequence encoding each polypeptide monomer comprises the amino acid sequence encoding the following: SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116 or SEQ ID NO: 118, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. In some embodiments, each polypeptide monomer may be encoded by the following: a nucleotide sequence of SEQ ID NO: 111, 113, 115, or 117, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 111, 113, 115, or 117. In some embodiments, each polypeptide monomer of the chimeric receptor may comprise the amino acid sequence of SEQ ID NO: 112, 114, 116, or 118. In some embodiments, each polypeptide monomer may be encoded by the nucleotide sequence of SEQ ID NO: 111, 113, 115, or 117.

[0241] In some embodiments, this disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise:

[0242] 1) Extracellular region, which contains a homodimeric leucine zipper motif;

[0243] 2) Transmembrane regions, which are derived from GHR, or a portion thereof or variant thereof, and

[0244] 3) Intracellular regions, which include signal transduction regions derived from GHR, or a portion thereof or variant thereof, and optionally signal transduction regions derived from MyD88, or a portion thereof or variant thereof.

[0245] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif may comprise the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the leucine zipper motif is encoded by the following: the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3. In some embodiments, the leucine zipper motif comprises the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0246] In some embodiments, the transmembrane region derived from GHR may comprise an amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from GHR comprises a variant encoding the following nucleotide sequence: SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142 or SEQ ID NO: 144, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.In some implementations, the transmembrane region derived from GHR may be encoded by the following nucleotide sequences: SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, or variants thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the transmembrane region derived from GHR comprises the amino acid sequence shown in SEQ ID NO: 52, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, or SEQ ID NO: 144. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from GHR comprises the nucleotide sequence shown in SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, or SEQ ID NO: 145.

[0247] In some embodiments, the signal transduction region derived from GHR may contain the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 44. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from the GHR comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 44. In some embodiments, the signal transduction region derived from GHR may be encoded by the nucleotide sequence of SEQ ID NO: 43, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 43. In some embodiments, the signal transduction region derived from GHR may comprise the amino acid sequence of SEQ ID NO: 44. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from GHR comprises the nucleotide sequence shown in SEQ ID NO: 43.

[0248] In some embodiments, the signal transduction region derived from MyD88 may contain the amino acid sequence of SEQ ID NO: 220, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 220. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from MyD88 comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 220, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 220. In some embodiments, the signal transduction region derived from MyD88 may be encoded by the nucleotide sequence of SEQ ID NO: 219, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 219. In some embodiments, the signal transduction region derived from MyD88 may comprise the amino acid sequence of SEQ ID NO: 220. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from MyD88 comprises the nucleotide sequence shown in SEQ ID NO: 219.

[0249] In some embodiments, each polypeptide monomer of the chimeric receptor may contain the amino acid sequence of SEQ ID NO: 120, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 120. In some embodiments, the nucleotide sequence encoding each polypeptide monomer comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 120, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 120. In some embodiments, each polypeptide monomer may be encoded by the nucleotide sequence of SEQ ID NO: 119, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 119. In some embodiments, each polypeptide monomer of the chimeric receptor may comprise the amino acid sequence of SEQ ID NO: 120. In some embodiments, each polypeptide monomer may be encoded by the nucleotide sequence of SEQ ID NO: 119.

[0250] In some embodiments, each polypeptide monomer of the chimeric receptor may contain the amino acid sequence of SEQ ID NO: 221, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 221. In some embodiments, the nucleotide sequence encoding each polypeptide monomer comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 221, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 221. In some embodiments, each polypeptide monomer may be encoded by: the nucleotide sequence of SEQ ID NO: 222, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 222. In some embodiments, each polypeptide monomer of the chimeric receptor may comprise the amino acid sequence of SEQ ID NO: 221. In some embodiments, each polypeptide monomer may be encoded by the nucleotide sequence of SEQ ID NO: 222.

[0251] In some embodiments, each polypeptide monomer of the chimeric receptor may comprise amino acids 28-611 of the amino acid sequence of SEQ ID NO: 221, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids 28-611 of SEQ ID NO: 221. In some embodiments, the nucleotide sequence encoding each polypeptide monomer comprises a nucleotide sequence encoding the following: amino acids 28-611 of the amino acid sequence of SEQ ID NO: 221, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids 28-611 of SEQ ID NO: 221. In some embodiments, each polypeptide monomer may be encoded by nucleotides 82-1833 of the nucleotide sequence of SEQ ID NO: 222, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 82-1833 of the SEQ ID NO: 222. In some embodiments, each polypeptide monomer of the chimeric receptor may comprise amino acids 28-611 of the amino acid sequence of SEQ ID NO: 221. In some embodiments, each polypeptide monomer may be encoded by nucleotides 82-1833 of the nucleotide sequence of SEQ ID NO: 222.

[0252] In some embodiments, this disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise:

[0253] 1) Extracellular region, which contains a homodimeric leucine zipper motif;

[0254] 2) Transmembrane regions derived from IL-18Rα, IL-18Rβ, or CD28, or portions thereof or variants thereof; and

[0255] 3) Intracellular region, which contains signal transduction regions derived from MyD88, or a portion thereof or variant thereof.

[0256] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif may comprise the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9. In some embodiments, the leucine zipper motif is encoded by the following: the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3. In some embodiments, the leucine zipper motif comprises the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0257] In some embodiments, the transmembrane region may contain an amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 42, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 42. In some embodiments, the nucleotide sequence encoding the transmembrane region comprises a nucleotide sequence encoding the following: an amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 42, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 42. In some embodiments, the transmembrane region may be encoded by the following: a nucleotide sequence of SEQ ID NO: 39, SEQ ID NO: 35, or SEQ ID NO: 41, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 39, SEQ ID NO: 35, or SEQ ID NO: 41. In some embodiments, the nucleotide sequence encoding the transmembrane region comprises the nucleotide sequence of SEQ ID NO: 39, SEQ ID NO: 35, or SEQ ID NO: 41.

[0258] In some embodiments, the signal transduction region derived from MyD88 may contain the amino acid sequence of SEQ ID NO: 38 or 220, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 38 or 220. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from MyD88 comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 38 or 220. In some embodiments, the signal transduction region derived from MyD88 may be encoded by the following: a nucleotide sequence of SEQ ID NO: 37 or 219, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 38 or 220. In some embodiments, the nucleotide sequence encoding the signal transduction region derived from MyD88 comprises the nucleotide sequence shown in SEQ ID NO: 37 or 219.

[0259] In some embodiments, each polypeptide monomer of the chimeric receptor may comprise an amino acid sequence of SEQ ID NO: 122, 124 or 126, or a sequence thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 122, SEQ ID NO: 124 or SEQ ID NO: 126. In some embodiments, the nucleotide sequence encoding each polypeptide monomer comprises a nucleotide sequence encoding the following: an amino acid sequence of SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID NO: 126, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID NO: 126. In some embodiments, each polypeptide monomer may be encoded by the following: a nucleotide sequence of SEQ ID NO: 121, 123, or 125, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 125. In some embodiments, each polypeptide monomer of the chimeric receptor may comprise the amino acid sequence of SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID NO: 126. In some embodiments, each polypeptide monomer may be encoded by a nucleotide sequence of SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 125.

[0260] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 112, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 112. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 111, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 111.

[0261] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 114, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 114. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 113, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 113.

[0262] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 116, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 116. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 115, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 115.

[0263] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 118, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 118. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 117, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 117.

[0264] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 120, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 120. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 119, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 119.

[0265] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 122, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 122. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 121, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 121.

[0266] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 124, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 124. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 123, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 123.

[0267] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 126, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 126. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 125, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 125.

[0268] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 146, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 146. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 147, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 147.

[0269] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 148, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 148. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 149, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 149.

[0270] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise the amino acid sequence shown in SEQ ID NO: 221, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 221. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleic acid sequence SEQ ID NO: 222, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 222.

[0271] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer may comprise amino acids 28-611 of the amino acid sequence shown in SEQ ID NO: 221, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids 28-611 of SEQ ID NO: 221. In some embodiments, each polypeptide monomer may comprise an amino acid sequence encoded by: nucleotide 82-1833 of nucleic acid sequence SEQ ID NO: 222, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotide 82-1833 of SEQ ID NO: 222.

[0272] In some embodiments, the chimeric receptor may comprise a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer comprises: 1) an extracellular region containing at least one homodimerization motif; 2) a transmembrane region; 3) an intracellular region containing at least one signal transduction region derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof; and optionally 4) at least one additional gene.

[0273] Table 2 shows non-limiting exemplary constructs that may include: 1) an extracellular region containing at least one homodimerization motif; 2) a transmembrane region; 3) an intracellular region containing at least one signal transduction region derived from a cell surface receptor or signal transduction adaptor protein, or a portion thereof or a variant thereof; and optionally 4) at least one additional gene.

[0274] Table 2: Exemplary combinations of polypeptide components

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] Table 3 shows the amino acid sequences of the domains of intracellular receptor candidates GHR and hEpoR. GHR and hEpoR are non-limiting examples of sources for intracellular signal transduction regions.

[0288] Table 3: hEpoR and GHR regions and constructs

[0289]

[0290] Polynucleotides

[0291] In one aspect, this disclosure provides polynucleotides capable of encoding any chimeric receptor disclosed herein. In some embodiments, the polynucleotide may be a DNA molecule. In some embodiments, the polynucleotide may be an RNA molecule.

[0292] In some implementations, the polynucleotide may comprise a nucleotide sequence that encodes any of the homodimeric polypeptides disclosed herein.

[0293] In some embodiments, the polynucleotide comprises a nucleotide sequence of any one of SEQ ID NO: 111, 113, 115, 117, 119, 121, 123, 125, 147, 149 or 222 (or nucleotides 82-1833 of SEQ ID NO: 222), or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 111, 113, 115, 117, 119, 121, 123, 125, 147, 149 or 222 (or nucleotides 82-1833 of SEQ ID NO: 222).

[0294] In some embodiments, the nucleotide sequence of the polypeptide encoding the chimeric receptor may be operatively linked via a sequence encoding a self-cleaving peptide and / or an internal ribosome entry site (IRES) to one or more additional nucleotide sequences encoding one or more additional polypeptide sequences.

[0295] In some embodiments, the self-cleaving peptide can be a 2A peptide. Non-limiting examples of self-cleaving peptide sequences include the 2A sequence of the *Thoseaasigna* virus (T2A; AEGRGGSLLTCGDVEENPGP (SEQ ID NO: 155), EGRGSLLTCGDVEENPGP (SEQ ID NO: 156), or GSGEGRGGSLLTCGDVEENPGP (SEQ ID NO: 157)); the 2A sequence of foot-and-mouth disease virus (FMDV) (F2A; GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 158)); the 2A sequence of the sponge (*Amphimedon queenslandica*) (LLCFLLLLLSGDVELNPGP (SEQ ID NO: 159); or HHFMFLLLLLAGDIELNPGP (SEQ ID NO: 160)); and the acorn worm. The 2A sequences include: Saccoglossus kowalevskii (WFLVLLSFILSGDIEVNPGP (SEQ ID NO: 161)); amphioxus (Florida amphioxus) 2A sequence (KNCAMYMLLLSGDVETNPGP (SEQ ID NO: 162); or MVISQLMLKLAGDVEENPGP (SEQ ID NO: 163)); porcine teschovirus-1 2A sequence (P2A; GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 11)); and equine rhinitis A virus 2A sequence (E2A; GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 164)). In some embodiments, the isolated sequences are naturally occurring or synthetic sequences. In some embodiments, the isolated sequence includes the 2A common sequence DXEX-NPGP (SEQ ID NO: 165), where X is any amino acid residue.

[0296] In some implementations, the 2A peptide can be a P2A peptide.

[0297] In some embodiments, the P2A peptide comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the P2A peptide comprises a nucleotide sequence encoding the following: the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the P2A peptide comprises the nucleotide sequence shown in SEQ ID NO: 5, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5. In some embodiments, the P2A peptide comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the P2A peptide comprises the nucleotide sequence shown in SEQ ID NO: 5.

[0298] In some implementations, nucleotide sequences (one or more) can be expressed in an inducible manner, for example, by inducible promoters, inducible expression systems, artificial signal transduction circuits, and / or drug-induced splicing.

[0299] In some embodiments, the nucleotide sequence(s) can be expressed in an inducible manner, such as by: i) an inducible promoter, such as, but not limited to, promoters that can be activated by T cells (e.g., NFAT, Nur66, IFNg) or by hypoxia; ii) an inducible expression system, such as, but not limited to, a doxycycline-inducible expression system or a tamoxifen-inducible expression system; iii) an artificial signal transduction circuit, including but not limited to SynNotch; and / or iv) drug-induced splicing. As a non-limiting example, drug-induced splicing methods and / or compositions useful in the practice of this disclosure may be based on those described, for example, in Monteys et al., 2021

[44] , the contents of which are incorporated herein by reference in their entirety for all purposes.

[0300] In some embodiments, the nucleotide sequence(s) may encode portions that allow the stability of the chimeric receptor to be modulated by small molecules, including but not limited to “SWIFF” technology or immunomodulatory drug (IMiD)-inducible degrons, as described, for example, in Juillerat et al., 2019

[17] , Carbonneau et al., 2021

[18] and Jan et al., 2021

[19] , the contents of which are incorporated herein by reference in their entirety for all purposes.

[0301] In some embodiments, the nucleotide sequence of the polypeptide encoding the chimeric receptor may be at least operatively linked to a regulatory element. The regulatory element may be capable of mediating the expression of the chimeric receptor polypeptide. Regulatory elements include, but are not limited to, promoters, enhancers, initiation sites, polyA tails, IRES elements, responsive elements, and termination signals. In some embodiments, the regulatory element regulates chimeric receptor expression. In some embodiments, the regulatory element increases the expression of the chimeric receptor construct. In some embodiments, the regulatory element increases the expression of the chimeric receptor construct upon activation of the host cell. In some embodiments, the regulatory element decreases the expression of the chimeric receptor construct. In some embodiments, the regulatory element decreases the expression of the chimeric receptor construct upon activation of the host cell.

[0302] In some implementations, the promoter is an inducible promoter. Non-limiting examples of inducible promoters are lac, sp6, T7, and promoters derived from Hsp70- and Hsp90-.

[0303] In some implementations, the inducible promoter is the tetracycline (Tc)-inducible promoter.

[0304] In some implementations, the promoter can be a T cell-specific promoter or an NK cell-specific promoter.

[0305] Additional genes

[0306] In addition to the chimeric receptor construct, the polynucleotide may further comprise at least one additional gene encoding an additional peptide. Examples of additional genes may include transduced host cell selection markers, in vivo tracking markers, cytokines, suicide genes, or some other functional gene. In some embodiments, the functional additional gene may induce the expression of another molecule. In some embodiments, the functional additional gene may increase the safety of the chimeric receptor. For example, the chimeric receptor construct may comprise an additional gene that is a truncated CD19 (tCD19). tCD19 can be used as a tag. Expression of tCD19 may also help determine transduction efficiency. In some embodiments, the functional additional gene may allow for the modulation of the stability of the chimeric receptor.

[0307] Non-limiting examples of additional gene classes that can be used to enhance the effector function of modified host cells include (a) secretible cytokines (e.g., but not limited to GM-CSF, IL-7, IL-12, IL-15, IL-18), (b) membrane-bound cytokines (e.g., but not limited to IL-15), (c) other chimeric receptors (e.g., but not limited to IL-2 / IL-7, IL-4 / IL-7), (d) constitutively active cytokine receptors (e.g., but not limited to C7R), and (e) dominant-negative receptors (DNRs; e.g., but not limited to TGFRII). (dNR), (f) ligands of co-stimulatory molecules (e.g., but not limited to CD80, 4-1BBL), (g) nuclear factors (NFAT) that activate T cells (e.g., NFAC1, NFAC2, NFAC3, NFAC4, and NFAC5), (h) antibodies, including fragments thereof and bispecific antibodies (e.g., but not limited to bispecific T cell connectives (BiTE)), (i) chimeric antigen receptors (CAR), or (j) safety switches or suicide genes (e.g., CD20, truncated EGFR or HER2, inducible caspase 9 molecules).

[0308] In some embodiments, the chimeric receptor construct may include an additional gene encoding GM-CSF. Expression of exogenous GM-CSF may further enhance the function of host cells expressing the chimeric receptor of this disclosure.

[0309] In some embodiments, the chimeric receptor construct may include at least one additional gene that encodes, for example, but not limited to, one or more cell markers, epitope tags, cytokines, safety switches, dimerization moieties, or degradation moieties.

[0310] In some implementations, the additional gene is an epitope tag. As a non-limiting example, the epitope tag is FLAG. As another non-limiting example, the epitope tag is Myc.

[0311] In some implementations, the additional gene is a cell marker. As a non-limiting example, the cell marker is mClover3. As another non-limiting example, the cell marker is mRuby.

[0312] In some implementations, additional genes may encode portions that allow the stability of the chimeric receptor to be modulated by small molecules, including but not limited to “SWIFF” technology or immunomodulatory drug (IMiD)-induced degraders, as described in, for example, Juillerat et al., 2019

[17] , Carbonneau et al., 2021

[18] and Jan et al., 2021

[19] , the contents of which are incorporated herein by reference in their entirety for all purposes.

[0313] In some embodiments, the functional additional gene may be a suicide gene. A suicide gene is a recombinant gene that causes host cells expressing the gene to undergo programmed cell death or antibody-mediated clearance at a desired time. Suicide genes can be used to increase the safety of chimeric receptors. In another embodiment, the additional gene may be an inducible suicide gene. Non-limiting examples of suicide genes include i) molecules expressed on the cell surface and that can be targeted by clinical-grade monoclonal antibodies, including CD20, EGFR or fragments thereof, HER2 or fragments thereof, and ii) inducible suicide genes (e.g., but not limited to inducible cystin 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; U.S. Publication No. 2011 / 0286980, each of which is incorporated herein by reference in its entirety for all purposes)).

[0314] In some respects, the chimeric receptors of this disclosure can be modulated by a safety switch. As used herein, the term "safety switch" refers to any mechanism that can remove or inhibit the action of the chimeric receptor from a system (e.g., a culture or a subject). Safety switches can be used to increase the safety of chimeric receptors.

[0315] The function of a safety switch can be inducible. Non-limiting examples of safety switches include (a) molecules expressed on the cell surface and targeted by clinical-grade monoclonal antibodies, including CD20, EGFR or fragments thereof, HER2 or fragments thereof, and (b) inducible suicide genes (e.g., but not limited to herpes simplex virus thymidine kinase (HSV-TK) and inducible cystin 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; US Publication No. 2011 / 0286980, each of which is incorporated herein by reference in its entirety for all purposes).

[0316] In some embodiments, the safety switch may be a CD20 peptide. Expression of human CD20 on the cell surface provides an attractive strategy for a safety switch. The inventors and others have demonstrated that cells expressing CD20 can be rapidly eliminated by the FDA-approved monoclonal antibody rituximab via complement-mediated and antibody-dependent cell-mediated cytotoxicity (see, for example, Griffioen, M. et al. Haematologica 94, 1316-1320 (2009), which is incorporated herein by reference in its entirety for all purposes). Rituximab is an FDA-approved anti-CD20 monoclonal antibody for chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), etc. (Storz, U. MAbs 6, 820-837 (2014), which is incorporated herein by reference in its entirety for all purposes). The CD20 safety switch is non-immunogenic and can be used as a reporter / selection marker in addition to being a safety switch (Bonifant, CL, et al. Mol Ther 24, 1615-1626 (2016); van Loenen, MM, et al. Gene Ther 20, 861-867 (2013); each is incorporated herein by reference in its entirety for all purposes).

[0317] In some embodiments, the chimeric receptor can be expressed in an inducible manner, such as by: i) an inducible promoter, such as, but not limited to, promoters that can be activated by T cells (e.g., NFAT, Nur66, IFNγ) or by hypoxia; ii) an inducible expression system, such as, but not limited to, a doxycycline-inducible expression system or a tamoxifen-inducible expression system; iii) an artificial signal transduction circuit, including but not limited to SynNotch; and / or iv) drug-induced splicing. As a non-limiting example, drug-induced splicing methods and / or compositions useful in the practice of this disclosure may be based on those described, for example, in Monteys et al., 2021

[20] , the contents of which are incorporated herein by reference in their entirety for all purposes.

[0318] In some embodiments, the chimeric receptor may contain at least one additional gene (i.e., a second gene). In some embodiments, the chimeric receptor may contain one second gene. In other embodiments, the chimeric receptor may contain two additional genes (i.e., a third gene). In yet another embodiment, the chimeric receptor may contain three additional genes (i.e., a fourth gene). In some embodiments, the additional genes may be separable from each other and from the chimeric receptor construct. For example, they may be separable via the 2A sequence and / or the internal ribosome entry site (IRES) as described above. In some instances, the chimeric receptor may be located at any position on the polynucleotide chain.

[0319] Recombinant vector

[0320] This disclosure provides recombinant vectors that may contain a polynucleotide encoding a chimeric receptor. Such recombinant vectors may contain a polynucleotide encoding a protein disclosed above. In some embodiments, the polynucleotide may be operatively linked to at least one regulatory element for the expression of the chimeric receptor.

[0321] In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be, but is not limited to, a retroviral vector, a lentiviral vector, adenovirus vector, adeno-associated virus vector, alphavirus vector, herpesvirus vector, baculovirus vector, or vaccinia virus vector.

[0322] In some implementations, the viral vector may be a retroviral vector.

[0323] In some embodiments, the vector may be a non-viral vector. Non-viral vectors suitable for use in this invention include, but are not limited to, microcircular plasmids, transposon systems (e.g., Sleeping Beauty transposon, piggyBac transposon), or single-stranded or double-stranded DNA molecules used as templates for homology-directed repair (HDR) based gene editing.

[0324] In some embodiments, the vector comprises a nucleotide sequence of any one of SEQ ID NO: 111, 113, 115, 117, 119, 121, 123, 125, 147, 149, or 222, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 111, 113, 115, 117, 119, 121, 123, 125, 147, 149, or 222.

[0325] Isolated host cells

[0326] On the other hand, this article provides an isolated host cell that may contain any of the various polynucleotides described herein or any of the recombinant vectors described herein.

[0327] In other respects, this article provides an isolated host cell that may contain a chimeric receptor encoded by the polynucleotide described herein.

[0328] In some implementations, the host cell may be an immune cell. In various implementations, the host cell is a T cell.

[0329] T cells may include, but are not limited to, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be helper T (Th) cells, such as type 1 helper T (Th1) or type 2 helper T (Th2) cells. T cells may be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+, CD8+ T cells, or any other subset of T cells. Other illustrative T cell populations suitable for a particular implementation include naive T cells, memory T cells, NKT cells, and iNKT cells.

[0330] In some implementations, the host cell may be a T cell, a natural killer (NK) cell, a mesenchymal stem cell (MSC), or a macrophage. In some implementations, the host cell may be a T cell.

[0331] In some implementations, the T cells may be selected from: αβ T cell receptor (TCR) T cells, γδ T cells, CD8+ T cells, CD4+ T cells, cytotoxic T cells, invariant natural killer T (iNKT) cells, memory T cells, and memory stem T cells (T cells). SCM ), naive T cells, effector T cells, helper T cells, or regulatory T cells (Treg).

[0332] In various implementation schemes, the host cell can be a natural killer (NK) cell. NK cells are differentiated lymphocytes with CD3-CD16+, CD3-CD56+, CD16+CD56+, and / or CD57+ TCR- phenotypes.

[0333] In some implementations, the host cell may be NK cells and / or cell lines derived from peripheral blood, umbilical cord blood, IPC, induced pluripotent stem (iPS) cells (iPSC) (e.g., NK-92 cells).

[0334] In some embodiments, the host cell may be an immune cell. In some embodiments, the immune cell may be derived from induced pluripotent stem (iPS) cells (iPSCs). Non-limiting examples of iPSCs that may be used according to this disclosure may be any such cells as described, for example, in Zhu et al., 2019

[21] and Iriguchi et al., 2021

[22] , the contents of which are incorporated herein by reference in their entirety for all purposes.

[0335] In various embodiments, other host immune cells may be selected, such as, but not limited to, macrophages. In various embodiments, the host immune cells may be dendritic cells, Langerhans cells, or B cells. In various embodiments, the host immune cells may be professional antigen-presenting cells (APCs). In various embodiments, the host immune cells may be non-professional antigen-presenting cells (APCs).

[0336] In some embodiments, the host cell may further express one or more antigen recognition molecules. In some embodiments, one or more antigen recognition molecules may be chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TRuCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell adaptors, natural or transgenic T-cell receptors, and antibodies, or combinations thereof.

[0337] In some implementations, host cells may be further genetically modified using gene-editing technologies (e.g., CRISPR-Cas9 or transcription activator-like effector nucleases (TALENs)) to enhance their function by a) expressing one or more additional genes (e.g., transcription factors (c-Jun)) or deleting one or more repressive genes (e.g., REGNASE-1, DNMT3A). Suitable methods for genetically modifying immune cells to knock out repressive genes (such as REGNASE-1 and DNMT3A) include those described, for example, in WO2020 / 219682 and WO2020 / 210365, which are incorporated herein by reference in their entirety.

[0338] In various implementation schemes, the host cells have been activated and / or expanded in vitro.

[0339] In various implementations, the host cell can be an allogeneic cell. In various implementations, the host cell can be an autologous cell.

[0340] In some embodiments, host cells may be isolated from a subject suffering from cancer. In some embodiments, host cells may be isolated from a subject with a tumor. In various embodiments, the cancer may be a solid tumor, a brain tumor, or leukemia. In some embodiments, the tumor may be present in, but is not limited to, breast tissue, prostate tissue, bladder tissue, oral and / or dental tissue, head and / or neck tissue, stomach tissue, liver tissue, colorectal tissue, lung tissue, brain tissue, ovary, cervix, esophagus, skin, lymph nodes, and / or bone. In some embodiments, the tumor may be cancer. In some implementations, cancer can be, for example, but not limited to, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma and other tumors of the Ewing sarcoma family, neuroblastoma, ganglioneurotic neuroblastoma, connective tissue proliferative small round cell tumor, malignant peripheral nerve sheath tumor, synovial sarcoma, undifferentiated sarcoma, adrenocortical carcinoma, hepatoblastoma, Wilms' tumor, rhabdomyosarcoma, high-grade glioma (glioblastoma multiforme), medulloblastoma, astrocytoma, glioma, ependymoma. Tumors, atypical teratoid rhabdoid tumors, meningiomas, craniopharyngiomas, primitive neuroectodermal tumors, diffuse endophytic pontine gliomas and other brain tumors, acute myeloid leukemia, multiple myeloma, lung cancer, mesothelioma, breast cancer, bladder cancer, gastric cancer, prostate cancer, colorectal cancer, endometrial cancer, cervical cancer, renal cancer, esophageal cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma and other liver cancers, head and neck cancers, leiomyosarcoma, and melanoma.

[0341] In some implementations, the host cells may be isolated from a subject with a tumor.

[0342] In some implementations, the host cells may be derived from blood, bone marrow, tissue, or tumor samples.

[0343] In some aspects, this disclosure provides a method for generating isolated host cells as described herein. The method includes genetically modifying the host cells with the polynucleotides or recombinant vectors described herein. In some embodiments, the method may further include genetically modifying the host cells to express one or more antigen recognition molecules. The one or more antigen recognition molecules may be selected from chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TRuCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell connectors, natural or transgenic T-cell receptors, and antibodies, or combinations thereof.

[0344] In some embodiments, the genetic modification step may be performed via viral gene delivery. In some embodiments, the genetic modification step may be performed via non-viral gene delivery. In some embodiments, the genetic modification step may be performed in vitro. In some embodiments, the method may further include in vitro activation and / or amplification of host cells before, after, and / or during the genetic modification step. In some embodiments, the method may further include in vitro activation and / or amplification of host cells before, after, and / or during the genetic modification step.

[0345] In addition to genetically modifying immune cells in vitro, polynucleotide or recombinant vectors encoding the chimeric receptors described herein can also be prepared in vitro and injected directly into patients using non-viral or viral delivery methods (i.e., in vivo gene delivery) (see, for example, Rurik et al., Science. 2022 January 07; 375(6576): 91-96, which is incorporated herein by reference in its entirety).

[0346] Separation / Enrichment

[0347] The host cell can be autologous / autogenetic (“self”) or non-self (“non-self”, such as allogeneic, syngeneic, or xenogeneic). In some embodiments, the host cell is obtained from a mammalian subject. In other embodiments, the host cell is obtained from a primate subject. In some embodiments, the host cell is obtained from a human subject.

[0348] Lymphocytes can be obtained from sources such as, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Lymphocytes can also be generated through differentiation of stem cells. In some embodiments, lymphocytes can be generated using techniques commonly known to those skilled in the art (such as sedimentation, e.g., FIL). TM (Separation) is obtained from blood collected from the subject.

[0349] In some embodiments, cells from a subject's circulating blood are obtained via apheresis. Apheresis devices typically contain lymphocytes (including T cells), monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected via apheresis may be washed to remove plasma fractions and placed in a suitable buffer or culture medium for subsequent processing. Cells may be washed with PBS or with another suitable solution lacking calcium, magnesium, and most (if not all) other divalent cations. The washing step may be performed by methods known to those skilled in the art, such as, but not limited to, using a semi-automatic flow-through centrifuge (e.g., the Cobe 2991 cell processor or the Baxter CytoMate). After washing, cells may be resuspended in various biocompatible buffers, cell culture media, or other saline solutions with or without buffers.

[0350] In some implementations, host cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes. As an example, cells can be separated by centrifugation via PERCOLL... TM Gradient sorting. In some implementations, after PBMC isolation, cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive T cell subsets, memory T cell subsets, and effector T cell subsets before or after activation, expansion, and / or genetic modification.

[0351] In some embodiments, T lymphocytes may be enriched. For example, specific subsets of T lymphocytes expressing one or more of the following markers, such as, but not limited to, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD27, CD28, CD34, CD36, CD45RA, CD45RO, CD56, CD62, CD62L, CD122, CD123, CD127, CD235a, CCR7, or HLA-DR, or combinations thereof, may be used. In some embodiments, the T lymphocytes used in the compositions of this disclosure do not express or substantially do not express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0352] In some implementations, NK cells may be enriched. For example, specific subsets of T lymphocytes expressing one or more of the following markers, such as but not limited to: CD2, CD16, CD56, CD57, CD94, or CD122, or combinations thereof, may be used with positive or negative selection techniques.

[0353] Stimulation / Activation

[0354] To achieve a sufficient therapeutic dose of the host cell composition, the host cells typically undergo one or more rounds of stimulation / activation. In some embodiments, a method for generating host cells for administration to a subject includes stimulating the host cells to activate them in the presence of one or more stimulating signals or substances (e.g., compounds, small molecules such as organic small molecules, nucleic acids, peptides, fragments thereof, isotypes, variants, analogs, or derivatives). In some embodiments, a method for generating host cells for administration to a subject includes stimulating the host cells to activate and proliferate them in the presence of one or more stimulating signals or agents.

[0355] Host cells (e.g., T lymphocytes and NK cells) can be activated by inducing changes in their biological state, leading to the expression of activation markers, production of cytokines, proliferation, and / or cytotoxicity against target cells. All of these changes can be generated by primary stimulation signals. Co-stimulatory signals amplify the amplitude of the primary signal after initial stimulation and inhibit cell death, resulting in a more persistent state of activation and thus greater cytotoxicity.

[0356] T cells can typically be activated using methods described in, for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0357] In some implementations, T-cell-based host cells can be activated by binding to substances that activate CD3ζ.

[0358] In other embodiments, CD2 binding agents can be used to deliver primary stimulatory signals to T cells. For example, CD2 agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, such as combinations of Tl 1.3 antibodies with Tl 1.1 or Tl 1.2 antibodies (Meuer, SC et al. (1984) Cell 36:897-906) and combinations of 9.6 antibodies (which recognize the same epitope as Tl 1.1) with 9-1 antibodies (Yang, SY et al. (1986) J. Immunol. 137:1097-1100). Other antibodies that bind to the same epitope as any of the above-mentioned antibodies may also be used.

[0359] In some embodiments, host cells are activated by administration of phorbolmyristate acetate (PMA) and ionomycine. In some embodiments, host cells are activated by administration of a suitable antigen that induces activation followed by amplification. In some embodiments, PMA, ionomycine, and / or a suitable antigen are administered together with CD3 to induce activation and / or amplification.

[0360] Typically, the activators used in this disclosure include, but are not limited to, antibodies, fragments thereof, and protein-binding molecules with antibody-like functions. Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single-chain Fv fragments (scFv), bivalent antibody fragments such as (Fab) 2'-fragments, double-chain antibodies, triple-chain antibodies (triabodies) (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94), and other domain antibodies (Holt, LJ, et al., Trends Biotechnol. (2003), 21, 11, 484-490). Bivalent antibody fragments can be (Fab) 2'-fragments or bivalent single-chain Fv fragments, while monovalent antibody fragments can be selected from the group consisting of Fab fragments, Fv fragments, and single-chain Fv fragments (scFv).

[0361] In some embodiments, one or more binding sites of the CD3ζ agent may be bivalent protein artificial binding molecules, such as dimer lipid carrier mutant proteins (i.e., duocalin). In some embodiments, the receptor binding agent may have a single second binding site (i.e., monovalent). Examples of monovalent agents include, but are not limited to, monovalent antibody fragments, protein-binding molecules with antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv), including bivalent single-chain Fv fragments.

[0362] Substances that specifically bind to CD3 include, but are not limited to, anti-CD3 antibodies, bivalent antibody fragments of anti-CD3 antibodies, monovalent antibody fragments of anti-CD3 antibodies, and protein CD3-binding molecules with antibody-like binding properties. Protein CD3-binding molecules with antibody-like binding properties can be aptamers, mutant proteins of peptides based on the lipid transporter family, glubody, ankyrin-based scaffold proteins, crystal scaffold-based proteins, adnectin, and avimer. They can also be coupled to beads.

[0363] In some embodiments, the activator (e.g., a CD3 binding agent) may be present at a concentration of about 0.1 to about 10 μg / ml. In some embodiments, the activator (e.g., a CD3 binding agent) may be present at concentrations of about 0.2 μg / ml to about 9 μg / ml, about 0.3 μg / ml to about 8 μg / ml, about 0.4 μg / ml to about 7 μg / ml, about 0.5 μg / ml to about 6 μg / ml, about 0.6 μg / ml to about 5 μg / ml, about 0.7 μg / ml to about 4 μg / ml, about 0.8 μg / ml to about 3 μg / ml, or about 0.9 μg / ml to about 2 μg / ml. In some embodiments, the activator (e.g., a CD3 binding agent) is applied at concentrations of about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, or about 10 μg / ml. In some embodiments, the CD3 binding agent may be present at a concentration of 1 μg / ml.

[0364] NK cells can typically be activated using methods described in, for example, U.S. Patents 7,803,376, 6,949,520, 6,693,086, 8,834,900, 9,404,083, 9,464,274, 7,435,596, 8,026,097, and 8,877,182; U.S. Patent Applications US2004 / 0058445, US2007 / 0160578, US2013 / 0011376, US2015 / 0118207, and US2015 / 0037887; and PCT Patent Application WO2016 / 122147, each of which is incorporated herein by reference in its entirety for all purposes.

[0365] In some implementations, NK-based host cells can be activated by, for example but not limited to, inhibiting inhibitory receptors on NK cells (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E, or LILRB5 receptors).

[0366] In some implementations, NK-based host cells can be activated by, for example but not limited to, feeder cells (e.g., naive K562 cells or K562 cells genetically modified to express 4-1BBL and cytokines such as IL-15 or IL-21).

[0367] In other embodiments, interferons or macrophage-derived cytokines can be used to activate NK cells. For example, without limitation, such interferons include, but are not limited to, interferon alpha and interferon gamma, and such cytokines include, but are not limited to, IL-15, IL-2, and IL-21.

[0368] In some embodiments, the NK activator may be present at a concentration of about 0.1 to about 10 μg / ml. In some embodiments, the NK activator may be present at concentrations of about 0.2 μg / ml to about 9 μg / ml, about 0.3 μg / ml to about 8 μg / ml, about 0.4 μg / ml to about 7 μg / ml, about 0.5 μg / ml to about 6 μg / ml, about 0.6 μg / ml to about 5 μg / ml, about 0.7 μg / ml to about 4 μg / ml, about 0.8 μg / ml to about 3 μg / ml, or about 0.9 μg / ml to about 2 μg / ml. In some embodiments, the NK activator is administered at concentrations of about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, or about 10 μg / ml. In some embodiments, the NK activator may be present at a concentration of 1 μg / ml.

[0369] In some embodiments, the activator is attached to a solid support such as, but not limited to, beads, an absorbent polymer present in a culture plate or well, or other matrix (such as, but not limited to, Sepharose or glass); which may be expressed on the cell surface of a natural or recombinant cell line by means known to those skilled in the art (such as in natural or recombinant form).

[0370] Polynucleotide transfer

[0371] In some embodiments, the host cells are genetically modified to express the chimeric receptor described above. In some embodiments, the host cells are further genetically modified to express the CAR, TCR, antibody, or fragments or derivatives thereof disclosed herein. The host cells may be genetically modified after stimulation / activation. In some embodiments, the host cells are modified within 12, 16, 24, 36, or 48 hours after stimulation / activation. In some embodiments, the cells are modified within 16 to 24 hours after stimulation / activation. In some embodiments, the host cells are modified within 24 hours.

[0372] To genetically modify host cells to express chimeric receptors or other related molecules (e.g., CARs, TCRs, antibodies, fragments of them, or derivatives), polynucleotide constructs must be transferred into the host cells. Polynucleotide transfer can be via viral or non-viral genetic methods. Suitable methods for polynucleotide delivery used in conjunction with current methods include any methods known to those skilled in the art, by which polynucleotides can be introduced into organelles, cells, tissues, or organisms.

[0373] In some embodiments, polynucleotides are transferred to cells in non-viral vectors. Non-viral vectors suitable for use in this invention include, but are not limited to, microcircular plasmids, transposon systems (e.g., Sleeping Beauty, piggyBac), or single-stranded or double-stranded DNA molecules used as templates for homology-directed repair (HDR) based gene editing.

[0374] Nucleic acid vaccines can be used to transfer polynucleotides into host cells. Such vaccines include, but are not limited to, non-viral polynucleotide vectors, "naked" DNA and RNA, and viral vectors. Methods for genetically modifying cells with these vaccines and for optimizing the expression of genes contained in these vaccines are known to those skilled in the art.

[0375] In some embodiments, the host cell may be genetically modified using methods commonly used by those skilled in the art. In some embodiments, the host cell may be transduced via retroviral transduction. References describing retroviral transduction of genes are Anderson et al., U.S. Patent No. 5,399,346; Mann et al., Cell 33:153 (1983); Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120 (1988); Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95 / 07358, Dougherty et al., March 16, 1995; and Kuo et al., Blood 82:845 (1993), each of which is incorporated herein by reference in its entirety.

[0376] One approach to genetic modification includes in vitro modification. Various methods can be used to transfect cells and tissues removed from a subject via in vitro modification. For example, in vitro retroviral gene transfer can be used to genetically modify cells removed from a subject and transfer those cells back into the subject. See, for example, Wilson et al., Science, 244:1344-1346, 1989 and Nabel et al., Science, 244(4910):1342-1344, 1989, both of which are incorporated herein by reference in their entirety. In some embodiments, host cells can be removed from a subject and transfected in vitro using polynucleotides (e.g., expression vectors) of this disclosure. In some embodiments, host cells obtained from a subject can be transfected or transduced using polynucleotides (e.g., expression vectors) of this disclosure and then administered back to the subject.

[0377] Another method of gene transfer includes injection. In some embodiments, cells, polynucleotides, or viral vectors can be delivered to cells, tissues, or organisms via one or more injections (e.g., needle injection). Non-limiting injection methods include injection compositions (e.g., saline-based compositions). Polynucleotides can also be introduced via direct microinjection. Non-limiting injection sites include subcutaneous, intradermal, intramuscular, intranodal (allowing direct delivery of antigens to lymphoid tissue), intravenous, intraprostatic, intratumoral, intralymphatic (allowing direct administration of DCs), and intraperitoneal. It should be understood that proper injection site preparation is necessary (e.g., shaving the injection site to observe appropriate needle placement).

[0378] Electroporation is another method for polynucleotide delivery. See, for example, Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81, 7161-7165 and Tur-Kaspa et al., (1986) Mol. Cell Biol., 6, 716-718, both incorporated herein by reference in their entirety for all purposes. Electroporation involves exposing a suspension of cells and DNA to a high-voltage discharge. In some embodiments, cell wall degrading enzymes (such as pectin-degrading enzymes) can be used to make host cells more readily genetically modified by electroporation than untreated cells. See, for example, U.S. Patent No. 5,384,253, which is incorporated herein by reference in its entirety for all purposes.

[0379] In vivo electroporation involves a basic injection technique in which a carrier is injected intradermally into the subject. Electrodes then apply electrical pulses to this intradermal site, causing cells located there (e.g., resident skin dendritic cells) to absorb the carrier. These dendritic cells, expressing tumor antigens, are activated through local inflammation and then migrate to lymph nodes.

[0380] Electroporation methods used in conjunction with the present invention include, for example, Sardesai, NY and Weiner, DB, Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro, B. et al., Human Vaccines 7:120-127 (2011), both of which are incorporated herein by reference in their entirety for all purposes.

[0381] Additional methods for polynucleotide transfer include liposome-mediated transfection (e.g., entrapped polynucleotides in lipid complexes suspended in excess aqueous solution. See, for example, Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. pp. 87-104). Also expected are polynucleotides complexed with Lipofectamine or Superfect; DEAE-glucan (e.g., using DEAE-glucan followed by polyethylene glycol to deliver polynucleotides into cells. See, for example, Gopal, TV, Mol Cell Biol. May 1985; 5(5):1188-90); calcium phosphate (e.g., using calcium phosphate precipitation to introduce polynucleotides into cells. See, for example, Graham and van der Eb, (1973) Virology, 52, 456-467; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and Rippe et al., Mol. Cell Biol., 10:689-695, 1990); sonication loading (introducing polynucleotides by direct sonication loading. See, for example, Fechheimer et al., (1987) Proc. Nat'l Acad. Sci. USA, 84, 8463-8467); microprojectile bombardment (e.g., one or more particles may be coated with at least one polynucleotide and delivered into cells by propulsion. See, for example, U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT application WO 94 / 09699; Klein et al., (1987) Nature, 327, 70-73; Yang et al., (1990) Proc. Nat'l Acad. Sci. USA, 87, 9568-9572); and receptor-mediated transfection (e.g., using cell type-specific distribution of various receptors, selective uptake of macromolecules by receptor-mediated endocytosis occurring in target cells).See, for example, Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994; Myers, EPO 0273085; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993; Nicolau et al. (1987) Methods Enzymol., 149, 157-176), and all references cited herein are incorporated herein by reference in their entirety for all purposes.

[0382] In a further embodiment, the host cell undergoes genetic modification using gene editing utilizing homology-directed repair (HDR). Homology-directed repair (HDR) is a mechanism by which cells repair double-strand DNA breaks. In HDR, a donor polynucleotide homologous to the double-strand DNA break site serves as a template to repair the cleaved DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to that DNA. Thus, new nucleic acid material can be inserted into or copied to the target DNA cleavage site. Double-strand DNA breaks in the host cell can be induced by site-specific nucleases. As used herein, the term "site-specific nuclease" refers to a nuclease capable of specifically recognizing and cleaving nucleic acid (DNA or RNA) sequences. Suitable site-specific nucleases for use in this invention include, but are not limited to, RNA-guided endonucleases (e.g., CRISPR-associated (Cas) proteins), zinc finger nucleases, TALEN nucleases, or mega-TALEN nucleases. For example, a site-specific nuclease (e.g., Cas9+ guide RNA) capable of inducing double-strand breaks in a target DNA sequence may be introduced into a host cell along with a donor polynucleotide encoding the chimeric receptor disclosed herein and optionally additional proteins (e.g., CAR, TCR, antibody, or fragments or derivatives thereof).

[0383] Amplification / Proliferation

[0384] After activation and transduction of host cells, cells are cultured to proliferate. T cells can be cultured for at least 1, 2, 3, 4, 5, 6 or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5 or 6 months or longer, with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more rounds of expansion.

[0385] Substances that can be used to amplify T cells may include interleukins, such as IL-2, IL-7, IL-15, or IL-21 (see, for example, Cornish et al. 2006, Blood. 108(2):600-8; Bazdar and Sieg, 2007, Journal of Virology, 2007, 81(22):12670-12674; Battalia et al., 2013, Immunology, 139(1):109-120). Other illustrative examples of substances that can be used to amplify T cells are substances that bind to CD8, CD45, or CD90, such as αCD8, αCD45, or αCD90 antibodies. Illustrative examples of T cell populations include antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells (an illustrative example of memory T cells is the CD62L|CD8| specific central memory T cell) or regulatory T cells (an illustrative example of Tregs is the CD4+CD25+CD45RA+ Treg cell).

[0386] Additional substances that can be used to expand T lymphocytes include methods as described in, for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0387] In some embodiments, the substance (one or more) used for amplification (e.g., IL-2) is administered at a concentration of about 20 units / ml to about 200 units / ml. In some embodiments, the substance (one or more) used for amplification (e.g., IL-2) is administered at a concentration of about 25 units / ml to about 190 units / ml, about 30 units / ml to about 180 units / ml, about 35 units / ml to about 170 units / ml, about 40 units / ml to about 160 units / ml, about 45 units / ml to about 150 units / ml, about 50 units / ml to about 140 units / ml, about 55 units / ml to about 130 units / ml, about 60 units / ml to about 120 units / ml, about 65 units / ml to about 110 units / ml, about 70 units / ml to about 100 units / ml, about 75 units / ml to about 95 units / ml, or about 80 units / ml to about 90 units / ml. In some embodiments, the substance (one or more) used for amplification (e.g., IL-2) is administered at concentrations of approximately 20 units / ml, approximately 25 units / ml, approximately 30 units / ml, 35 units / ml, 40 units / ml, 45 units / ml, approximately 50 units / ml, approximately 55 units / ml, approximately 60 units / ml, approximately 65 units / ml, approximately 70 units / ml, approximately 75 units / ml, approximately 80 units / ml, approximately 85 units / ml, approximately 90 units / ml, approximately 95 units / ml, approximately 100 units / ml, or approximately 105 units / ml. Approximately 110 units / ml, approximately 115 units / ml, approximately 120 units / ml, approximately 125 units / ml, approximately 130 units / ml, approximately 135 units / ml, approximately 140 units / ml, approximately 145 units / ml, approximately 150 units / ml, approximately 155 units / ml, approximately 160 units / ml, approximately 165 units / ml, approximately 170 units / ml, approximately 175 units / ml, approximately 180 units / ml, approximately 185 units / ml, approximately 190 units / ml, approximately 195 units / ml, or approximately 200 units / ml are administered. In some embodiments, the substance (one or more) used for amplification (e.g., IL-2) is administered at approximately 5 mg / ml to approximately 10 ng / ml. In some embodiments, the substance (one or more) used for amplification (e.g., IL-2) is administered at a concentration of about 5.5 ng / ml to about 9.5 ng / ml, about 6 ng / ml to about 9 ng / ml, about 6.5 ng / ml to about 8.5 ng / ml, or about 7 ng / ml to about 8 ng / ml. In some embodiments, the substance (one or more) used for amplification (e.g., IL-2) is administered at a concentration of about 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml.

[0388] After activating and transducing the host cells, the cells are cultured to proliferate. The host cells may be cultured for at least 1, 2, 3, 4, 5, 6 or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5 or 6 months or longer, with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more rounds of expansion.

[0389] Substances that can be used to amplify NK cells may include substances that bind CD16 or CD56, such as, for example, αCD16 or αCD56 antibodies. In some embodiments, the binder includes antibodies (see, for example, Hoshino et al., Blood. 15 Dec. 1991; 78(12):3232-40). Other substances that can be used to amplify NK cells may be IL-15 (see, for example, Vitale et al. 2002. The Anatomical Record. 266:87-92, which is incorporated herein by reference in its entirety for all purposes).

[0390] Suitable conditions for T cell culture include appropriate culture media (e.g., Minimal Essential Medium (MEM), RPMI 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM-V, DMEM, a-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20, Optimizer, which contain added amino acids, sodium pyruvate, and vitamins, and are serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones and / or sufficient amounts of cytokines (one or more) for growth and expansion).

[0391] Examples of other additives used for host cell expansion include, but are not limited to, surfactants, piasmanate, pH buffers (such as HEPES), and reducing agents (such as N-acetylcysteine ​​and 2-mercaptoethanol), and antibiotics (e.g., penicillin and streptomycin), which are contained only in the experimental culture and not in the culture of the cells to be infused into the subject. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2).

[0392] In some embodiments, the host cells of this disclosure may be modified to reduce or eliminate the expression of endogenous TCR, MHC molecules, or other immunogenic molecules. When allogeneic cells are used, rejection of therapeutic cells can be a problem, as it can cause serious complications such as graft-versus-host disease (GvHD). While not wishing to be bound by theory, immunogenic molecules (e.g., endogenous TCR and / or MHC molecules) are typically expressed on the cell surface and are involved in self vs. non-self-discrimination. Reducing or eliminating the expression of such molecules can decrease or eliminate the ability of therapeutic cells to induce GvHD.

[0393] In some embodiments, the expression of endogenous TCRs in host cells is reduced or eliminated. In specific embodiments, the expression of endogenous TCRs (e.g., αβ TCRs) in host cells is reduced or eliminated. The expression of endogenous TCRs can be reduced or eliminated by disrupting the TRAC locus, the TCRbeta invariant locus, and / or the CD3 locus. In some embodiments, the expression of endogenous TCRs can be reduced or eliminated by disrupting one or more of the TRAC, TRBC1, TRBC2, CD3E, CD3G, and / or CD3D loci.

[0394] In some embodiments, the expression of one or more endogenous MHC molecules in host cells is reduced or eliminated. The modified MHC molecule may be an MHC class I or class II molecule. In some embodiments, the expression of endogenous MHC molecules may be reduced or eliminated by disrupting one or more of the MHC, β2M, TAP1, TAP2, CIITA, RFX5, RFXAP, and / or RFXANK loci.

[0395] The expression of endogenous TCRs, MHC molecules, and / or any other immunogenic molecules in host cells can be disrupted using genome editing techniques such as clustered regularly spaced short palindromic repeats (CRISPR) / Cas, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases. These genome editing methods can disrupt target genes by completely knocking out all outputs or partially knocking down their expression. In a particular embodiment, CRISPR / Cas technology is used to disrupt the expression of endogenous TCRs, MHC molecules, and / or any other immunogenic molecules in host cells.

[0396] Pharmaceutical Composition

[0397] In some embodiments, the composition comprises one or more polypeptides and other related molecules (e.g., CARs, TCRs, or antibodies, or fragments or derivatives thereof) of chimeric receptors as disclosed herein, polynucleotides, a carrier comprising said polynucleotides, and a cellular composition. The compositions disclosed herein include, but are not limited to, pharmaceutical compositions.

[0398] In some embodiments, the pharmaceutical composition comprises any host cell and pharmaceutically acceptable carrier and / or excipient disclosed herein.

[0399] In one aspect, this disclosure provides a pharmaceutical composition comprising a polynucleotide or recombinant vector encoding the chimeric receptor described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0400] In another aspect, this disclosure provides a pharmaceutical composition comprising a modified host cell including the chimeric receptor described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0401] Examples of drug carriers include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Aqueous or aqueous solutions, saline solutions, dextrose solutions, and glycerol solutions are preferred as carriers, particularly for injectable solutions.

[0402] Compositions containing the modified host cells disclosed herein may include buffers, such as neutral buffered saline and phosphate buffered saline; carbohydrates, such as glucose, mannose, sucrose or dextrose, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0403] Compositions containing the modified host cells disclosed herein may include one or more of the following: sterile diluents such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixing oils such as synthetic monoglycerides or diglycerides (which may be used as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol or other solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for regulating tension such as sodium chloride or dextrose.

[0404] In some embodiments, the composition is formulated for parenteral administration, such as intravascular (intravenous or intra-arterial), intraperitoneal, intratumoral, intraventricular, intrapleural, or intramuscular administration. The parenteral preparation may be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile. In some embodiments, the composition is reconstituted from a lyophilized preparation prior to administration.

[0405] In some implementations, the modified host cells may be mixed with the attached or permeated material (e.g., but not limited to nanoparticles) prior to their application.

[0406] Treatment

[0407] In one aspect, this disclosure provides a method for enhancing the effector function of immune cells, the method comprising genetically modifying cells with a polynucleotide or recombinant vector encoding a chimeric receptor. In some embodiments, the immune cells may express one or more antigen recognition molecules selected from: chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TRuCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell connectors, natural or transgenic T-cell receptors, and antibodies, or combinations thereof.

[0408] In some embodiments, this disclosure provides a method for enhancing the effector function of immune cells, wherein the immune cells express chimeric antigen receptors (CARs), the method comprising genetically modifying the cells with the polynucleotides disclosed herein or the recombinant vectors disclosed herein.

[0409] In some implementations, the effector function may be one or more of amplification, persistence, and / or cytotoxicity (e.g., antitumor activity).

[0410] The term "expand" (or "expansion"), when used in relation to immune cells, refers to the ability of immune cells to undergo cell proliferation (i.e., increase their cell number). The terminology used in this article encompasses both in vivo and in vitro immune cell expansion.

[0411] The terms “persistence” or “persistence” in relation to the use of immune cells refer to the ability of immune cells (and / or their progeny) to remain in a recipient (e.g., a subject) for a period of time. The terminology used herein encompasses both in vivo and in vitro immune cell persistence.

[0412] As used in this article, the term "tumor killing activity" refers to the ability of immune cells to inhibit tumor growth and / or kill tumor cells (e.g., cancer cells).

[0413] In one aspect, this disclosure provides a method of treating a disease, the method comprising administering to a subject an effective amount of a host cell comprising a chimeric receptor described herein, or a pharmaceutical composition comprising a host cell. In some embodiments, the disease may be cancer, such as, but not limited to, a solid tumor. In some embodiments, the disease may be an infection. In some embodiments, the disease may be an autoimmune disease.

[0414] In one aspect, this disclosure provides a method for treating a tumor in a subject in need. A therapeutically effective amount of a modified host cell comprising the chimeric receptor described herein, or a pharmaceutical composition comprising the host cell, is administered to the subject.

[0415] The term "tumor" refers to a benign or malignant abnormal growth of tissue. The term "tumor" includes cancer. Examples of tumors include, but are not limited to, soft tissue tumors (e.g., lymphoma), tumors of blood and blood-forming organs (e.g., leukemia), and solid tumors that grow in anatomical locations outside the bloodstream (e.g., carcinoma). Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (e.g., osteosarcoma or rhabdomyosarcoma), and leukemia or malignant lymphomas. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), adenosquamous cell carcinoma, lung cancer (e.g., including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (e.g., including gastrointestinal cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, primary or metastatic melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, brain (e.g., high-grade glioma, diffuse pontine glioma, ependymoma, neuroblastoma, or glioblastoma), and head and neck cancers, and related metastases. Additional examples of tumors can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition. on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, On Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition of Merck Manuals’ Internet website); and SEER Program Coding and Staging Manual 2016, each incorporated herein by reference in its entirety for all purposes.

[0416] In some embodiments, the cancer treated by the method of the present invention is HER2-positive cancer. In some embodiments, HER2-positive cancer is brain, breast, stomach, ovary, serous endometrial cancer, colon, bladder, lung, cervix, head and neck cancer, sarcoma, bone tumor, or esophageal cancer.

[0417] In some embodiments, the cancer treated by the method of the present invention is EphA2-positive cancer. In some embodiments, EphA2-positive cancer is breast, prostate, bladder, skin, lung, ovarian, sarcoma, bone tumor, or brain cancer.

[0418] In some embodiments, the cancer treated by the method of the present invention is B7-H3 positive cancer. In some embodiments, B7-H3 positive cancer includes osteosarcoma, rhabdomyosarcoma, Ewing sarcoma and other tumors of the Ewing sarcoma family, neuroblastoma, ganglioneurotic neuroblastoma, connective tissue proliferative small round cell tumor, malignant peripheral nerve sheath tumor, synovial sarcoma, undifferentiated sarcoma, adrenocortical carcinoma, hepatoblastoma, Wilms' tumor, rhabdomyosarcoma, high-grade glioma (glioblastoma multiforme), medulloblastoma, and astrocytoma. Gliomas, ependymomas, atypical teratoid rhabdoid tumors, meningiomas, craniopharyngiomas, primitive neuroectodermal tumors, diffuse endophytic pontine gliomas and other brain tumors, acute myeloid leukemia, multiple myeloma, lung cancer, mesothelioma, breast cancer, bladder cancer, gastric cancer, prostate cancer, colorectal cancer, endometrial cancer, cervical cancer, kidney cancer, esophageal cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma and other liver cancers, head and neck cancers, leiomyosarcoma, or melanoma.

[0419] In some embodiments, the treatment method of this disclosure may include one or more of the following steps: a) isolating immune cells (e.g., T cells or NK cells) from a subject or donor; b) in vitro modifying the immune cells (e.g., T cells or NK cells) with a polynucleotide or recombinant vector encoding the chimeric receptor described herein; c) optionally, in vitro modifying the immune cells (e.g., T cells or NK cells) to express a chimeric antigen receptor (CAR), an antigen-specific T cell receptor (TCR), and / or an antibody, or a derivative or fragment thereof, and said CAR, TCR, or antibody specifically binding to a disease-associated antigen; d) optionally, expanding and / or activating the modified immune cells (e.g., T cells or NK cells) before, after, and / or during step b) or c); and e) introducing a therapeutically effective amount of the modified immune cells (e.g., T cells or NK cells) into a subject. In some embodiments, the immune cells express GR-CSF after activation. In some embodiments, the immune cells are αβ TCRT cells, γδ T cells, or iNKT cells.

[0420] In some implementations, one or more antigen recognitions may be selected from: chimeric antigen receptors (CARs), T-cell receptor fusion constructs (TRuCs), HLA-independent T-cell receptors (HITs), synthetic T-cell receptors and antigen receptors (STARs), T-cell antigen couplers (TACs), bispecific T-cell adapters, natural or transgenic T-cell receptors, and antibodies, or combinations thereof.

[0421] In some embodiments, the treatment methods of this disclosure may include one or more of the following steps: a) isolating T cells or NK cells from a subject or donor; b) in vitro modifying the T cells or NK cells with a polynucleotide encoding the chimeric receptor described herein or a recombinant vector encoding said polynucleotide; c) optionally, in vitro modifying the T cells or NK cells to express a chimeric antigen receptor (CAR) that binds to an antigen associated with the disease; d) optionally, expanding and / or activating the modified T cells or NK cells before, after, and / or during step b) or c); and e) introducing a therapeutically effective amount of the modified T cells or NK cells into the subject.

[0422] In some embodiments, the modified host cell is an autologous cell. In some embodiments, the modified host cell is an allogeneic cell. In cases where the host cell is isolated from a donor, the method may further include methods for preventing graft-versus-host disease (GVHD) and host cell rejection.

[0423] In some embodiments, the modified host cells may also express the CD20 peptide as a safety switch. Therefore, the method may further include administering an anti-CD20 antibody to the subject to remove isolated host cells. The anti-CD20 antibody is administered in an amount effective for adequately removing isolated host cells from the subject. In some embodiments, the anti-CD20 antibody is administered in an amount effective for removing more than 50% of isolated host cells from the subject. For example, the anti-CD20 antibody may be administered in an amount effective for removing more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, or about 100% of isolated host cells from the subject. The anti-CD20 antibody may be administered in an amount effective for removing about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100% of isolated host cells from the subject.

[0424] Non-limiting examples of anti-CD20 antibodies that can be used to remove isolated host cells include rituximab, tiuxetan, tositumomab, offatumumab, ocrelizumab, TRU-015, veltuzumab, AME-133v, PRO131921, and ocinutuzumab. In some embodiments, the anti-CD20 antibody is rituximab.

[0425] In some embodiments of any of the above-described treatment methods, the composition is administered at a therapeutically effective amount. The dosage of the composition administered in the methods of the present invention will vary widely depending on the subject's physical parameters, frequency of administration, method of administration, and clearance rate, etc. The initial dose may be large, followed by a smaller maintenance dose. The dose may be administered infrequently once a week or every two weeks, or divided into smaller doses and administered once daily, twice weekly, etc., to maintain an effective dose level. Various dosages are expected to effectively achieve in vivo persistence of the modified host cells. Various dosages are also expected to effectively improve the in vivo effector function of the modified host cells.

[0426] In some embodiments, the composition comprising modified host cells manufactured by the methods described herein can be in the form of 10 2 Up to 10 10 Cells / kg body weight, 10 5 Up to 10 9 Cells / kg body weight, 10 5 Up to 10 8Cells / kg body weight, 10 5 Up to 10 7 Cells / kg body weight, 10 7 Up to 10 9 cells / kg body weight, or 10 7 Up to 10 8 Dosage administration is in cells / kg body weight, including all integer values ​​within these ranges. The number of modified host cells will depend on the intended therapeutic use of the composition.

[0427] Modified host cells can be administered multiple times at the doses listed above. The modified host cells for the patients undergoing the therapy can be allogeneic, syngeneic, allogeneic, or autologous.

[0428] The compositions and methods described in this disclosure can be used in combination with other types of therapies for tumors, such as chemotherapy, surgery, radiation, and gene therapy.

[0429] It is also anticipated that, when used to treat various diseases / conditions, the compositions and methods of this disclosure can be used in conjunction with other treatment methods / agents applicable to the same or similar diseases / conditions. Such other treatment methods / agents can be administered concurrently (simultaneously or sequentially) to produce an additive or synergistic effect. Due to the additive or synergistic effect, the appropriate therapeutically effective dose of each agent may be reduced.

[0430] In some embodiments of any of the above treatment methods, the method further includes administering to a subject one or more additional compounds selected from the group consisting of: immunosuppressants, biologicals, probiotics, prebiotics, and cytokines (e.g., GM-CSF, IFN, or IL-2).

[0431] In some embodiments, in addition to GM-CSF produced by immune cells, the methods described herein further include providing exogenous GM-CSF to enhance the function of immune cells expressing the chimeric receptor of this disclosure. Exogenous GM-CSF can be provided, for example, but not limited to, by: i) injection of an FDA-approved GM-CSF drug, sargramostin (Leukine™), or ii) using a non-viral or viral vector to express GM-CSF (e.g., an FDA-approved oncolytic virus expressing GM-CSF, talimogene laherparepvec [TVEC, Imlygic™]). These drugs can be administered before, concurrently with, or after the administration (e.g., infusion) of immune cells expressing the chimeric receptor of this disclosure to the patient.

[0432] As a non-limiting example, the compositions disclosed herein can be combined with other therapies that block inflammation (e.g., by blocking IL-1, IFNα / β, IL-6, TNF, IL-23, etc.).

[0433] The methods and compositions disclosed herein can be combined with other immunomodulatory therapies, such as, for example, therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.), or activators (including but not limited to agents that enhance 4-1BB, OX40, etc.). The methods of the present invention can also be combined with other therapies possessing the ability to modulate NKT function or stability, including but not limited to CD1d, CD1d fusion proteins, CD1d dimers, or larger polymers of CD1d (unloaded or loaded with antigen), CD1d chimeric antigen receptors (CD1d-CAR), or any other isoform of the five known CD1 isoforms (CD1a, CD1b, CD1c, CD1e) present in humans. The methods of the present invention can also be combined with other therapies (such as midostaurin or enasidenib, or combinations thereof).

[0434] The treatment methods disclosed herein can be combined with additional immunotherapies and other treatments. For example, when used to treat tumors, the compositions of the present invention can be used in combination with conventional therapies (such as, for example, surgery, radiation therapy, chemotherapy, or combinations thereof), depending on the type of tumor, the patient's condition, other health problems, and various factors. In some aspects, other therapeutic agents used in combination with the inhibitors of the present invention for tumor therapy include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, platelet-reactive protein-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, soluble bFGF receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferation protein-associated protein, and those listed by Carmeliet and Jain (2000). In one embodiment, the modified host cell of the present invention can be used in combination with a VEGF antagonist or a VEGF receptor antagonist, such as an anti-VEGF antibody, a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, an inhibitor of VEGFR tyrosine kinase, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab, or ranibizumab).

[0435] Non-limiting examples of chemotherapeutic compounds that can be used in combination therapy according to this disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, BCG, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, and actinomycin D. (dactinomycin), daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil Rouracil), fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustineNitrogen mustard (mechlorethamine), medroxyprogesterone, megestrol, melphalan, mecaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel Pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0436] These chemotherapeutic compounds can be classified according to their mechanisms of action into groups such as: antimetabolites / antitumor agents, such as pyrimidine analogs (5-fluorouracil, fluxuridine, capecitabine, gemcitabine, and tarasporin) and purine analogs, folate antagonists, and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine cladribine); antiproliferative / antimitotic agents, including natural products such as vincaalkaloids (vincrine, vincristine, and vinorelbine); microtubule disruptors such as taxanes (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilone, and navelbine; and epidermal agents. Epidipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycins, acridine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, actinomycin D, daunorubicin, doxorubicin, epirubicin, hexamethystine oxaliplatin, iphosphamide, melphalan, nitrogen mustard, mitomycin, mitoxantrone, nitrosourea, procainamide, procarbazine, taxol, taxotere, teniposide, triethylene thiophosphamide, and etoposide (VP16)); antibiotics, such as actinomycin D. (dactinomycin or actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, procainox (mithramycin), and mitomycin; enzymes (L-asparaginase, which systematically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustard. Mustards (nitrogen mustard, cyclophosphamide and analogues, melphalan, chlorambucil), ethylenimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates-bustanoic acid, nitrosoureas (carmustine (BCNU and analogues, streptozotocin), trazenes-dacarbazinine (DTIC); antiproliferative / antimitotic metabolites, such as folic acid analogues (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;Hormones, hormone analogues (estrogens, tamoxifen, goserelin, bicalutamide, nilumid), and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigration agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine, cyclosporine (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil). henolatemofetil; anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (doxacin), acridine, camptothecin, daunorubicin, actinomycin D, teniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone and prednisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0437] In various implementations of the methods described herein, the subjects are human beings. Subjects can be adolescents or adults of any age or sex.

[0438] According to the present invention, there are many tools and techniques available within the scope of the art, such as those commonly used in molecular biology, pharmacology and microbiology. Such tools and techniques are described in detail in, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. (ed.) (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. (ed.) (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. (ed.) (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. (ed.) (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. (ed.) (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. Edited. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ.

[0439] Example

[0440] The following examples are provided to further describe some embodiments disclosed herein. These examples are intended to be illustrative and not to limit the disclosed embodiments.

[0441] Example 1. Jun.MyD88 homodimer activates NFκB signaling and improves CAR T cell antitumor activity sex

[0442] This generates a homodimer IL-18 ZipR utilizing either an IL-18Rα chain or an IL-18Rbeta chain conjugated to human cJun leucine. These constructs activate NFκB signaling in NFκB reporter cells only when expressed together, rather than as homodimer molecules. Figure 1A IL-18R signaling involves MyD88 aggregation to trigger downstream signaling activation; therefore, human MyD88 was subsequently fused to cJun via the CD28 transmembrane™ domain (Jun.CD28™.MyD88), the IL-18Rα chain™ domain (Jun.18α™.MyD88), or the IL-18Rbeta chain™ domain (Jun.18β™.MyD88). All three constructs are functional, as determined by NFκB activation in reporter cells. Figure 1B When co-expressed with EphA2-specific CARs in human T cells, all three constructs improved the ability of CAR T cells to repeatedly kill A673 tumor cells and expand. Figure 1C-1D ).

[0443] Example 2. Jun leucine zipper-based homodimer activation of STAT5, leading to improved T cell viability. And it is inhibited by ruxolitinib.

[0444] Generate and test the following builds:

[0445] i) Jun.IL-2Rbeta chain constructs with 0 to 6 alanine insertions to explore all potential intracellular orientations;

[0446] ii) Jun.EpoR constructs with 0, 1, 3 or 5 amino acid TM domain deletions to explore receptors with 0, 100, 300 or 500 degrees of rotation;

[0447] iii) Jun.GHR; and

[0448] iv) Jun.18β.MyD88 fused to EpoR or GHR (Jun.MyD88.EpoR or Jun.MyD88.GHR).

[0449] The STAT5 signaling pathway improves CAR T cell function. When expressed in human T cells, all Jun.EpoR ZipR and Jun.GHR ZipR phosphorylate (activate) STAT5. Figure 2A Conversely, the homodimers Jun.IL-2Rβ ZipR and Jun.MyD88.EpoR or Jun.MyD88.GHR ZipR cannot ( Figure 2B ).

[0450] As a potential safety switch for the constitutively active homodimer ZipR, T cells expressing Jun.EpoR ZipR or Jun.GHRZipR were treated with increased concentrations of ruxotinib (an FDA-approved JAK1 / 2 inhibitor). All ZipRs were inhibited by ruxotinib. Figure 2C-2E As can be seen from their ability to phosphorylate STAT5. The half-maximal effective concentration (EC50) of ruxotinib for each ZipR ranges from 6.3 to 668.3 nM (Table 4), indicating that the signaling strength of each ZipR is different, allowing for customized STAT5 signaling in T cells.

[0451] In addition to STAT5 activation, the ability of Jun ZipR to improve T cell function was also determined. Human T cells expressing Jun.EpoR ZipR or Jun.GHR ZipR were cultured in vitro for 7 days without cytokines, and the frequencies of live cells, dead cells, and dying cells were then quantified. ZipR signaling allowed cells to survive 7 days of cytokine starvation, which was eliminated by co-treatment with ruxolitinib. Figure 2F ).

[0452] Table 4: EC50 values ​​of ruxolitinib

[0453]

[0454] Example 3. Jun homodimer ZipR does not alter the human T cell immune phenotype.

[0455] Finally, the effects of ZipR signaling on T cell phenotype and differentiation were evaluated. Although ZipR is expressed in human T cells ( Figure 3A However, they do not change the frequency of CD4+ or CD8+ T cells. Figure 3B ) or their immunophenotype ( Figure 3C ).

[0456] In summary, the data provided in Figures 1 to 3 demonstrate that ZipR, generated based on EpoR (ZipEpoR) and GHR (ZipGHR), activates STAT5 in reporter cell lines and in primary human T cells.

[0457] The following are the methods used in the above embodiments.

[0458] Production of viral vectors. The production of RD114 pseudotyped gamma-retroviral vectors (pSFG) encoding second-generation EphA2-specific CARs (4H5.CD28z) has been previously described. [29, 30] The pSFG vectors encoding Jun.IL2Rβ, Jun.EpoR, Jun.GHR, Jun.IL18R, and Jun.MyD88 ZipR were produced by synthesizing gene fragments via In-Fusion cloning (Takara) (Thermo Fisher). They consist of (i) human cJun (amino acids 277-315) linked to (ii) an intracellular domain indicating the cytokine receptor chain, (iii) a P2A sequence, and (iv) an mClover for transduction detection. The sequences of the final constructs were validated by sequencing (Hartwell Center, St. Jude Children's Research Hospital). As previously described. 4 RD114 pseudotyped retroviral particles were generated through transient transfection of 293T cells. The supernatant was collected after 48 hours, filtered, and rapidly frozen.

[0459] Generation of EphA2-CAR T cells. Human peripheral blood mononuclear cells (PBMCs) were isolated from a labeling-free panning chamber of leukapheresis products obtained from the St. Jude donor center using Lymphoprep (Abbott Laboratories). On day 0, PBMCs were stimulated overnight in 24-well plates pre-coated with CD3 and CD28 antibodies (Miltenyi) in non-tissue culture. On day 1, cytokines IL-7 (10 ng / mL) and IL-15 (5 ng / mL) (PeproTech, Rocky Hill, NJ, USA) were added to the culture. On day 2, T cells were transferred to retrovirus-coated (Clontech) plates containing retroviral particles for 2 to 4 days. On day 3, T cells were transferred to fresh 24-well plates pre-coated with tissue culture and subsequently expanded with IL-7 and IL-15. All experiments were performed 7 to 14 days post-transduction. Biological replication was performed using PBMCs from different healthy donors.

[0460] Production of ZipR-transduced T cells. To transduce T cells using EphA2-CAR and ZipR, T cells were transduced as described in the section on the generation of EphA2-CAR-CAR cells, but using a mixture of EphA2-CAR and ZipR gamma-retroviral vectors. To transduce T cells using ZipR alone, PBMCs were stimulated for 48 hours on plates coated with anti-CD3 and anti-CD28. Recombinant human IL-7 (10 ng / mL, Peprotech) and IL-15 (5 ng / mL, Peprotech) were added 24 hours after initial stimulation and maintained in culture until functional studies. Cells were then seeded onto retrovirus-coated (Clontech) plates containing retroviral particles for 2 to 4 days. T cells were transferred to fresh 24-well tissue culture-treated plates and subsequently expanded with IL-7 and IL-15. Untransduced T cells were prepared similarly, except that the retrovirus was not included in the retrovirus wells. All experiments were performed 7 to 14 days post-transduction. Non-transduced (NT) T cells were prepared similarly, except that the retrovirus was not included in the retronectin wells. Biological replicates were performed using PBMCs from different healthy donors.

[0461] Cell lines. A673 (Ewing sarcoma) were purchased from the American Type Culture Collection (ATCC). Cell lines were identified using the ATCC Human Short Tandem Repeat Spectrum Cell Identification Service and routinely tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza). Once thawed, cell lines were kept in culture for up to 3 months before thawing new reference vials. Cell lines were maintained and expanded in Dulbecco modified Eagle medium (GE Healthcare Life Sciences HyClone Laboratories) supplemented with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences HyClone Laboratories) and 2 mmol / L Glutamax (Invitrogen).

[0462] Flow cytometry. Flow cytometry data were acquired using FACSCanto II (BD Biosciences) and analyzed using FlowJo v10 (BD Biosciences). For surface staining, samples were washed with PBS (Lonza) containing 1% FBS (GE Healthcare) and stained. For all experiments, matched isotypes or known negative (e.g., NT T cells) were used as gated controls. CAR detection was performed using an F(ab')2 fragment-specific antibody (polyclonal, Jackson Immuno Research, West Grove, PA, USA). T cells were stained with antibodies conjugated with fluorescent dyes using a combination of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone 2-L1-A, BD Biosciences), and CD45RO (clone HI100, BD Biosciences).

[0463] Intracellular staining. Intracellular staining was performed using BD Phosflow Protocol III to detect pSTAT5. Briefly, T cells were either unstimulated or stimulated with 5 ng / mL IL-15 for 15 min, followed by lysis / fixation with 1X lysis / fixation buffer (BD Biosciences) at 37°C for 12 min. Cells were washed with PBS + 1% FBS and then permeabilized with pre-chilled Perm buffer III (BD Biosciences). Cells were then washed three times and stained with an antibody against pSTAT5 (clone 47, BD Biosciences).

[0464] Cytokine starvation. To measure cell survival after 7 days of cytokine withdrawal, 1×10⁻⁶ cells were used. 6 Cells were plated in 1 mL of cytokine-free, complete RPMI in 48-well plates treated with tissue culture. After 7 days, cells were harvested and stained for flow cytometry. To quantify apoptosis, cells were labeled with annexin V (BD Biosciences) and eBioscience immobilizable viability dye (Invitrogen).

[0465] Repeated stimulation assay. In a 24-well plate treated with tissue culture, 1×10⁻⁶... 6 5 × 10 T cells and 5 × 10 5Tumor cells were co-cultured in pure RPMI. Cells were fed with fresh pure RPMI and split as needed. After 7 days, T cells were harvested, counted, and replated with fresh tumor cells in the same proportion, provided they killed tumor cells as determined by microscopic examination.

[0466] Example 4. Modification of the homochimera Jun.GHR ZipR for activating additional signal transduction pathways

[0467] This is publicly available. Figures 2A to 2B (See also Example 2 above) This demonstrates that the homochimeric Jun.GHR ZipR is functional, as determined by STAT5 phosphorylation. To demonstrate that this receptor can be further modified to activate additional signaling pathways, a novel molecule, Jun.GHR.MyD88 ZipR, was created, in which the MyD88 signaling domain is fused to Jun.GHR ZipR. The ability of this ZipR (Jun.GHR.MyD88) to activate STAT5 and the MyD88 signaling pathway was evaluated using reporter cell lines. Figure 4A The successful transduction of the STAT5 reporter cell line using retroviral constructs encoding Jun.GHR and Jun.GHR.MyD88 is demonstrated. A retroviral vector encoding the IL-2 receptor-based heterodimer ZipR (Zip2R; Bell et al., Nat Biomed Eng 2023) was used as a positive control. Figure 4B The diagram shows that Jun.GHR and Jun.GHR.MyD88 phosphorylate STAT5. Therefore, adding the MyD88 domain to Jun.GHR does not interfere with the ability of the GHR domain to phosphorylate STAT5.

[0468] To evaluate the function of the MyD88 domain in Jun.GHR.MyD88 ZipR, the Ramos Blue MyD88 signaling reporter cell line was transduced with Jun.GHR and Jun.GHR.MyD88. Figure 5A Cells transduced with Jun.MyD88 ZipR were used as a positive control. Figure 5B It is shown that, unlike Jun.GHR, Jun.GHR.MyD88 activates MyD88 signaling. Therefore, this embodiment demonstrates for the first time the ability to generate a homodimer ZipR that activates both the STAT5 and MyD88 signaling pathways. This method is applicable to the EpoR-based homodimer ZipR (also described herein) Figure 2A ).

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[0486] 17. Juillerat A, Tkach F, Busser BW, Temburni S, Valton J, Duclert A,Poirot L, Depil S, and Duchateau P. Modulation of chimeric antigen receptorsurface expression by a small molecule switch.BMC Biotechnol.2019; 19:44.Published online 2019 Jul 3. doi: 10.1186 / s12896-019-0537-3.

[0487] 18. Carbonneau S, Sharma S, Peng L, Rajan V, Hainzl D, Henault M,Yang C, Hale J, Shulok J, Tallarico J, Porter J, Brogdon JL, Dranoff G,Bradner JE, Hild M, and Guimaraes CP.An IMiD-inducible degron providesreversible regulation for chimeric antigen receptor expression andactivity.Cell Chemical Biology.Volume 28, Issue 6, 17 June 2021, Pages 802-812.e6

[0488] 19. Jan M, Scarfò I, Larson RC,Walker A, Schmidts A, Guirguis AA,Gasser JA, , Bouffard AB, Castano AP, Kann MC, Cabral ML, TepperA, Grinshpun DE, Sperling AS, Kyung T, Sievers QL, Birnbaum ME, Maus MV,Ebert BL.Reversible ON- and OFF-switch chimeric antigen receptors controlledby lenalidomide.Sci Transl Med. 2021 Jan 6;13(575):eabb6295. doi: 10.1126 / scitranslmed.abb6295.

[0489] [ PubMed ] 20. Monteys AM, Hundley AA, Ranum PT, Tecedor L, Muehlmatt A, Lim E,Lukashev D, Sivasankaran R, Davidson BL.Nature.2021 Aug;596(7871):291-295. doi: 10.1038 / s41586-021-03770-2.Epub 2021 Jul 28.

[0490] [ PubMed ] 21. Zhu H and Kaufman DS.An Improved Method to Produce Clinical-ScaleNatural Killer Cells from Human Pluripotent Stem Cells.Methods Mol Biol.2019;2048:107-119. doi: 10.1007 / 978-1-4939-9728-2_12.

[0491] 22. Iriguchi S, Yasui Y, Kawai Y, Arima S, Kunitomo M, Sato T, UedaT, Minagawa A, Mishima Y, Yanagawa N, Baba Y, Miyake Y, Nakayama K, TakiguchiM, Shinohara T, Nakatsura T, Yasukawa M, Kassai Y, Hayashi A & Kaneko S.Aclinically applicable and scalable method to regenerate T cells from iPSCsfor off-the-shelf T-cell immunotherapy.Nature Communications volume 12,Article number: 430 (2021).

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[0493] 24. Trinchieri G. Interleukin-12 and the Regulation of InnateResistance and Adaptive Immunity. Nat Rev Immunol (2003) 3(2):133–46. doi:10.1038 / nri1001.

[0494]

[0495] This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention beyond those described herein will become apparent to those skilled in the art based on the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0496] All patents, applications, publications, test methods, documents and other materials cited herein are incorporated herein in their entirety as if they were actually present in this specification.

[0497] sequence list

[0498] SEQ ID NO: 1 Leader nucleotide sequence

[0499] ATGGATTGGATCTGGCGGATCCTGTTCCTTGTGGGAGCTGCCACAGGCGCCCACAGC

[0500] SEQ ID NO: 2 FLAG nucleotide sequence

[0501] GACTACAAGGACGACGACGACAAG

[0502] SEQ ID NO: 3 cJun nucleotide sequence

[0503] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCAC

[0504] SEQ ID NO: 4 cJunzip.hEpoRmut.2A.mClover nucleotide sequence

[0505] CTGATCCTGACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0506] SEQ ID NO: 5 P2A nucleotide sequence

[0507] GGCAGCGGCGCCACCAACTTTAGCCTGCTGAAACAAGCCGGCGACGTGGAAGAGAATCCCGGACCT

[0508] SEQ ID NO: 6 mClover3 nucleotide sequence

[0509] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTCCGCGGCGAaGGtGAGGGaGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGGCCTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCTTTCAAGGACGACGGTACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTTCAACAGCCACTACGTCTATATCACGGCCGACAAGCAGAAGAACTGCATCAAGGCTAACTTCAAGATCCGCCACAACGTTGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCCATCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATTACACATGGCATGGACGAGCTGTACAAGTAA

[0510] SEQ ID NO: 7 Leader protein sequence

[0511] MDWIWRILFLVGAATGAHS

[0512] SEQ ID NO: 8 FLAG protein sequence

[0513] DYKDDDDK

[0514] SEQ ID NO: 9 cJun protein sequence

[0515] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNH

[0516] SEQ ID NO: 10 cJunzip.hEpoRmut.2A.mClover protein sequence

[0517] LILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0518] SEQ ID NO: 11 P2A protein sequence

[0519] GSGATNFSLLKQAGDVEENPGP

[0520] SEQ ID NO: 12 mClover3 protein sequence

[0521] MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0522] SEQ ID NO: 13 cJunzip.hEpoRmut.del1.2A.mClover nucleotide sequence

[0523] ATCCTGACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0524] SEQ ID NO: 14 cJunzip.hEpoRmut.del1.2A.mClover protein sequence

[0525] ILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0526] SEQ ID NO: 15 cJunzip.hEpoRmut.del3.2A.mClover nucleotide sequence

[0527] ACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0528] SEQ ID NO: 16 cJunzip.hEpoRmut.del3.2A.mClover protein sequence

[0529] TLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0530] SEQ ID NO: 17 cJunzip.hEpoRmut.del5.2A.mClover nucleotide sequence

[0531] AGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0532] SEQ ID NO: 18 cJunzip.hEpoRmut.del5.2A.mClover protein sequence

[0533] SLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0534] SEQ ID NO: 19 cJunzip.hGHR.2A.mClover nucleic acid

[0535]

[0536] SEQ ID NO: 20 cJunzip.hGHR.2A.mClover protein sequence

[0537] FPWLLIIIFGIFGLTVMLFVFLFSKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLDQKNQNNSPYHDACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMP

[0538] SEQ ID NO: 21 Jun_IL2Rb nucleotide sequence

[0539] ATCCCCTGGCTGGGACATCTGCTCGTTGGACTGTCTGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCAACTGCCGGAACACAGGCCCTTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTCCTGCAGCAGGATAAGGTGCCAGAACCTGCCAGCCTGTCCAGCAATCACAGCCTGACCAGCTGCTTTACCAACCAGGGCTACTTCTTCTTCCATCTGCCTGACGCTCTCGAGATCGAGGCCTGCCAGGTGTACTTCACCTACGATCCCTACAGCGAGGAAGATCCCGACGAAGGCGTTGCAGGCGCTCCTACAGGATCTTCTCCACAGCCTCTGCAGCCACTGTCCGGCGAGGATGATGCCTACTGCACCTTTCCAAGCCGGGACGACCTGCTGCTGTTTAGCCCTAGTCTGCTCGGCGGACCTTCTCCACCATCTACAGCTCCAGGTGGAAGCGGAGCCGGCGAAGAAAGAATGCCTCCTAGCCTGCAAGAGCGGGTGCCCAGAGATTGGGATCCTCAACCTCTCGGACCTCCTACACCAGGCGTGCCAGACCTGGTGGATTTTCAGCCTCCTCCAGAGCTGGTGCTGAGAGAAGCTGGCGAAGAAGTGCCAGACGCCGGACCTAGAGAGGGCGTTAGCTTTCCTTGGAGCAGACCTCCTGGACAGGGCGAGTTCAGAGCCCTGAATGCTAGACTGCCCCTGAACACCGATGCCTACCTGTCTCTGCAAGAACTGCAGGGACAAGACCCCACACACCTCGTT

[0540] SEQ ID NO: 22 Jun_IL2Rb protein sequence

[0541] IPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0542] SEQ ID NO: 23 Nucleotide sequence of Jun_IL2Rb_1Ala

[0543] ATCCCCTGGCTGGGACATCTGCTCGTTGGACTGTCTGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCGCCAACTGCCGGAACACAGGCCCTTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTCCTGCAGCAGGATAAGGTGCCAGAACCTGCCAGCCTGTCCAGCAATCACAGCCTGACCAGCTGCTTTACCAACCAGGGCTACTTCTTCTTCCATCTGCCTGACGCTCTCGAGATCGAGGCCTGCCAGGTGTACTTCACCTACGATCCCTACAGCGAGGAAGATCCCGACGAAGGCGTTGCAGGCGCTCCTACAGGATCTTCTCCACAGCCTCTGCAGCCACTGTCCGGCGAGGATGATGCCTACTGCACCTTTCCAAGCCGGGACGACCTGCTGCTGTTTAGCCCTAGTCTGCTCGGCGGACCTTCTCCACCATCTACAGCTCCAGGTGGAAGCGGAGCCGGCGAAGAAAGAATGCCTCCTAGCCTGCAAGAGCGGGTGCCCAGAGATTGGGATCCTCAACCTCTCGGACCTCCTACACCAGGCGTGCCAGACCTGGTGGATTTTCAGCCTCCTCCAGAGCTGGTGCTGAGAGAAGCTGGCGAAGAAGTGCCAGACGCCGGACCTAGAGAGGGCGTTAGCTTTCCTTGGAGCAGACCTCCTGGACAGGGCGAGTTCAGAGCCCTGAATGCTAGACTGCCCCTGAACACCGATGCCTACCTGTCTCTGCAAGAACTGCAGGGACAAGACCCCACACACCTCGTT

[0544] SEQ ID NO: 24 Jun_IL2Rb_1Ala protein sequence

[0545] IPWLGHLLVGLSGAFGFIILVYLLIANCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0546] SEQ ID NO: 25 Nucleotide sequence of Jun_IL2Rb_2Ala

[0547] ATCCCCTGGCTGGGACATCTGCTCGTTGGACTGTCTGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCGCCGCTAACTGCCGGAACACAGGCCCTTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTCCTGCAGCAGGATAAGGTGCCAGAACCTGCCAGCCTGTCCAGCAATCACAGCCTGACCAGCTGCTTTACCAACCAGGGCTACTTCTTCTTCCATCTGCCTGACGCTCTCGAGATCGAGGCCTGCCAGGTGTACTTCACCTACGATCCCTACAGCGAGGAAGATCCCGACGAAGGCGTTGCAGGCGCTCCTACAGGATCTTCTCCACAGCCTCTGCAGCCACTGTCCGGCGAGGATGATGCCTACTGCACCTTTCCAAGCCGGGACGACCTGCTGCTGTTTAGCCCTAGTCTGCTCGGCGGACCTTCTCCACCATCTACAGCTCCAGGTGGAAGCGGAGCCGGCGAAGAAAGAATGCCTCCTAGCCTGCAAGAGCGGGTGCCCAGAGATTGGGATCCTCAACCTCTCGGACCTCCTACACCAGGCGTGCCAGACCTGGTGGATTTTCAGCCTCCTCCAGAGCTGGTGCTGAGAGAAGCTGGCGAAGAAGTGCCAGACGCCGGACCTAGAGAGGGCGTTAGCTTTCCTTGGAGCAGACCTCCTGGACAGGGCGAGTTCAGAGCCCTGAATGCTAGACTGCCCCTGAACACCGATGCCTACCTGTCTCTGCAAGAACTGCAGGGACAAGACCCCACACACCTCGTT

[0548] SEQ ID NO: 26 Jun_IL2Rb_2Ala protein sequence

[0549] IPWLGHLLVGLSGAFGFIILVYLLIAANCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0550] SEQ ID NO: 27 Jun_IL2Rb_3Ala nucleotide sequence

[0551] ATCCCCTGGCTGGGACATCTGCTCGTTGGACTGTCTGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCGCCGCTGCCAACTGCCGGAACACAGGCCCTTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTCCTGCAGCAGGATAAGGTGCCAGAACCTGCCAGCCTGTCCAGCAATCACAGCCTGACCAGCTGCTTTACCAACCAGGGCTACTTCTTCTTCCATCTGCCTGACGCTCTCGAGATCGAGGCCTGCCAGGTGTACTTCACCTACGATCCCTACAGCGAGGAAGATCCCGACGAAGGCGTTGCAGGCGCTCCTACAGGATCTTCTCCACAGCCTCTGCAGCCACTGTCCGGCGAGGATGATGCCTACTGCACCTTTCCAAGCCGGGACGACCTGCTGCTGTTTAGCCCTAGTCTGCTCGGCGGACCTTCTCCACCATCTACAGCTCCAGGTGGAAGCGGAGCCGGCGAAGAAAGAATGCCTCCTAGCCTGCAAGAGCGGGTGCCCAGAGATTGGGATCCTCAACCTCTCGGACCTCCTACACCAGGCGTGCCAGACCTGGTGGATTTTCAGCCTCCTCCAGAGCTGGTGCTGAGAGAAGCTGGCGAAGAAGTGCCAGACGCCGGACCTAGAGAGGGCGTTAGCTTTCCTTGGAGCAGACCTCCTGGACAGGGCGAGTTCAGAGCCCTGAATGCTAGACTGCCCCTGAACACCGATGCCTACCTGTCTCTGCAAGAACTGCAGGGACAAGACCCCACACACCTCGTT

[0552] SEQ ID NO: 28 Jun_IL2Rb_3Ala protein sequence

[0553] IPWLGHLLVGLSGAFGFIILVYLLIAAANCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0554] SEQ ID NO: 29 Jun_IL2Rb_4Ala nucleotide sequence

[0555] ATCCCCTGGCTGGGACATCTGCTCGTTGGACTGTCTGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCGCCGCTGCCGCTAACTGCCGGAACACAGGCCCTTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTCCTGCAGCAGGATAAGGTGCCAGAACCTGCCAGCCTGTCCAGCAATCACAGCCTGACCAGCTGCTTTACCAACCAGGGCTACTTCTTCTTCCATCTGCCTGACGCTCTCGAGATCGAGGCCTGCCAGGTGTACTTCACCTACGATCCCTACAGCGAGGAAGATCCCGACGAAGGCGTTGCAGGCGCTCCTACAGGATCTTCTCCACAGCCTCTGCAGCCACTGTCCGGCGAGGATGATGCCTACTGCACCTTTCCAAGCCGGGACGACCTGCTGCTGTTTAGCCCTAGTCTGCTCGGCGGACCTTCTCCACCATCTACAGCTCCAGGTGGAAGCGGAGCCGGCGAAGAAAGAATGCCTCCTAGCCTGCAAGAGCGGGTGCCCAGAGATTGGGATCCTCAACCTCTCGGACCTCCTACACCAGGCGTGCCAGACCTGGTGGATTTTCAGCCTCCTCCAGAGCTGGTGCTGAGAGAAGCTGGCGAAGAAGTGCCAGACGCCGGACCTAGAGAGGGCGTTAGCTTTCCTTGGAGCAGACCTCCTGGACAGGGCGAGTTCAGAGCCCTGAATGCTAGACTGCCCCTGAACACCGATGCCTACCTGTCTCTGCAAGAACTGCAGGGACAAGACCCCACACACCTCGTT

[0556] SEQ ID NO: 30Jun_IL2Rb_4Ala Protein Sequence

[0557] IPWLGHLLVGLSGAFGFIILVYLLIAAAANCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0558] SEQ ID NO: 31 Jun_IL2Rb_5Ala Nucleotide Sequence

[0559] ATCCCCTGGCTGGGACATCTGCTCGTTGGACTGTCTGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCGCCGCTGCCGCTGCTAACTGCCGGAACACAGGCCCTTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTCCTGCAGCAGGATAAGGTGCCAGAACCTGCCAGCCTGTCCAGCAATCACAGCCTGACCAGCTGCTTTACCAACCAGGGCTACTTCTTCTTCCATCTGCCTGACGCTCTCGAGATCGAGGCCTGCCAGGTGTACTTCACCTACGATCCCTACAGCGAGGAAGATCCCGACGAAGGCGTTGCAGGCGCTCCTACAGGATCTTCTCCACAGCCTCTGCAGCCACTGTCCGGCGAGGATGATGCCTACTGCACCTTTCCAAGCCGGGACGACCTGCTGCTGTTTAGCCCTAGTCTGCTCGGCGGACCTTCTCCACCATCTACAGCTCCAGGTGGAAGCGGAGCCGGCGAAGAAAGAATGCCTCCTAGCCTGCAAGAGCGGGTGCCCAGAGATTGGGATCCTCAACCTCTCGGACCTCCTACACCAGGCGTGCCAGACCTGGTGGATTTTCAGCCTCCTCCAGAGCTGGTGCTGAGAGAAGCTGGCGAAGAAGTGCCAGACGCCGGACCTAGAGAGGGCGTTAGCTTTCCTTGGAGCAGACCTCCTGGACAGGGCGAGTTCAGAGCCCTGAATGCTAGACTGCCCCTGAACACCGATGCCTACCTGTCTCTGCAAGAACTGCAGGGACAAGACCCCACACACCTCGTT

[0560] SEQ ID NO: 32Jun_IL2Rb_5Ala Protein Sequence

[0561] IPWLGHLLVGLSGAFGFIILVYLLIAAAAANCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0562] SEQ ID NO: 33 Jun_IL2Rb_6Ala Nucleotide Sequence

[0563] ATCCCCTGGCTGGGACATCTGCTCGTTGGACTGTCTGGCGCCTTCGGCTTCATCATCCTGGTGTACCTGCTGATCGCCGCTGCCGCTGCTGCCAACTGCCGGAACACAGGCCCTTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTCCTGCAGCAGGATAAGGTGCCAGAACCTGCCAGCCTGTCCAGCAATCACAGCCTGACCAGCTGCTTTACCAACCAGGGCTACTTCTTCTTCCATCTGCCTGACGCTCTCGAGATCGAGGCCTGCCAGGTGTACTTCACCTACGATCCCTACAGCGAGGAAGATCCCGACGAAGGCGTTGCAGGCGCTCCTACAGGATCTTCTCCACAGCCTCTGCAGCCACTGTCCGGCGAGGATGATGCCTACTGCACCTTTCCAAGCCGGGACGACCTGCTGCTGTTTAGCCCTAGTCTGCTCGGCGGACCTTCTCCACCATCTACAGCTCCAGGTGGAAGCGGAGCCGGCGAAGAAAGAATGCCTCCTAGCCTGCAAGAGCGGGTGCCCAGAGATTGGGATCCTCAACCTCTCGGACCTCCTACACCAGGCGTGCCAGACCTGGTGGATTTTCAGCCTCCTCCAGAGCTGGTGCTGAGAGAAGCTGGCGAAGAAGTGCCAGACGCCGGACCTAGAGAGGGCGTTAGCTTTCCTTGGAGCAGACCTCCTGGACAGGGCGAGTTCAGAGCCCTGAATGCTAGACTGCCCCTGAACACCGATGCCTACCTGTCTCTGCAAGAACTGCAGGGACAAGACCCCACACACCTCGTT

[0564] SEQ ID NO: 34Jun_IL2Rb_6Ala protein sequence

[0565] IPWLGHLLVGLSGAFGFIILVYLLIAAAAAANCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0566] SEQ ID NO: 35 Jun_IL18RbTM nucleotide sequence

[0567] GGCGTGCTGCTGTACATCCTGCTGGGCACAATCGGAACACTGGTGGCTGTGCTGGCTGCC

[0568] SEQ ID NO: 36 Jun_IL18RbTM protein sequence

[0569] GVLLYILLGTIGTLVAVLAA

[0570] SEQ ID NO: 37 hMyD88 nucleotide sequence

[0571] CTCGAGGCGGCTGGAGGGCCAGGAGCTGGCTCTGCCGCGCCGGTGTCTTCCACCAGTTCACTGCCTTTGGCGGCTCTTAACATGCGGGTCAGGCGAAGATTGAGTTTGTTTCTGAATGTTAGGACTCAAGTAGCTGCCGACTGGACGGCCCTTGCGGAAGAAATGGACTTCGAGTATCTaGAGATTCGCCAACTTGAAACCCAGGCaGATCCTACAGGCAGACTCCTCGATGCGTGGCAGGGTCGGCCTGGAGCATCTGTGGGAAGGTTGCTGGAACTGTTGACTAAACTTGGTCGAGATGACGTACTGCTGGAATTGGGTCCAAGCATCGAAGAAGATTGTCAAAAATACATCCTCAAGCAGCAACAAGAGGAAGCAGAGAAACCATTGCAAGTAGCGGCAGTAGACTCTAGCGTACCGCGCACTGCTGAGCTTGCTGGAATCACGACCCTTGACGATCCTCTCGGGCATATGCCAGAGCGGTTCGACGCATTCATATGTTATTGTCCAAGCGATATC

[0572] SEQ ID NO: 38 hMyD88 protein sequence

[0573] LEAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI

[0574] SEQ ID NO: 39 Jun_IL18RaTM nucleotide sequence

[0575] ATGATCATTGCCGTGCTGATCCTGGTGGCCGTCGTGTGTCTGGTCACCGTGTGCGTGATC

[0576] SEQ ID NO: 40 Jun_IL18RaTM protein sequence

[0577] MIIAVLILVAVVCLVTVCVI

[0578] SEQ ID NO: 41 Jun_CD28TM nucleotide sequence

[0579] TGCCCAAGTCCTTTGTTTCCGGGTCCTAGCAAGCCGTTCTGGGTCCTCGTTGTCGTAGGAGGAGTGCTTGCGTGCTATAGTCTCCTTGTAACGGTTGCTTTCATTATCTTTTGGGTA

[0580] SEQ ID NO: 42 Jun_CD28TM protein sequence

[0581] CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0582] SEQ ID NO: 43 hGHR nucleotide sequence

[0583]

[0584] SEQ ID NO: 44 hGHR protein sequence

[0585] KQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLDQKNQNNSPYHDACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMP

[0586] SEQ ID NO: 45 EpoRmut nucleotide sequence

[0587] CATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0588] SEQ ID NO: 46 EpoRmut protein sequence

[0589] HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0590] SEQ ID NO: 47 hEpoR EC domain protein sequence

[0591] APPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPRYHRVIHINEVVLLDAPVGLVARLADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDP

[0592] SEQ ID NO: 48 hEpoR TM domain protein sequence

[0593] LILTLSLILVVILVLLTVLALLS

[0594] SEQ ID NO: 49 hEpoR IC domain protein sequence

[0595] HRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS

[0596] SEQ ID NO: 50 Geneblock order A protein sequence

[0597] WSEPVSLLTPSDLDPLILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS

[0598] SEQ ID NO: 51 GHR EC domain protein sequence

[0599] FSGSEATAAILSRAPWSLQSVNPGLKTNSSKEPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQMSQFTCEEDFY

[0600] SEQ ID NO: 52 GHR TM domain protein sequence

[0601] FPWLLIIIFGIFGLTVMLFVFLFS

[0602] SEQ ID NO: 54 Geneblock order B protein sequence

[0603] TLPQMSQFTCEEDFYFPWLLIIIFGIFGLTVMLFVFLFSKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLDQKNQNNSPYHDACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMP

[0604] SEQ ID NO: 166 ATF-2 protein sequence

[0605] DLERNRAAASRCRQKRKVWVQSLEKKAEDLSSLNGQLQSEVTLLRNEVAQLKQLLLAHKDCPVTAMQKKSG

[0606] SEQ ID NO: 167 ATF-7 protein sequence

[0607] KRKLWVSSLEKKAEELTSQNIQLSNEVTLLRNEVAQLKQLLLAHKDCPVTALQK

[0608] SEQ ID NO: 168 CREBPA protein sequence

[0609] QRKVWVMSLEKKAEELTQTNMQLQNEVSMLKNEVAQLKQLLLTHKDCPITAMQKES

[0610] SEQ ID NO: 169 ATF-6 protein sequence

[0611] ACQSRKKKKEYMLGLEARLKAALSENEQLKKENGTLKRQLDEVVSENQRLKVPSPKRRV

[0612] SEQ ID NO:59 CREBL1 protein sequence

[0613] EYLQGLEARLQAVLADNQQLRRENAALRRRLEALLAENSELKLGSGNRK

[0614] SEQ ID NO:170 ZF protein sequence

[0615] CRLNRLKKKEYVMGLESRVRGLAAENQELRAENRELGKRVQALQEESRYLRAVLANETGL

[0616] SEQ ID NO:171 B-ATF protein sequence

[0617] QKADTLHLESEDLEKQNAALRKEIKQLTEELKYFTSVLNSHE

[0618] SEQ ID NO:172 p21SNFT protein sequence

[0619] RSRKKQTQKADKLHEEYESLEQENTMLRREIGKLTEELKHLTEALKEHEKMCPLLLCPMNFVH

[0620] SEQ ID NO:63 BAC04846 protein sequence

[0621] DKADALHQQHESLEKDNLALRKEIQSLQAELAWWSRTLHVHE

[0622] SEQ ID NO:173 C / EBPα protein sequence

[0623] QRNVETQQKVLELTSDDNDRLRKRVEQLSRELDTLRGIFRQL

[0624] SEQ ID NO:174 C / EBPβ protein sequence

[0625] SDEYKIRRERNNIAVRKSRDKAKMRNLETQHKVLELTAENERLQKKVEQLSRELSTLRNLFKQLPEPLLASSGHC

[0626] SEQ ID NO:175 C / EBPγ protein sequence

[0627] GERNNMAVKKSRLKSKQKAQDTLQRVNQLKEENERLEAKIKLLTKELSVLKDLFLEHAHNLADNVQSISTENTTADG

[0628] SEQ ID NO:176 C / EBPδ protein sequence

[0629] RRNQEMQQKLVELSAENEKLHQRVEQLTRDLAGLRQFFKQL

[0630] SEQ ID NO:177 C / EBPε protein sequence

[0631] HLEYRLRRERNNIAVRKSRDKAKRRILETQQKVLEYMAENERLRSRVEQLTQELDTLRNLFRQIPEAANLIKGV

[0632] SEQ ID NO:178 CHOP protein sequence

[0633] RMKEKEQENERKVAQLAEENERLKQEIERLTREVEATRRALIDRMVNLHQA

[0634] SEQ ID NO:179 NFE2 protein sequence

[0635] QRKLETIVQLERELERLTNERERLLRARGEADRTLEVMRQQLTELYRDIFQHLRDESGNS

[0636] SEQ ID NO:180 ATF-1 protein sequence

[0637] DPQLKREIRLMKNREAARECRRKKKEYVKCLENRVAVLENQNKTLIEELKTLKDLYSNKSV

[0638] SEQ ID NO:181 CREM-Ia protein sequence

[0639] KEEATRKRELRLMKNREAARECRRKKKEYVKCLENRVAVLENQNKTLIEELKALKDLYCHKVE

[0640] SEQ ID NO:182 CREM-Ia(K324R) protein sequence

[0641] KEEATRKRELRLMKNREAARECRRKKKEYVKCLENRVAVLENQNKTLIEELKALRDLYC

[0642] SEQ ID NO:183 CREM-Ib protein sequence

[0643] EYVKCLESRVAVLEVQNKKLIEELETLKDICS

[0644] SEQ ID NO:184 CREB protein sequence

[0645] KEAARKREVRLMKNREAARECRRKKKEYVKCLENRVAVLENQNKTLIEELKALKDLYCHKSD

[0646] SEQ ID NO:185 E4BP4 protein sequence

[0647] EKRRLNDLVLENKLIALGEENATLKAELLSLKLKFGLISSTAYAQEIQKLSNSTAVYFQDYQTSKSNV

[0648] SEQ ID NO:77 AC012616 protein sequence

[0649] LNDAAIAGRLAALMEENALLKGELKALKLRFGLLPLT

[0650] SEQ ID NO:186 cJun protein sequence

[0651] QRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQY

[0652] SEQ ID NO:187 cMaf protein sequence

[0653] QRVQQRHVLESEKNQLLQQVDHLKQEISRLVRERDAYKEKYEKLVSSGFRENGSSSDNPSSPEFFM

[0654] SEQ ID NO:188 MafB protein sequence

[0655] QYKRVQQKHHLENEKTQLIQQVEQLKQEVSRLARERDAYKVKCEKLANSGFREAGSTSDSPSSPEFFL

[0656] SEQ ID NO:81 MafA protein sequence

[0657] QQRHILESEKCQLQSQVEQLKLEVGRLAKERRDLYKEKYEKLAGRGG

[0658] SEQ ID NO:82 NRL protein sequence

[0659] QQRRGLEAERARLAAQLDALRAEVARLARERDLYKARCDRLTSSG

[0660] SEQ ID NO:189 OASIS protein sequence

[0661] EYVECLEKKVETFTSENNELWKKVETLENANRTLLQQLQKLQTLVTNKISR

[0662] SEQ ID NO:190 CREB-H protein sequence

[0663] EYIDGLETRMSACTAQNQELQRKVLHLEKQNSLSLEQLKKLQAIVVQSTS

[0664] SEQ ID NO:191 CREB3 protein sequence

[0665] WRRKIRNKRSAQESRRKKKVYVGGLESRVLKYTAQNMELQNKVQLLEEQNLSLLDQLRKLQAMVIEISNKTSS

[0666] SEQ ID NO:192 CREB4 protein sequence

[0667] WRKIRNKQSAQDSRRRKKEYIDGLESRVAACSAQNQELQKKVQELERHNISLVAQLRQLQTLIAQTSNKAAQTSTCVL

[0668] SEQ ID NO:87 AC022173 protein sequence

[0669] EYMDSLEDDVESCSTENLELRKKVEVLENTNRASHGELV

[0670] SEQ ID NO:193 DBP protein sequence

[0671] EEQKDEKYWSRRYKNNEAAKRSRDARRLKENQISVRAAFLEKENALLRQEVVAVRQELSHYRAVLSRYQAQHGAL

[0672] SEQ ID NO:194 HLF protein sequence

[0673] LKENQIAIRASFLEKENSALRQEVADLRKELGKCKNILAKYEARH

[0674] SEQ ID NO:195 TEF protein sequence

[0675] NWTRRKKNNVAAKRSRDARRLKENQITIRAAFLEKENTALRTEVAELRKEVGKCKTIVSKYETKYGPL

[0676] SEQ ID NO:196 MafG protein sequence

[0677] GVTQKEELEKQKAELQQEVEKLASENASMKLELDALRSKYEALQTFARTVARSPVAPARGPLAAGLGPLVPGKVAATSVITIVKSKTDA

[0678] SEQ ID NO:197 MafK protein sequence

[0679] TQKEELERQRVELQQEVEKLARENSSMRLELDALRSKYEALQTFARTVAR

[0680] SEQ ID NO:93 MafF protein sequence

[0681] CQKEELQKQKSELEREVDKLARENAAMRLELDALRGKCEALQGFARSVAAARG

[0682] SEQ ID NO:198 XBP-1 protein sequence

[0683] QTARDRKKARMSELEQQVVDLEEENQKLLLENQLLREKTHGLVVENQELRQRLGMDALVAEEEAEAKGNEV

[0684] SEQ ID NO:199 ARR1 protein sequence

[0685] PSQNAFRKRKLERLEELEKKEAQLTVTNDQIHILKKENELLHFMLRSLLTERNMP

[0686] SEQ ID NO:200 CIN5 protein sequence

[0687] KRAAQNRSAQKAFRQRREKYIKNLEEKSKLFDGLMKENSELKKMIESLKSKLKE

[0688] SEQ ID NO:201 YAP2 protein sequence

[0689] EDTPPNPKKEIEFPGGSPGRKRIDSEAKSRRTAQNRAAQRAFRDRKEAKMKSLQERVELLEQKDAQNKTTTDFLLCSLKSLLSEITKYRAKNSDDERILAFLDDL

[0690] SEQ ID NO:202 YAP3 protein sequence

[0691] EDTPPNPKKEIEFQAKKKAQNRAAQKAFRERKEARMKELQDKLLESERNRQSLLKEIEELRKANTEINAENRLLLRSGN

[0692] SEQ ID NO:203 YAP5 protein sequence

[0693] EDTPPNPKKEIEFPKRQNRDAQRAYRERKNNKLQVLEETIESLSKVVKNYETKLNRLQNELQAKESENHALKQKLETLTLKQASVPAQDPILQN

[0694] SEQ ID NO:204 YAP7 protein sequence

[0695] QQNRDAQRAYRERRTTRIQVLEEKVEMLHNLVDDWQRKYKLLESEFSDTKENLQKSIALNNELQKALPLIVNTPFQQQPEN

[0696] SEQ ID NO:205 GCN4 protein sequence

[0697] EDTPPNPKKEIEFHPAALKRARNTEAARRSRARKLQRMKQLEDKVEELLSKNYHLENEVARLKKLVGDAAR

[0698] SEQ ID NO:206 MET4 protein sequence

[0699] EDTPPNPKKEIEFDDDEDLLIQSKKSHQKKKLKELESSIHELTEIAASLQKRIHTLETENKLLKNLVLSSGET

[0700] SEQ ID NO:103 hEpoR™ domain nucleotide sequence

[0701] CTGATCCTGACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCT

[0702] SEQ ID NO:104 hEpor™ domain del1 protein sequence

[0703] ILTLSLILVVILVLLTVLALLS

[0704] SEQ ID NO:105 hEpoR™ domain del1 nucleotide sequence

[0705] ATCCTGACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCT

[0706] SEQ ID NO:106 hEpoR™ domain del3 protein sequence

[0707] TLSLILVVILVLLTVLALLS

[0708] SEQ ID NO:107 hEpoR™ domain del3 nucleotide sequence

[0709] ACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCT

[0710] SEQ ID NO:108 hEpoR™ domain del5 protein sequence

[0711] SLILVVILVLLTVLALLS

[0712] SEQ ID NO:109 hEpoR™ del5 nucleotide sequence

[0713] AGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCT

[0714] SEQ ID NO:111 cJunzip.hEpoRmut nucleotide sequence

[0715] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACCTGATCCTGACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0716] SEQ ID NO:112 cJunzip.hEpoRmut protein sequence

[0717] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHLILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0718] SEQ ID NO:113 Nucleotide sequence of cJunzip.hEpoRmut del1

[0719] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACATCCTGACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0720] SEQ ID NO:114 cJunzip.hEpoRmut del1 protein sequence

[0721] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHILTLLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTE DPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0722] SEQ ID NO:115 cJunzip.hEpoRmut del3 nucleotide sequence

[0723] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0724] SEQ ID NO:116 cJunzip.hEpoRmut del3 protein sequence

[0725] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0726] SEQ ID NO:117 cJunzip.hEpoRmut del5 nucleotide sequence

[0727] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCTCATAGAAGGGCCCTGAAGCAGAAGATCTGGCCTGGCATCCCATCTCCAGAGAGCGAGTTCGAGGGCCTGTTCACCACACACAAGGGCAACTTCCAGCTGTGGCTGTACCAGAACGATGGCTGCCTTTGGTGGTCCCCTTGCACACCCTTTACCGAGGATCCACCAGCCAGCCTGGAAGTGCTGAGCGAGAGATGTTGGGGCACAATGCAGGCCGTGGAACCCGGCACAGATGATGAAGGACCTCTGCTGGAACCTGTGGGCTCTGAACATGCCCAGGACACCTATCTGGTGCTGGACAAGTGGCTGCTGCCCCGGAATCCTCCATCTGAGGATTTGCCTGGACCTGGCGGCTCCGTGGATATCGTGGCTATGGATGAGGGCAGCGAGGCCAGCTCTTGTTCTTCTGCCCTGGCCAGCAAGCCTTCTCCAGAAGGCGCTTCTGCCGCCAGCTTCGAGTACACCATTCTGGACCCTAGCAGCCAGCTGCTGAGGCCT

[0728] SEQ ID NO:118 cJunzip.hEpoRmut del5 protein sequence

[0729] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHSLILVVILVLLTVLLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0730] SEQ ID NO:119 cJunzip.hGHR

[0731]

[0732] SEQ ID NO:120 cJunzip.h GHR protein sequence

[0733] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHFPWLLIIIFGIFGLTVMLFVFLFSKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLDQKNQNNSPYHDACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMP

[0734] SEQ ID NO:121 Jun_IL18RaTM_MyD88 nucleotide sequence

[0735] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACATGATCATTGCCGTGCTGATCCTGGTGGCCGTCGTGTGTCTGGTCACCGTGTGCGTGATCCTCGAGGCGGCTGGAGGGCCAGGAGCTGGCTCTGCCGCGCCGGTGTCTTCCACCAGTTCACTGCCTTTGGCGGCTCTTAACATGCGGGTCAGGCGAAGATTGAGTTTGTTTCTGAATGTTAGGACTCAAGTAGCTGCCGACTGGACGGCCCTTGCGGAAGAAATGGACTTCGAGTATCTaGAGATTCGCCAACTTGAAACCCAGGCaGATCCTACAGGCAGACTCCTCGATGCGTGGCAGGGTCGGCCTGGAGCATCTGTGGGAAGGTTGCTGGAACTGTTGACTAAACTTGGTCGAGATGACGTACTGCTGGAATTGGGTCCAAGCATCGAAGAAGATTGTCAAAAATACATCCTCAAGCAGCAACAAGAGGAAGCAGAGAAACCATTGCAAGTAGCGGCAGTAGACTCTAGCGTACCGCGCACTGCTGAGCTTGCTGGAATCACGACCCTTGACGATCCTCTCGGGCATATGCCAGAGCGGTTCGACGCATTCATATGTTATTGTCCAAGCGATATC

[0736] SEQ ID NO:122 Jun_IL18RaTM_MyD88 protein sequence

[0737] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHMIIAVLILVAVVCLVTVCVILEAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI

[0738] SEQ ID NO:123 Nucleotide sequence of Jun_IL18RbTM_MyD88

[0739] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACGGCGTGCTGCTGTACATCCTGCTGGGCACAATCGGAACACTGGTGGCTGTGCTGGCTGCCCTCGAGGCGGCTGGAGGGCCAGGAGCTGGCTCTGCCGCGCCGGTGTCTTCCACCAGTTCACTGCCTTTGGCGGCTCTTAACATGCGGGTCAGGCGAAGATTGAGTTTGTTTCTGAATGTTAGGACTCAAGTAGCTGCCGACTGGACGGCCCTTGCGGAAGAAATGGACTTCGAGTATCTaGAGATTCGCCAACTTGAAACCCAGGCaGATCCTACAGGCAGACTCCTCGATGCGTGGCAGGGTCGGCCTGGAGCATCTGTGGGAAGGTTGCTGGAACTGTTGACTAAACTTGGTCGAGATGACGTACTGCTGGAATTGGGTCCAAGCATCGAAGAAGATTGTCAAAAATACATCCTCAAGCAGCAACAAGAGGAAGCAGAGAAACCATTGCAAGTAGCGGCAGTAGACTCTAGCGTACCGCGCACTGCTGAGCTTGCTGGAATCACGACCCTTGACGATCCTCTCGGGCATATGCCAGAGCGGTTCGACGCATTCATATGTTATTGTCCAAGCGATATC

[0740] SEQ ID NO:124 Jun_IL18RbTM_MyD88 protein sequence

[0741] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHGVLLYILLGTIGTLVAVLAALEAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI

[0742] SEQ ID NO:125 Nucleotide sequence of Jun_CD28TM_MyD88

[0743] ATCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCGCCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACTGCCCAAGTCCTTTGTTTCCGGGTCCTAGCAAGCCGTTCTGGGTCCTCGTTGTCGTAGGAGGAGTGCTTGCGTGCTATAGTCTCCTTGTAACGGTTGCTTTCATTATCTTTTGGGTACTCGAGGCGGCTGGAGGGCCAGGAGCTGGCTCTGCCGCGCCGGTGTCTTCCACCAGTTCACTGCCTTTGGCGGCTCTTAACATGCGGGTCAGGCGAAGATTGAGTTTGTTTCTGAATGTTAGGACTCAAGTAGCTGCCGACTGGACGGCCCTTGCGGAAGAAATGGACTTCGAGTATCTaGAGATTCGCCAACTTGAAACCCAGGCaGATCCTACAGGCAGACTCCTCGATGCGTGGCAGGGTCGGCCTGGAGCATCTGTGGGAAGGTTGCTGGAACTGTTGACTAAACTTGGTCGAGATGACGTACTGCTGGAATTGGGTCCAAGCATCGAAGAAGATTGTCAAAAATACATCCTCAAGCAGCAACAAGAGGAAGCAGAGAAACCATTGCAAGTAGCGGCAGTAGACTCTAGCGTACCGCGCACTGCTGAGCTTGCTGGAATCACGACCCTTGACGATCCTCTCGGGCATATGCCAGAGCGGTTCGACGCATTCATATGTTATTGTCCAAGCGATATC

[0744] SEQ ID NO:126 Jun_CD28TM_MyD88 protein sequence

[0745] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVLEAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAAD WTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI

[0746] SEQ ID NO:127 hEpoR™ domain del2 nucleotide sequence

[0747] CTGACACTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCT

[0748] SEQ ID NO:128 hEpoR™ domain del2 protein sequence

[0749] LTLSLILVVILVLLTVLALLS

[0750] SEQ ID NO:129 hEpoR™ domain del4 nucleotide sequence

[0751] CTGAGCCTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCT

[0752] SEQ ID NO:130 hEpoR™ domain del4 protein sequence

[0753] LSLILVVILVLLTVLALLS

[0754] SEQ ID NO:131 hEpoR™ domain del6 nucleotide sequence

[0755] CTGATCCTGGTGGTCATCCTGGTGCTGCTGACAGTGCTGGCCCTGCTGTCT

[0756] SEQ ID NO:132 hEpoR™ domain del6 protein sequence

[0757] LILVVILVLLTVLALLS

[0758] SEQ ID NO:133 GHR™ domain nucleotide sequence

[0759] TTCCCCTGGCTGCTGATCATCATCTTCGGCATCTTTGGCCTGACCGTGATGCTGTTCGTGTTCCTGTTCAGC

[0760] SEQ ID NO:134 GHR™ del1 protein sequence

[0761] PWLLIIIFGIFGLTVMLFVFLFS

[0762] SEQ ID NO:135 GHR™ del1 nucleotide sequence

[0763] CCCTGGCTGCTGATCATCATCTTCGGCATCTTTGGCCTGACCGTGATGCTGTTCGTGTTCCTGTTCAGC

[0764] SEQ ID NO:136 GHR™ del2 protein sequence

[0765] WLLIIIFGIFGLTVMLFVFLFS

[0766] SEQ ID NO:137 GHR™ del2 nucleotide sequence

[0767] TGGCTGCTGATCATCATCTTCGGCATCTTTGGCCTGACCGTGATGCTGTTCGTGTTCCTGTTCAGC

[0768] SEQ ID NO:138 GHR™ del3 protein sequence

[0769] LLIIIFGIFGLTVMLFVFLFS

[0770] SEQ ID NO:139 GHR™ del3 nucleotide sequence

[0771] CTGCTGATCATCATCTTCGGCATCTTTGGCCTGACCGTGATGCTGTTCGTGTTCCTGTTCAGC

[0772] SEQ ID NO:140 GHR™ del4 protein sequence

[0773] LIIIFGIFGLTVMLFVFLFS

[0774] SEQ ID NO:141 GHR™ del4 nucleotide sequence

[0775] CTGATCATCATCTTCGGCATCTTTGGCCTGACCGTGATGCTGTTCGTGTTCCTGTTCAGC

[0776] SEQ ID NO:142 GHR™ del5 protein sequence

[0777] IIIFGIFGLTVMLFVFLFS

[0778] SEQ ID NO:143 GHR™ del5 nucleotide sequence

[0779] ATCATCATCTTCGGCATCTTTGGCCTGACCGTGATGCTGTTCGTGTTCCTGTTCAGC

[0780] SEQ ID NO:144 GHR™ del6 protein sequence

[0781] IIFGIFGLTVMLFVFLFS

[0782] SEQ ID NO:145 GHR™ del6 nucleotide sequence

[0783] ATCATCTTCGGCATCTTTGGCCTGACCGTGATGCTGTTCGTGTTCCTGTTCAGC

[0784] SEQ ID NO:146 Jun_IL18RbTM_MyD88_GHR protein sequence

[0785] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHGVLLYILLGTIGTLVAVLAALEAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLDQKNQNNSPYHDACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMP

[0786] SEQ ID NO:147 Nucleotide sequence of Jun_IL18RbTM_MyD88_GHR

[0787]

[0788] SEQ ID NO:148 Jun_IL18RbTM_MyD88_EpoR protein sequence

[0789] IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHGVLLYILLGTIGTLVAVLAALEAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPEGASAASFEYTILDPSSQLLRP

[0790] SEQ ID NO:149 Jun_IL18RbTM_MyD88_EpoR nucleotide sequence

[0791]

[0792] SEQ ID NO:150 adapter protein sequence

[0793] (GGGGS)n

[0794] SEQ ID NO:151 adapter protein sequence

[0795] GGGGSGGGGSGGGGS

[0796] SEQ ID NO:152 Linker nucleotide sequence

[0797] GGTGGCGGAGGTTCTGGCGGAGGCGGCTCTGGTGGTGGTGGATCT

[0798] SEQ ID NO:153 Linker nucleotide sequence

[0799] GGTGGCGGAGGTTCTGGCGGAGGCGGAAGTGGTGGTGGCGGATCT

[0800] SEQ ID NO:154 hEpoR IC domain nucleotide sequence

[0801] CATCGCCGCGCGCTGAAACAGAAAATTTGGCCGGGCATTCCGAGCCCGGAAAGCGAATTTGAAGGCCTGTTTACCACCCATAAAGGCAACTTTCAGCTGTGGCTGTATCAGAACGATGGCTGCCTGTGGTGGAGCCCGTGCACCCCGTTTACCGAAGATCCGCCGGCGAGCCTGGAAGTGCTGAGCGAACGCTGCTGGGGCACCATGCAGGCGGTGGAACCGGGCACCGATGATGAAGGCCCGCTGCTGGAACCGGTGGGCAGCGAACATGCGCAGGATACCTATCTGGTGCTGGATAAATGGCTGCTGCCGCGCAACCCGCCGAGCGAAGATCTGCCGGGCCCGGGCGGCAGCGTGGATATTGTGGCGATGGATGAAGGCAGCGAAGCGAGCAGCTGCAGCAGCGCGCTGGCGAGCAAACCGAGCCCGGAAGGCGCGAGCGCGGCGAGCTTTGAATATACCATTCTGGATCCGAGCAGCCAGCTGCTGCGCCCGTGGACCCTGTGCCCGGAACTGCCGCCGACCCCGCCGCATCTGAAATATCTGTATCTGGTGGTGAGCGATAGCGGCATTAGCACCGATTATAGCAGCGGCGATAGCCAGGGCGCGCAGGGCGGCCTGAGCGATGGCCCGTATAGCAACCCGTATGAAAACAGCCTGATTCCGGCGGCGGAACCGCTGCCGCCGAGCTATGTGGCGTGCAGC

[0802] SEQ ID NO:155 T2A sequence protein sequence

[0803] AEGRGSLLTCGDVEENPGP

[0804] SEQ ID NO:156 T2A sequence protein sequence

[0805] EGRGSLLTCGDVEENPGP

[0806] SEQ ID NO:157 T2A sequence protein sequence

[0807] GSGEGRGSLLTCGDVEENPGP

[0808] SEQ ID NO:158 F2A sequence protein sequence

[0809] GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP

[0810] SEQ ID NO:159 Great Barrier Reef Sponge 2A Sequence Protein Sequence

[0811] LLCFLLLLLSGDVELNPGP

[0812] SEQ ID NO:160 Great Barrier Reef Sponge 2A Sequence Protein Sequence

[0813] HHFMFLLLLLAGDIELNPGP

[0814] SEQ ID NO:161 Acorn 2A sequence protein sequence

[0815] WFLVLLSFILSGDIEVNPGP

[0816] SEQ ID NO:162 Amphioxus (Florida amphioxus) 2A sequence protein sequence

[0817] KNCAMYMLLLSGDVETNPGP

[0818] SEQ ID NO:163 Amphioxus (Florida amphioxus) 2A sequence protein sequence

[0819] MVISQLMLKLAGDVEENPGP

[0820] SEQ ID NO:164 Equine rhinitis A virus 2A sequence protein sequence

[0821] GSGQCTNYALLKLAGDVESNPGP

[0822] SEQ ID NO:165 2A common sequence protein sequence

[0823] DXEX-NPGP, where X is any amino acid

[0824] SEQ ID NO:219 MyD88 intracellular domain protein sequence:

[0825] GCGGCTGGAGGGCCAGGAGCTGGCTCTGCCGCGCCGGTGTCTTCCACCAGTTCACTGCCTTTGGCGGCTCTTAACATGCGGGTCAGGCGAAGATTGAGTTTGTTTCTGAATGTTAGGACTCAAGTAGCTGCCGACTGGACGGCCCTTGCGGAAGAAATGGACTTCGAGTATCTaGAGATTCGCCAACTTGAAACCCAGGCaGATCCTACAGGCAGACTCCTCGATGCGTGGCAGGGTCGGCCTGGAGCATCTGTGGGAAGGTTGCTGGAACTGTTGACTAAACTTGGTCGAGATGACGTACTGCTGGAATTGGGTCCAAGCATCGAAGAAGATTGTCAAAAATACATCCTCAAGCAGCAACAAGAGGAAGCAGAGAAACCATTGCAAGTAGCGGCAGTAGACTCTAGCGTACCGCGCACTGCTGAGCTTGCTGGAATCACGACCCTTGACGATCCTCTCGGGCATATGCCAGAGCGGTTCGACGCATTCATATGTTATTGTCCAAGCGATATC

[0826] SEQ ID NO:220 Nucleotide sequence of the MyD88 intracellular domain:

[0827] AAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI

[0828] SEQ ID NO:221 Protein sequences of Jun, GHR, and MyD88

[0829] MDWIWRILFLVGAATGAHSDYKDDDDKIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHFPWLLIIIFGIFGLTVMLFVFLFSKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTEESDTDRLLSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLDQKNQNNSPYHDACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMPAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI

[0830] SEQ ID NO:222 Jun.GHR.MyD88 nucleotide sequence

[0831]

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