Chimeric receptor

Abstract

This application relates to chimeric receptors, particularly chimeric receptors comprising homodimeric polypeptides. The use of these chimeric receptors in tumor immunotherapy (e.g., adoptive cell therapy) is also disclosed. This application further relates to a method for genetically modifying therapeutic immune cells to enhance the immune response to a target antigen. This application also relates to therapeutic cells expressing the chimeric receptor, and a method for treating a patient using the modified therapeutic cells.

Description

Cross-reference of related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 497,140, ​​filed on 19 April 2023, the disclosures thereof being incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy created on April 19, 2024, is filenamed 243734_000199_SL.xml and has a size of 235,430 bytes. Government support

[0003] Statement on federally funded research and development This invention was made with government support under CA250401 granted by the National Institutes of Health. The government has certain rights to this invention. [Technical Field]

[0004] This application relates to chimeric receptors, and more specifically, to chimeric receptors comprising a homodimer polypeptide 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 a method for genetically modifying therapeutic immune cells to enhance the immune response to a target antigen. This application further relates to therapeutic cells expressing chimeric receptors, and a method for treating a patient using the modified therapeutic cells. [Background technology]

[0005] Immunotherapy using immune cells, including T cells expressing chimeric antigen receptors (CARs), has already revolutionized the treatment approach for patients with hematological malignancies. However, immune cells such as CAR T cells are not very effective against solid tumors and brain tumors. While the lack of effectiveness is most likely multifactorial, the main obstacles have emerged as limited persistence without stimulation and inability to function in situations of chronic antigen exposure.

[0006] To enhance the ability of CAR T cells to maintain viability without antigen exposure and to function under chronic antigen exposure, gamma (γ) cytokines such as IL-2, IL-7, IL-15, and IL-21, or members of other cytokine families (e.g., IL-18), were overexpressed in CAR T cells. While cytokine-expressing CAR T cells showed improved antitumor activity in preclinical models, there are safety concerns because these secreted or membrane-bound cytokines may activate bystander immune cells. Therefore, the development of chimeric receptors that can signal genetically modified immune cells is needed. [Overview of the project] [Means for solving the problem]

[0007] This specification discloses chimeric receptors, particularly chimeric receptors comprising a homodimer polypeptide containing at least one homodimerizing motif. It also discloses the use of chimeric receptors in tumor immunotherapy (e.g., adoptive cell therapy). Furthermore, it discloses methods for genetically modifying therapeutic immune cells to enhance the immune response to a target antigen. It also discloses therapeutic cells expressing chimeric receptors, and methods for treating patients using the modified therapeutic cells.

[0008] In one embodiment, a chimeric receptor which is a homodimer comprising two polypeptide monomers, wherein each polypeptide monomer is i) Extracellular region containing at least one homodimerizing motif; ii) Transmembrane region; and iii) An intracellular region comprising at least one signaling region derived from a cell surface receptor or signaling adapter protein, or a portion or variant thereof. This provides a chimeric receptor that includes [the specified component].

[0009] In some embodiments, the extracellular region 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.

[0010] In some embodiments, the extracellular region includes at least one homodimerized leucine zipper motif. In some embodiments, the homodimerized leucine zipper motif includes at least five heptad repeats of amino acids having leucine every 7 positions. In some embodiments, the homodimerized leucine zipper motif is derived from the transcription factor c-Jun. In some embodiments, the homodimerized leucine zipper motif includes the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the homodimerized leucine zipper motif is encoded by the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 3.

[0011] In some embodiments, the ligand-dependent homodimerizing motif undergoes homodimerization in the presence of a chemical inducer. In some embodiments, the ligand-dependent homodimerizing motif is FKBP12 F36V Includes the domain.

[0012] In some embodiments, signaling regions derived from cell surface receptors or signaling adapter proteins, or portions thereof or variants, activate the Janus kinase (JAK) signaling transducer and activator of transcription (STAT) pathway, the kappa-light-chain-enhancer of activated B cell (NFκB) pathway, and / or further signaling pathways activated by the MyD88 signaling complex (Myddosome).

[0013] In some embodiments, the further signaling pathways activated by the MyD88 signaling complex (Myddosome) are one or more of the following pathways: interleukin-1 receptor-associated kinase (IRAK), TNF receptor-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).

[0014] In some embodiments, the intracellular region includes a first signaling region derived from a cell surface receptor, or a portion or variant thereof, and a second signaling region derived from a signaling adapter protein, or a portion or variant thereof.

[0015] In some embodiments, the signaling region is derived from a cytokine receptor, or a portion or variant thereof. In some embodiments, the cytokine receptor, or a portion or variant thereof, is 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 β subunit (IL-10R2), IL-6β chain (gp130), IL-2Rβ, IL-18Rα, or IL-18Rβ.

[0016] In some embodiments, the signaling region is derived from EpoR, or a portion or variant thereof. In some embodiments, the signaling region derived from EpoR does not include a Src homology region 2 domain-containing phosphatase-1 (SHP1) binding site. In some embodiments, the signaling region derived from EpoR includes the amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from EpoR is encoded by the nucleotide sequence of SEQ ID NO: 45, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from EpoR includes the amino acid sequence of SEQ ID NO: 49, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from EpoR is encoded by the nucleotide sequence of SEQ ID NO: 154, or a sequence having at least 80% identity thereto.

[0017] In some embodiments, the signaling region is derived from the GHR, or a portion or variant thereof. In some embodiments, the signaling region derived from the GHR includes the amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from the GHR is encoded by the nucleotide sequence of SEQ ID NO: 43, or a sequence having at least 80% identity thereto.

[0018] In some embodiments, the signaling region is derived from a receptor tyrosine kinase, or a portion or variant thereof. In some embodiments, the receptor tyrosine kinase is the epidermal growth factor receptor (EGFR).

[0019] In some embodiments, the signaling region is derived from a Toll-like receptor, or a portion or variant thereof. In some embodiments, the Toll-like receptor is TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10.

[0020] In some embodiments, the signaling region is derived from a signal transduction adapter protein, or a portion or variant thereof. In some embodiments, the signal transduction adapter protein is MyD88. In some embodiments, the signaling region derived from MyD88 includes the amino acid sequence of SEQ ID NO: 38 or 220, or a sequence having at least 80% identity thereto. In some embodiments, the signaling 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 thereto.

[0021] In some embodiments, the transmembrane region is derived from the same cell surface receptor as the signaling region. In some embodiments, the transmembrane region is derived from EpoR, or a portion or variant thereof. In some embodiments, the transmembrane region derived from EpoR includes 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 thereto. 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 thereto.

[0022] In some embodiments, the transmembrane region is derived from GHR, or a portion or variant thereof. In some embodiments, the transmembrane region derived from GHR includes 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 thereto. 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 thereto.

[0023] In some embodiments, the transmembrane region originates from IL-18Rα, IL-18Rβ, CD28, IL-2Rβ, CD8, CD4, CD3ζ, CD40, CD134(OX-40), CD19, or CD7. In some embodiments, the transmembrane region originates from IL-18Rα, or a portion or variant thereof.

[0024] In some embodiments, the transmembrane region derived from IL-18Rα includes the amino acid sequence of SEQ ID NO: 40, or a sequence having at least 80% identity thereto. 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 thereto.

[0025] In some embodiments, the transmembrane region is derived from IL-18Rβ, or a portion or variant thereof. According to some embodiments, the transmembrane region derived from IL-18Rβ includes the amino acid sequence of SEQ ID NO: 36, or a sequence having at least 80% identity thereto. 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 thereto.

[0026] In some embodiments, the transmembrane region is derived from CD28, or a portion or variant thereof. In some embodiments, the transmembrane region derived from CD28 includes the amino acid sequence of SEQ ID NO: 42, or a sequence having at least 80% identity thereto. 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 thereto.

[0027] In some embodiments, each polypeptide monomer of the chimeric receptor is i) Extracellular region containing a homodimerized leucine zipper motif; ii) Transmembrane regions derived from EpoR, or a portion or variant thereof; and iii) Intracellular regions containing signal transduction regions derived from EpoR, or a portion thereof or a variant thereof. Includes.

[0028] In some embodiments, the signaling region derived from EpoR does not include a Src homologous domain 2-domain-containing phosphatase-1 (SHP1) binding site. In some embodiments, the signaling region derived from EpoR includes the amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from EpoR is encoded by the nucleotide sequence of SEQ ID NO: 45, or a sequence having at least 80% identity thereto. In some embodiments, the transmembrane region derived from EpoR includes 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 thereto. 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 thereto. In some embodiments, each polypeptide monomer includes the amino acid sequence of SEQ ID NO: 112, 114, 116, or 118, or a sequence having at least 80% identity thereto. 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 thereto.

[0029] In some embodiments, each polypeptide monomer of the chimeric receptor is i) Extracellular region containing a homodimerized leucine zipper motif; ii) Transmembrane regions derived from GHR, or a portion or variant thereof; and iii) Intracellular regions containing signal transduction regions derived from GHR, or a portion thereof or a variant thereof. Includes.

[0030] In some embodiments, the signaling region derived from GHR includes the amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from GHR is encoded by the nucleotide sequence of SEQ ID NO: 43, or a sequence having at least 80% identity thereto. In some embodiments, the transmembrane region derived from GHR includes 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 thereto. 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 includes the amino acid sequence of SEQ ID NO: 120, or a sequence having at least 80% identity thereto. In some embodiments, each polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 119, or a sequence having at least 80% identity thereto.

[0031] In some embodiments, each polypeptide monomer of the chimeric receptor is i) Extracellular region containing a homodimerized leucine zipper motif; ii) Transmembrane regions derived from IL-18Rα, IL-18Rβ, or CD28, or a portion or variant thereof; and iii) Intracellular regions containing signal transduction regions derived from MyD88, or a portion thereof or a variant thereof. Includes.

[0032] In some embodiments, the signaling region derived from MyD88 includes the amino acid sequence of SEQ ID NO: 38 or 220, or a sequence having at least 80% identity thereto. In some embodiments, the signaling 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 thereto. In some embodiments, the transmembrane region includes 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 thereto. 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 thereto. In some embodiments, each polypeptide monomer includes the amino acid sequence of SEQ ID NO: 122, 124, or 126, or a sequence having at least 80% identity thereto. 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 thereto.

[0033] In some embodiments, each polypeptide monomer of the chimeric receptor is i) Extracellular region containing a homodimerized leucine zipper motif; ii) Transmembrane regions derived from GHR, or a portion or variant thereof; and iii) An intracellular region comprising a signaling region derived from GHR, or a portion or variant thereof, and a signaling region derived from MyD88, or a portion or variant thereof. Includes.

[0034] In some embodiments, the signaling region derived from GHR includes the amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from GHR is encoded by the nucleotide sequence of SEQ ID NO: 43, or a sequence having at least 80% identity thereto. In some embodiments, the signaling region derived from MyD88 includes the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 220, or a sequence having at least 80% identity thereto. In some embodiments, the signaling 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 thereto. In some embodiments, the transmembrane region derived from GHR includes 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 thereto. 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 contains amino acids 28-611 of the amino acid sequence of SEQ ID NO: 221, or a sequence having at least 80% identity thereto. 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 thereto.

[0035] 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 of SEQ ID NO: 7.

[0036] 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 signaling region. In some embodiments, the hinge region is derived from IgG1, IgG2, IgG3, IgG4, CD28, or CD8α.

[0037] In some embodiments, the polypeptide monomer further comprises one or more additional polypeptide sequences. In some embodiments, one or more additional polypeptide sequences are selected from one or more cell markers, epitope tags, cytokines, safety switches, dimerization moieties, and degradation moieties.

[0038] In another embodiment, provided herein are polynucleotides encoding the chimeric receptor described herein. In some embodiments, the nucleotide sequence is expressed in an inducible manner achieved using an inducible promoter, an inducible expression system, an artificial signaling circuit, and / or drug-induced splicing. In some embodiments, the nucleotide sequence is operably ligated 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.

[0039] 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 operably ligated to one or more additional nucleotide sequences encoding one or more additional polypeptide sequences via a self-cleaving peptide and / or a sequence encoding an internal ribosomal 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 of SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto.

[0040] In some embodiments, the polynucleotides described herein are DNA molecules.

[0041] In some embodiments, the polynucleotides described herein are RNA molecules.

[0042] In another embodiment, this specification provides recombinant vectors comprising polynucleotides as 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 vacciniavirus vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the vector is a nonviral vector. In some embodiments, the nonviral vector is a minicircle plasmid, a Sleeping Beauty transposon, a piggyBac transposon, or a single-stranded or double-stranded DNA molecule used as a template for gene editing based on homologous recombination repair (HDR).

[0043] In another embodiment, isolated host cells comprising the polynucleotide or recombinant vector described herein are provided herein. In yet another embodiment, isolated host cells comprising the chimeric receptor described herein are provided herein.

[0044] In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell, a natural killer (NK) cell, a mesenchymal stem cell (MSC), or a 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, a γδ T cell, a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an invariant natural killer T (iNKT) cell, a memory T cell, or a stem cell-like memory T cell (iNKT). SCMThese are naive T cells, effector T cells, helper T cells, or regulatory T cells (Treg). In some embodiments, the host cells are NK cells. In some embodiments, the host cells are NK cells derived from peripheral blood, umbilical cord blood, induced pluripotent stem (iPS) cells (iPSCs), and / or cell lines (e.g., NK92 cells).

[0045] In some embodiments, the host cell further expresses one or more antigen-recognizing molecules. In some embodiments, one or more antigen-recognizing 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, native or transgenic T cell receptors, and antibodies, or combinations thereof.

[0046] In some embodiments, host cells are further genetically modified to enhance their function by expressing one or more additional genes using gene editing techniques (e.g., CRISPR-Cas9 or transcription activator-like effector nucleases: TALENs) or by deleting one or more inhibitory 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 inhibitory genes are REGNASE-1 or DNMT3A. In some embodiments, the gene editing technique is CRISPR-Cas9 or transcription activator-like effector nucleases (TALENs).

[0047] In some embodiments, host cells are activated and / or enlarged ex vivo.

[0048] In some embodiments, the host cells are allogeneic cells. In some embodiments, the host cells are autologous.

[0049] In some embodiments, immune cells are derived from induced pluripotent stem (iPS) cells (iPSCs).

[0050] In another embodiment, the Specified herein provides a pharmaceutical composition comprising a host cell as described herein and a pharmaceutically acceptable carrier and / or excipient.

[0051] In another embodiment, methods for enhancing the effector function of immune cells are provided herein, comprising the immune cells expressing one or more antigen-recognizing molecules and the cells being genetically modified with polynucleotides or recombinant vectors as described herein. In some embodiments, one or more antigen-recognizing 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, innate or transgenic T cell receptors, and antibodies, or combinations thereof. In some embodiments, the effector function is one or more of expansion, persistence, and / or cytotoxicity (e.g., antitumor activity).

[0052] In another embodiment, a method for producing isolated host cells as described herein is provided herein, comprising genetically modifying the host cells with polynucleotides or recombinant vectors as described herein. In some embodiments, the method further comprises genetically modifying the host cells to express one or more antigen-recognizing molecules. In some embodiments, the one or more antigen-recognizing 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, innate or transgenic T cell receptors, and antibodies, or combinations thereof. In some embodiments, the genetic modification step is carried out via viral gene delivery. In some embodiments, the genetic modification step is carried out via non-viral gene delivery. In some embodiments, the genetic modification step is carried out ex vivo. In some embodiments, the method further comprises activating and / or expanding the host cells ex vivo before, after, and / or during the genetic modification.

[0053] In another embodiment, a method for treating a disease is provided herein, comprising administering an effective amount of a host cell or a pharmaceutical composition described herein to a target. In some embodiments, the method is: a) Isolating T cells or NK cells from the subject or donor; b) Ex vivo modification of T cells or NK cells with the polynucleotides or recombinant vectors described herein; c) Optionally, modifying T cells or NK cells ex vivo to express one or more antigen-recognizing molecules that bind to disease-related antigens; d) optionally, before, after, and / or in between steps b) or c); and e) Introducing a therapeutically effective dose of modified T cells or NK cells into the target cells. Includes.

[0054] In some embodiments, one or more antigen-recognizing 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, native or transgenic T cell receptors, and antibodies, or combinations thereof.

[0055] In some embodiments, the disease is cancer, an infection, or an autoimmune disease.

[0056] In some embodiments, the subject is human.

[0057] In another embodiment, a chimeric receptor is a homodimer comprising two polypeptide monomers, where each polypeptide monomer is i) An extracellular region comprising means for homodimerizing two polypeptide monomers; ii) A transmembrane region including means for joining an intracellular region with an extracellular region; and iii) Intracellular regions including means for enhancing the effector function of immune cells Chimeric receptors, including those mentioned herein, are provided herein.

[0058] In some embodiments, means for homodimerizing two polypeptide monomers include at least one homodimerizable leucine zipper motif, at least one ligand-dependent homodimerizable motif, or at least one single-chain variable region fragment, or a combination thereof. In some embodiments, means for homodimerizing two polypeptide monomers include at least one homodimerizable leucine zipper motif.

[0059] In some embodiments, the homodimerized leucine zipper motif comprises at least five heptad repeats of amino acids having leucine at every 7th position. In some embodiments, the homodimerized leucine zipper motif is derived from the transcription factor c-Jun. In some embodiments, the homodimerized leucine zipper motif comprises the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 80% identity thereto. In some embodiments, the homodimerized leucine zipper motif is encoded by the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 80% identity thereto.

[0060] In some embodiments, ligand-dependent homodimerization motifs undergo homodimerization in the presence of a chemical inducer.

[0061] In some embodiments, means for enhancing effector function include at least one signaling region derived from a cell surface receptor or signaling adapter protein, or a portion or variant thereof. In some embodiments, means for enhancing effector function include a signaling region derived from EpoR, or a portion or variant thereof. In some embodiments, means for enhancing effector function include a signaling region derived from GHR, or a portion or variant thereof. In some embodiments, means for enhancing effector function include a signaling region derived from MyD88, or a portion or variant thereof. In some embodiments, means for enhancing effector function include a signaling region derived from GHR, or a portion or variant thereof, and a signaling region derived from MyD88, or a portion or variant thereof.

[0062] In another embodiment, a chimeric receptor is a homodimer comprising two polypeptide monomers, where each polypeptide monomer is i) An extracellular region comprising means for homodimerizing two polypeptide monomers; ii) Transmembrane region; and iii) An intracellular region comprising at least one signaling region derived from a cell surface receptor or signaling adapter protein, or a portion or variant thereof. Chimeric receptors, including those mentioned herein, are provided herein.

[0063] In another embodiment, a chimeric receptor is a homodimer comprising two polypeptide monomers, where each polypeptide monomer is i) Extracellular region containing at least one homodimerized motif; ii) Transmembrane region; and iii) Intracellular regions including means for enhancing the effector function of immune cells Chimeric receptors, including those mentioned herein, are provided herein.

[0064] In another embodiment, a chimeric receptor is provided herein comprising i) an extracellular domain; ii) a transmembrane domain; and iii) an intracellular domain comprising at least one signaling domain derived from a cell surface receptor or signaling adapter protein, or a portion or variant thereof, the improvement comprising the extracellular domain comprising at least one homodimerized motif.

[0065] In another embodiment, a chimeric receptor is provided herein, the improvement comprising each of two transmembrane polypeptide monomers comprising i) an extracellular region comprising at least one homodimerization motif, and ii) an intracellular region comprising at least one signaling region derived from a cell surface receptor or signaling adapter protein, or a portion or variant thereof.

[0066] In some embodiments, the improvement includes the extracellular region comprising at least one homodimerized leucine zipper motif, at least one ligand-dependent homodimerized motif, or at least one single-chain variable region fragment, or a combination thereof. In some embodiments, the improvement includes the extracellular region comprising at least one homodimerized leucine zipper motif. In some embodiments, the improvement includes the homodimerized leucine zipper motif comprising at least five heptad repeats of amino acids having leucine at every 7 position. In some embodiments, the improvement includes the homodimerized leucine zipper motif being derived from the transcription factor c-Jun. In some embodiments, the improvement includes the homodimerized leucine zipper motif comprising the amino acid sequence of SEQ ID NO: 9 or a sequence having at least 80% identity thereto. In some embodiments, the improvement includes the homodimerized leucine zipper motif being encoded by the nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 80% identity thereto. In some embodiments, the improvement includes the ligand-dependent homodimerization motif undergoing homodimerization in the presence of a chemical inducer. In some embodiments, the improvement further includes the intracellular region comprising a signaling region derived from EpoR, or a portion or variant thereof. In some embodiments, the improvement further includes the intracellular region comprising a signaling region derived from GHR, or a portion or variant thereof. In some embodiments, the improvement further includes the intracellular region comprising a signaling region derived from MyD88, or a portion or variant thereof. In some embodiments, the improvement further includes the intracellular region comprising a signaling region derived from GHR, or a portion or variant thereof, and a signaling region derived from MyD88, or a portion or variant thereof.

[0067] In another embodiment, polynucleotides encoding the chimeric receptor described herein are provided herein.

[0068] In another embodiment, recombinant vectors comprising the polynucleotides described herein are provided herein.

[0069] In another embodiment, immune cells expressing the chimeric receptor described herein are provided herein.

[0070] In another embodiment, immune cells comprising the polynucleotides or recombinant vectors described herein are provided herein.

[0071] In another embodiment, a method for enhancing the effector function of immune cells is provided herein, the improvement comprising the immune cells expressing the chimeric receptor described herein.

[0072] In another embodiment, the present invention provides a method for improving the expansion, persistence, or cytotoxicity of antigen-activated immune cells, wherein the improvement comprises the immune cells expressing the chimeric receptor described herein.

[0073] In another embodiment, the present invention provides a method for treating a disease, comprising administering an effective amount of immune cells to a target, wherein improvement comprises the immune cells expressing a chimeric receptor as described herein. [Brief explanation of the drawing]

[0074] [Figure 1A] Figures 1A–1D show that Jun.MyD88 homodimers activate NFκB signaling and improve the antitumor activity of CAR T cells. NFκB activity in Ramos Blue NFκB reporter cells transduced with the shown construct (N=3 technical replicates, paired ANOVA, *p<0.05). [Figure 1B] NFκB activity in Ramos Blue NFκB reporter cells transduced with the indicated construct (technical replicates of N=5, paired ANOVA, ****p<0.0001). [Figure 1C] Transduction efficiency in human T cells (N=1). [Figure 1D] Repeated stimulation assay using A673 tumor cells and EphA2-CAR T cells (N=1). [Figure 2A] Figures 2A–2F show that Jun leucine zipper-based homodimers activate STAT5, leading to improved T cell viability, and that these are inhibited by ruxolitinib. Geometric mean fluorescence intensity of pSTAT5 in JunZipR+ cells (biological replicates of N=4, one-way ANOVA, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 2B] Positive percentage of pSTAT5 in JunZipR+ cells (biological replicates of N=4, one-way ANOVA, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 2C] Frequency of pSTAT5+ cells after 48 hours of in vitro treatment with ruxolitinib at the prescribed concentration (biological replicates of N=3). [Figure 2D] Frequency of pSTAT5+ cells after 48 hours of in vitro treatment with ruxolitinib at the prescribed concentration (biological replicates of N=3). [Figure 2E] Frequency of pSTAT5+ cells after 48 hours of in vitro treatment with ruxolitinib at the prescribed concentration (biological replicates of N=3). [Figure 2F] Frequency of dead cells (Annexin V+ Dead+), apoptotic cells (Annexin V+ Dead-), necrotic cells (Annexin V- Dead+), and viable cells (Annexin V- Dead-) 7 days after cytokine starvation with or without treatment using 5 μM ruxolitinib in vitro (N=2 biological replicates, two-way ANOVA, p<0.0001 in the viable cell population). [Figure 3A]Figures 3A-3C show that the Jun homodimer ZipR does not alter the immunophenotype of human T cells. Transduction efficiency in human T cells (N=4, biological repeat). [Figure 3B] Frequency of CD4+ or CD8+ cells (N=4, biological replication). [Figure 3C] T cell immunophenotypes in CD4+ cells (left) or CD8+ cells (right) (N=4, biological replication, TEMRA: effector memory [CD45+CCR7-] reexpressing CD45RA; TEM: effector memory [CD45RA-CCR7-], TCM: central memory [CD45RA-CCR7+]; TN-Like: naive-like [CD45RA+CCR7+]). [Figure 4A] Figures 4A-4B show the successful transduction of reporter cell lines by retroviral constructs encoding Jun.GHR and Jun.GHR.MyD88, as well as the corresponding phosphorylation of STAT5. This demonstrates that STAT5 reporter cell lines were successfully transduced by retroviral constructs encoding Jun.GHR and Jun.GHR.MyD88. [Figure 4B] Jun.GHR and Jun.GHR.MyD88 demonstrate phosphorylation of STAT5. [Figure 5A] Figures 5A-5B show the evaluation of the functionality of the MyD88 domain in Jun.GHR.MyD88 ZipR. Transduction of Ramos Blue MyD88 signaling reporter cell lines by Jun.GHR and Jun.GHR.MyD88 is shown. [Figure 5B] This demonstrates that Jun.GHR.MyD88 activates MyD88 signaling, in contrast to Jun.GHR. [Modes for carrying out the invention]

[0075] Despite recent advances in cancer treatment, patients with relapsed or refractory disease continue to have poor outcomes, highlighting the need for novel approaches. T cells genetically modified to express chimeric antigen receptors (CARs) can kill chemotherapy-resistant tumor cells and thus have the potential to improve outcomes and reduce treatment-related toxicity from conventional therapies [1, 2].

[0076] CARs typically consist of four components: i) an extracellular antigen recognition domain, most commonly a single-chain variable region fragment (scFv); ii) structural components such as hinges and transmembrane domains; iii) a costimulatory domain that provides signals to maintain the effector function of CAR T cells; and iv) a CD3ζ activation domain.[1-3]

[0077] First-generation CARs provide only signal 1 via CD3ζ. Second-generation CARs, in most cases, also provide signal 2 via CD28 or 4-1BB co-stimulation to maintain the expansion of CAR T cells after activation. Activated CAR T cells produce cytokines such as interleukin-2 (IL-2), but production decreases after repeated exposure to tumor cells [9], and several 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, it is necessary to manipulate CAR T cells to increase cytokine-mediated signaling.

[0078] This application provides, in particular, a chimeric receptor capable of providing signals to 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 simplifies the generation of immune cells expressing the chimeric receptor, and therefore their clinical translation.

[0079] While the data disclosed herein (see the Examples section below) focus on expressing CARs in T cells, the methods and / or compositions disclosed herein may be applicable to a variety of other actively investigated cell therapy platforms, including but not limited to NK cells, NK92 cells, NKT cells, and γδT cells. Similarly, while the methods and / or compositions disclosed herein may be useful in the treatment of cancer, the present invention may also be broadly applicable to adoptive immunotherapy for non-malignant diseases (including, but not limited to, infectious diseases (e.g., viral infections) and autoimmune diseases).

[0080] definition As used herein, the term “chimeric receptor” may refer to a cell surface receptor that has been engineered to have at least a portion of one domain derived from one or more sequences of different origins (e.g., an extracellular domain, a transmembrane domain, or an intracellular domain which may include a signaling domain (i.e., an intracellular signaling domain)).

[0081] As used herein, the terms “chimeric antigen receptor” or “CAR” are defined as cell surface receptors comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular domain comprising a lymphocyte-activating domain and optionally at least one costimulatory signaling domain, all in a combination not found naturally together on a single protein. This particularly includes receptors in which the extracellular and intracellular domains are not found naturally together on a single receptor protein. The chimeric antigen receptors (CARs) of the present invention can be used with lymphocytes such as T cells and natural killer (NK) cells.

[0082] A "homodimerizing motif" is any molecule that has the ability to associate (covalently or noncovalently) with molecules of the same composition.

[0083] The terms “T cell” and “T lymphocyte” are interchangeable and are used 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 may be helper T (Th) cells, e.g., helper T1 (Th1) or helper T2 (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 exemplary populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. "NKT cells" are also included, referring to a specialized population of T cells that express semi-invariant αβ T cell receptors but also express various molecular markers typically 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 in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. "γδ T cells" are also included, referring to a specialized population of a small subset of T cells that have a distinct TCR on their surface, unlike most T cells whose TCR consists of two glycoprotein chains called α-TCR chains and β-TCR chains; in γδ T cells, the TCR consists of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immunomodulation, are an important source of IL-17, and have been found to induce robust CD8+ cytotoxic T cell responses. "Regulatory T cells" or "Tregs" are also included, referring to 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, but may also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0084] The terms “natural killer cells” and “NK cells” are used interchangeably and synonymously herein. As used herein, NK cells refer to differentiated lymphocytes having the CD16+CD56+ and / or CD57+TCR- phenotype. NKs are characterized by their ability to bind to and kill cells that do not express “self” MHC / HLA antigens by activation of specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or suppress the immune response.

[0085] As used herein, the term “antigen” refers to any substance molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, part thereof, or combination thereof) that can be bound by a T cell receptor. Antigens can also induce an immune response. Examples of immune responses, but not limited to, include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will understand that antigens do not necessarily need to be encoded by a “gene.” It is readily apparent that antigens can be synthesized and produced, or derived from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or other biological components, organisms, protein / antigen subunits, dead or inactivated whole cells, or fluids containing lysates.

[0086] The term “antigen-recognizing molecule” refers to any molecule capable of recognizing the antigens described herein. Non-exclusive examples of antigen-recognizing molecules include T cell receptors (TCRs) (e.g., αβ TCRs), synthetic T cell receptors and antigen receptors (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 engagers, and antibodies (e.g., bispecific antibodies) or antibody fragments.

[0087] The terms “antibody” and “antibody” refer 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), intrabodies, minibody diabodies, and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against antigen-specific TCRs), as well as any of the epitope-binding fragments described above. The term “antibody” also refers to covalent diabodies, 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 useful as TCR-binding molecules include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. The immunoglobulin molecule may 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.

[0088] The term "host cell" refers to any cell containing heterologous nucleic acids. Heterologous nucleic acids can be vectors (e.g., expression vectors). For example, a host cell can be a cell of any organism that is selected, modified, transformed, grown, used, or manipulated in any way for the production of substances by the cell, such as the expression of genes, DNA or RNA sequences, proteins, or enzymes by the cell. A suitable host can be determined. For example, a host cell may 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 DH5α, JM109, and KCB, SURE® competent cells, and SOLOPACK Gold Cells, etc., can be used as host cells for vector replication and / or expression. Furthermore, bacterial cells such as E. coli LE392 strain can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insect, and mammalian cells. Examples of mammalian eukaryotic host cells for vector replication and / or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. In certain embodiments, the host cells are autologous. In certain embodiments, the host cells are allogeneic.

[0089] The host cells of this disclosure 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 (iPSCs)) that contain DNA or RNA sequences encoding the chimeric receptor described herein and express the chimeric receptor on their cell surface. The host cells may be used for enhancing immune cell activity (e.g., effector function), treating tumors, treating infectious diseases, and treating autoimmune diseases.

[0090] The terms "activation" or "stimulation" refer to the induction of changes in the biological state of cells (e.g., T cells and NK cells) that result in the expression of activation markers, the production of cytokines, proliferation, and / or cytotoxicity against target cells. All of these changes can be brought about by a primary stimulatory signal. Co-stimulatory signals can amplify the magnitude of the primary signal, suppress cell death after initial stimulation, and result in a more durable activated state, and therefore higher cytotoxic capacity. A "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, results in the proliferation of T cells and / or NK cells, as well as the upregulation or downregulation of key molecules.

[0091] The terms "express" and "expression" mean enabling or causing the production of information in a gene or DNA sequence, for example, by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence, thereby producing a protein. A DNA sequence is expressed within or by a cell to form "expression products," such as proteins. Alternatively, the expression product itself, for example, the resulting protein, can also be said to be "expressed" by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0092] The term "tumor" refers to the benign or malignant abnormal growth of tissue. The term "tumor" includes cancer.

[0093] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity (e.g., tumor-killing activity) or helper activity, including cytokine secretion.

[0094] 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 subject).

[0095] As used herein, the term “site-specific nuclease” refers to a nuclease that can specifically recognize and cleave a nucleic acid (DNA or RNA) sequence.

[0096] The terms "genetically modified" or "genetically engineered" refer to adding extra genetic material, in the form of DNA or RNA, to a cell.

[0097] As used herein, the terms “derivative” or “derived from” in the context of a protein or polypeptide (e.g., a chimeric receptor or its domain) mean: (a) a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to the polypeptide from which it is derived; (b) a nucleotide sequence with respect to the polypeptide from which it is derived. (c) 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; (c) a polypeptide having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more amino acid mutations (i.e., additions, deletions) relative to the polypeptide from which it is derived. (d) a polypeptide comprising (and / or substitution); (e) a polypeptide encoded by a nucleotide sequence that can hybridize to a nucleic acid encoding the polypeptide from which it is derived under high, moderate, or typical stringency hybridization conditions; (f) a fragment of the polypeptide from which it is derived, encoded by a nucleotide sequence that can hybridize to a nucleotide sequence encoding a fragment of 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 under high, moderate, or typical stringency hybridization conditions; or (f) a fragment of the polypeptide from which it is derived.

[0098] The sequence identity percentage can be determined using any method known to those skilled in the art. In certain embodiments, the identity percentage is determined using the “Best Fit” or “Gap” program of the sequence analysis software package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (e.g., high, moderate, and typical stringency conditions) is described and should be seen, for example, in U.S. Patent Application Publication 2005 / 0048549 (e.g., paragraphs 72-73) (the contents thereof are incorporated herein by reference in their entirety for all purposes).

[0099] When used in relation to polypeptides or proteins, the term "portion" refers to any component of a polypeptide or protein, including but not limited to subunits, domains, or fragments. For example, a portion of a dimeric cell surface receptor may be a subunit (or chain) of the receptor.

[0100] As used herein, the term "functional fragment" refers to a fragment of a polypeptide or protein, or a polynucleotide encoding a polypeptide or protein, that retains at least one function of a full-length polypeptide or protein. A functional fragment may consist of one, two, three, or more fragments of a full-length polypeptide or protein, or a polynucleotide encoding a 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 amino acid sequence of the full-length polypeptide or protein.

[0101] The terms “vector,” “cloning vector,” “recombinant vector,” and “expression vector” refer to a vehicle that can introduce a DNA or RNA sequence (e.g., a foreign gene) 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, phages, viruses, and the like. In certain embodiments, vectors are viral vectors, including but not limited to retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors, herpesvirus vectors, baculovirus vectors, and vacciniavirus vectors.

[0102] As used herein, the terms “operably linked” or “operatively linked” and similar phrases refer, when used in relation to nucleic acids or amino acids, to the operable linkage of nucleic acid sequences or amino acid sequences that are functionally related to each other, respectively. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the precise generation of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of an open reading frame, ultimately leading to the generation of a polypeptide (i.e., expression of the open reading frame). As another example, operably linked peptides have functional domains positioned at appropriate distances from each other to confer the intended function of each domain.

[0103] As used herein, the term “promoter” refers to any sequence capable of driving the transcription of a coding sequence in a cell. Therefore, promoters used in the vectors of this disclosure may include cis-acting transcriptional control elements and regulatory sequences involved in regulating or modulating the timing and / or rate of gene transcription. In non-limiting examples, promoters may include cis-acting transcriptional control elements, such as enhancers, promoters, transcriptional terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences, that are involved in transcriptional regulation. Such cis-acting sequences typically interact with proteins or other biomolecules to perform transcription (e.g., turning it on / off, regulating it, or modulating it). “Constitutive” promoters drive expression continuously under most environmental conditions and the developmental and / or differentiated states of the cell. “Inducible” or “regulatable” 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 high temperatures, drought, the presence of light, and anaerobic conditions.

[0104] "Enhance," "promote," "increase," "expand," or "improve" generally refer to the ability of a composition intended herein to produce, induce, or cause a greater physiological response (i.e., downstream effect) compared to the response caused by either the vehicle or the control molecule / composition. Measurable physiological responses may include, among other things, increased proliferation, activation, effector function, and persistence of immune cells, and / or increased tumor cell killing ability, as is evident from the understanding in the art and the description herein. In certain embodiments, the amount “increased” or “enhanced” may be a “statistically significant” amount and may include increases of 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, or more (e.g., 500 times, 1000 times) (including all integers and decimals between 1 and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0105] "Decrease," "lower," "lessen," "reduce," or "abate" generally refers to the ability of a composition intended herein to produce, induce, or cause a less physiological response (i.e., downstream effect) compared to the response caused by either the vehicle or the control molecule / composition. In certain embodiments, the amount "decreased" or "abaten" may be a "statistically significant" amount and may include a reduction to 1 / 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500, 1000) (including all integers and decimals between and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the vehicle, the control composition, or the response in a particular cell line (reference response).

[0106] The terms “treat” or “treatment” a condition, disease, disorder, or state include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the onset of at least one clinical or subclinical symptom of a condition, disease, disorder, or state in a subject who is already suffering from or susceptible to a condition, disease, disorder, or state but has not yet experienced or shown any clinical or subclinical symptoms of the condition, disease, disorder, or state; or (2) suppressing a condition, disease, disorder, or state, i.e., stopping, reducing, or delaying the onset of a condition, disease, disorder, or state or its recurrence or at least one clinical or subclinical symptom; or (3) mitigating a condition, disease, disorder, or state, i.e., causing the regression of at least one clinical or subclinical symptom of the condition, disease, disorder, or state. The benefit to the subject being treated must be statistically significant or at least perceptible to the patient or physician.

[0107] The term "effective" as applied to dosage or quantity refers to the amount of compound or pharmaceutical composition sufficient to produce the desired activity when administered to the target requiring it. Note that when a combination of active ingredients is administered, the effective dose of the combination may or may not include the amount of each individual ingredient that would have been effective if administered individually. The exact amount required varies from target to target, depending on the species, age, and general condition of the target, the severity of the condition being treated, the specific drug(s) used, and the mode of administration.

[0108] As used herein, the term “pharmaceutical composition” refers to a composition comprising a polynucleotide, vector, peptide, composition, or host cell described herein, formulated for administration to a subject for the treatment, alleviation, or prevention of a disease.

[0109] As used herein in reference to the compositions, the term “pharmaceutically acceptable” refers to the molecular entities and other components of such compositions that are physiologically tolerable and do not typically produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term “pharmaceutically acceptable” means that it is approved by a federal or state government regulatory authority or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeia for use in mammals, more specifically in humans.

[0110] As used herein, the term “protein” includes all kinds of natural and synthetic proteins, including protein fragments (or protein pieces) of any length, fusion proteins, and modified proteins (including, but not limited to, glycoproteins, and all other kinds of modified proteins, such as proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0111] The terms “nucleic acid,” “nucleotide,” and “polynucleotide” encompass both DNA and RNA unless otherwise specified. “Nucleic acid sequence” or “nucleotide sequence” means a nucleic acid sequence that codes for an amino acid; this term can also refer to a nucleic acid sequence that includes any amino acid that codes for a linker, or any amino acid that is added as a cloning artifact.

[0112] The terms “patient,” “individual,” “subject,” and “animal” are used interchangeably herein and refer to, but are not limited to, humans and veterinary animals (e.g., cats, dogs, cattle, horses, sheep, pigs, etc.) as well as mammals, including experimental animal models. In preferred embodiments, the subject is human.

[0113] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers may 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 saline solutions, as well as aqueous solutions of dextrose and glycerol, are preferred as carriers, particularly for injectable solutions. Alternatively, the carrier may be a solid dosage form carrier containing, but not limited to, one or more of the following: binders (for compressed pills), flow enhancers, encapsulating agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by EW Martin.

[0114] The singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Therefore, for example, a reference to “method” includes one or more methods and / or steps of the type described herein and / or steps that would become apparent to a person skilled in the art upon reading this disclosure.

[0115] The terms “about” or “approximately” include being within a statistically meaningful range of values. Such a range may be within a given number of decimal places of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the terms “about” or “approximately” depends on the particular system under study and can be readily understood by those skilled in the art.

[0116] Where any aspect of this disclosure is described as "comprising" or a variation thereof (e.g., "comprise") a certain feature, the embodiments are also intended to "consist of" or "consisting essentially of" that feature.

[0117] Unless otherwise indicated, the implementation of this disclosure will utilize conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the scope of the art. Such tools and techniques are available, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Further details are described in John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Further technical details are described, for example, in U.S. Patent No. 7,912,698 and U.S. Patent Application Publications 2011 / 0202322 and 2011 / 0307437.

[0118] The techniques described herein by reference can be adequately implemented in the absence of any elements not specifically disclosed herein.

[0119] The terms and expressions adopted are used as descriptive terms, not restrictive ones, and the use of such terms and expressions does not preclude any equivalents of the features or parts thereof shown or described, and various modifications are permitted within the scope of the claimed technology.

[0120] Chimeric receptor In some embodiments, the Disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, where each polypeptide monomer is i. Extracellular region containing at least one homodimerized motif; ii. Transmembrane region; and iii. Intracellular region containing at least one signaling region It may include.

[0121] In some embodiments, at least one signaling region may originate from a cell surface receptor or signaling adapter protein, or a portion or variant thereof.

[0122] extracellular region In some embodiments, the extracellular domain of the chimeric receptor may contain at least one homodimerization motif. In some embodiments, the extracellular domain of the chimeric receptor may contain multiple homodimerization motifs, such as two, three, four, five or more homodimerization motifs.

[0123] In some embodiments, the homodimerized motif may be a homodimerized leucine zipper motif, a ligand-dependent homodimerized motif, or a homodimerized motif (e.g., a single-chain variable fragment) that can otherwise bring the signaling region into proximity.

[0124] In some embodiments, the extracellular region may include at least one leucine zipper motif. In some embodiments, the extracellular region may include multiple leucine zipper motifs, such as two, three, four, five or more leucine zipper motifs.

[0125] In some embodiments, the leucine zipper motif may comprise at least two, three, four, or five heptad repeats of amino acids having leucine every seven positions.

[0126] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif may include the amino acid sequence of SEQ ID NO: 9, 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 with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9, or a nucleotide sequence encoding 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 nucleotide sequence of SEQ ID NO: 3, or by 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 includes the amino acid sequence shown in SEQ ID NO: 9.In some embodiments, the nucleotide sequence encoding the leucine zipper motif includes the nucleotide sequence shown in SEQ ID NO: 3.

[0127] In some embodiments, the leucine zipper motif may include any homodimerized leucine zipper motif, such as those described in Moll et al., 2001 [6], Reike et al., 2013 [7], and Newman et al., 2003 [8], the contents of each of these are incorporated herein by reference in whole for all purposes.

[0128] In certain embodiments, the zipper motifs that can be used pursuant to this disclosure (e.g., the leucine zipper motif) may be derived from any number of human proteins within the scope of the knowledge of those skilled in the art. A non-limiting example of human proteins containing zipper motifs that can be used pursuant to this disclosure is listed in Table 1. With respect to Table 1 (see also Newman JRS and Keating AE. Comprehensive identification of human bZIP interactions with coiled-coil arrays. Science. 2003 Jun 27;300(5628):2097-101 (the whole thereof is incorporated herein by reference for any intended purpose)), a: another name for the protein identified on the left, or the name of another protein that shares the same coiled-coil sequence as used herein. b: The sequence used in the array experiment is divided into five sections. From left to right: [cloning vector sequence] - [basic region included for cloning] - [coiled-coil domain] - [additional presence to facilitate cloning] - [cloning vector]. The N-terminus (left) of the coiled-coil domain was determined by referencing the Fos / Jun crystal structure, and the C-terminus was determined by the point where the PAIRCOIL probability drops to less than 10%. The register of the coiled-coil domain is shown at the bottom of the column, starting from position f. The sequences were aligned according to the sequence similarity of the coiled-coil domains using CLUSTALX.Further protein sequences indicated by numbers in square brackets ([]) are as follows:[1]~[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: 215);

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

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

[20] -PAEIYES(SEQ ID NO: 218). The first methionine in sequences [1]~[4] is cleaved in vivo during expression. [Table 1] TIFF2026514805000002.tif248170TIFF2026514805000003.tif254170TIFF2026514805000004.tif98170

[0129] In some embodiments, the extracellular domain of the chimeric receptor disclosed herein may include at least one ligand-dependent homodimerization motif. For example, the ligand-dependent homodimerization motif may include a chemical inducer of dimerization (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 each are incorporated herein by reference in their entirety for all purposes)). In one embodiment, the ligand-dependent homodimerization motif is FKBP12 v36It includes a domain. In one embodiment, the ligand-dependent homodimerization motif includes a single-chain variable region fragment.

[0130] In some embodiments, the extracellular domain of the chimeric receptor disclosed herein may include other homodimerizing motifs that can bring the signaling domain into proximity, for example, single-chain variable region fragments (see, for example, Katsarou et al., 2021

[10] (the contents of which are incorporated herein by reference in their entirety for all purposes)). Single-chain variable region fragments may induce homodimerization by binding to a single ligand, which is another example of a “ligand-dependent homodimerizing motif”, or single-chain variable region fragments may induce homodimerization by hydrophobic interaction between two variable fragments.

[0131] In some embodiments, when two or more homodimerization motifs are used, the two or more homodimerization motifs may be operably linked to one another via any of a variety of linkers. An exemplary linker sequence may include (GGGGS)n (SEQ ID NO: 150) (wherein n is any integer (e.g., 1, 2, 3, 4, 5)).

[0132] In some embodiments, the linker includes 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 linker includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 151, or a nucleotide sequence encoding 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 linker includes 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 linker includes 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 linker includes the amino acid sequence shown in SEQ ID NO: 151. In some embodiments, the nucleotide sequence encoding the linker includes the nucleotide sequence shown in SEQ ID NO: 152. In some embodiments, the nucleotide sequence encoding the linker includes the nucleotide sequence described in SEQ ID NO: 153.

[0133] Hinge area In some embodiments, the extracellular region of the chimeric receptor disclosed herein may include a hinge region.

[0134] The hinge region may be derived from all or part of a naturally occurring molecule, for example, all or part of the extracellular region of CD8, CD4, or CD28, or all or part of the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge region sequence, or it may be a completely synthetic hinge region sequence.

[0135] Non-limiting examples of hinge regions that may be used according to the present invention include a portion of human CD8α, a partial extracellular region of CD28, the FcyRIIIa 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 CD8α-stoke, CD28, or IgG1. In certain embodiments, the hinge region is derived from CD8α-stoke. In various embodiments, the hinge region is derived from CD28. The hinge region can provide flexibility and accessibility between the extracellular region and the transmembrane region.

[0136] In some embodiments, the hinge may be derived from, for example, IgG1, IgG2, IgG3, IgG4, CD28, or CD8α.

[0137] In some embodiments, the hinge region may originate from the same cell surface receptor as the intracellular signaling region. In some embodiments, the hinge region may originate from a different molecule than the cell surface receptor from which the intracellular signaling region originates.

[0138] The hinge region may contain up to 300 amino acids, 10 to 100 amino acids, or 25 to 50 amino acids.

[0139] Leader Array In various embodiments, the extracellular domain of the chimeric receptor disclosed herein may include a leader sequence. The leader sequence may be located at the N-terminus of the extracellular domain. The leader sequence may optionally be cleaved from the extracellular domain during cell processing and localization of the chimeric receptor to the cell membrane. Various leader sequences known to those skilled in the art may be used as the leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include FcεR, the variable region of the human immunoglobulin heavy chain, CD8α, or various other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In certain embodiments, the leader sequence is derived from the human immunoglobulin heavy chain.

[0140] In some embodiments, the leader sequence may be derived from the immunoglobulin heavy chain variable region or the colony-stimulating factor 2 receptor alpha chain (CSF2RA).

[0141] In some embodiments, the leader sequence includes 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 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7, or a nucleotide sequence encoding 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 includes 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 includes the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the leader sequence includes the nucleotide sequence shown in SEQ ID NO: 1.

[0142] transmembrane region In some embodiments, the chimeric receptors disclosed herein include a transmembrane region.

[0143] In some embodiments, the transmembrane region may originate from the same cell surface receptor as the intracellular signaling region.

[0144] In some embodiments, the transmembrane domain may originate from EpoR, or a portion or variant thereof.

[0145] In some embodiments, the transmembrane region derived from EpoR may include 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 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 includes a nucleotide sequence encoding 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 nucleotide sequence encoding 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 transmembrane region derived from EpoR can be coded 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 by 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 includes 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 includes 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.

[0146] In some embodiments, the transmembrane region may originate from the GHR, or a portion or variant thereof.

[0147] In some embodiments, the transmembrane region derived from GHR may include 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 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: 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 includes a nucleotide sequence encoding 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 nucleotide sequence encoding 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: 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 transmembrane region derived from GHR can be coded by the nucleotide sequences 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, SEQ ID NO: 145, or by 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 with 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, the transmembrane region derived from EpoR includes 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 includes 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.

[0148] In some embodiments, the transmembrane domain may originate from IL-18Rα, IL-18Rβ, CD28, IL-2Rβ, CD8, CD4, CD3ζ, CD40, CD134(OX-40), CD19, or CD7.

[0149] In some embodiments, the transmembrane domain may originate from IL-18Rα, or a portion or variant thereof.

[0150] In some embodiments, the transmembrane region derived from IL-18Rα may include 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 may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 40, or a nucleotide sequence encoding 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 by 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 includes the amino acid sequence shown in SEQ ID NO: 40.In some embodiments, the nucleotide sequence encoding the transmembrane region derived from IL-18Rα includes the nucleotide sequence shown in SEQ ID NO: 39.

[0151] In some embodiments, the transmembrane region may originate from IL-18Rβ, or a portion or variant thereof.

[0152] In some embodiments, the transmembrane region derived from IL-18Rβ may include 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 may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36, or a nucleotide sequence encoding 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 by 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 includes the amino acid sequence shown in SEQ ID NO: 36.In some embodiments, the nucleotide sequence encoding the transmembrane region derived from IL-18Rβ includes the nucleotide sequence shown in SEQ ID NO: 35.

[0153] In some embodiments, the transmembrane region may originate from CD28, or a portion or variant thereof.

[0154] In some embodiments, the transmembrane region derived from CD28 may include 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 may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 42, or a nucleotide sequence encoding 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 by 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 includes the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the nucleotide sequence encoding the transmembrane region derived from CD28 includes the nucleotide sequence shown in SEQ ID NO: 41.

[0155] In certain embodiments, the transmembrane region is derived from CD8α, CD28, CD8, CD4, CD3ζ, CD40, CD134(OX-40), CD19, or CD7. In certain embodiments, the transmembrane region is derived from CD8α. In certain embodiments, the transmembrane region is derived from CD28. The transmembrane region may be fused in frame or operably linked between an extracellular region and an intracellular signaling region.

[0156] In some embodiments, the transmembrane region may be modified by the deletion or addition of amino acids for optimized intracellular orientation. In some embodiments, the transmembrane region may be modified by the deletion or addition of 1, 2, 3, 4, 5, or 6 amino acids for optimized intracellular orientation. For example, the transmembrane region described herein may be modified by the deletion of 1, 2, 3, 4, 5, or 6 amino acids at either the N-terminus or C-terminus of the transmembrane region. For example, the transmembrane region described herein may be modified by the addition (e.g., alanine addition) of 1, 2, 3, 4, 5, or 6 amino acids at either the N-terminus or C-terminus of the transmembrane region.

[0157] In some cases, the transmembrane region can be modified by amino acid substitution, deletion, or insertion to avoid the binding of proteins that naturally bind to the transmembrane region. In certain embodiments, the transmembrane region includes additional amino acids that allow for flexibility and / or optimal distance between regions connected to the transmembrane region.

[0158] The transmembrane region may originate from a natural or synthetic source. If the source is natural, the region may originate from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane regions particularly useful in this disclosure may originate from 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 (i.e., including at least their transmembrane regions). Alternatively, the transmembrane region may be synthetic, in which case it mainly consists of hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan, and / or valine may be found at each end of a synthetic transmembrane region.

[0159] In some embodiments, it is desirable to utilize the transmembrane domains of the ζ, η, or FcεR1γ chains containing cysteine ​​residues capable of disulfide bonding, and the resulting chimeric protein can form disulfide dimers with itself or with unmodified versions of the ζ, η, or FcεR1γ chains or related proteins. In some cases, the transmembrane domains are selected or modified by amino acid substitutions to minimize interaction with other members of the receptor complex by avoiding binding of such domains to the transmembrane domains of the same or different surface membrane proteins. In other cases, it is desirable to use the transmembrane domains of ζ, η, or FcεR1γ and -β, MB1(Igα), B29, or CD3-γ, ζ, or η.

[0160] Intracellular signal transduction regions In some embodiments, the chimeric receptors disclosed herein may include an intracellular region comprising at least one signaling region (also referred to herein as an intracellular signaling region). The at least one intracellular signaling region may originate from a cell surface receptor or a signaling adapter protein, or a portion or variant thereof.

[0161] In some embodiments, the intracellular region may comprise multiple signaling regions, e.g., two, three, four, or more signaling regions. In some embodiments, the intracellular region may comprise a first signaling region derived from a cell surface receptor, or a portion or variant thereof, and a second signaling region derived from a signaling adapter protein, or a portion or variant thereof.

[0162] In some embodiments, the signaling region is derived from a native homodimeric cell surface receptor or signaling adapter protein. In some embodiments, the signaling region is derived from a non-native homodimeric cell surface receptor or signaling adapter protein.

[0163] In some embodiments, signaling regions derived from cell surface receptors or signaling adapter proteins, or portions or variants thereof, can activate Janus kinase (JAK) signaling and transcription activator (STAT) pathways, nuclear factor κ-activated B cell light chain enhancer (NFκB) pathways, and / or further signaling pathways activated by the MyD88 signaling complex (Myddosome).

[0164] In some embodiments, further signaling pathways activated by the MyD88 signaling complex (Myddosome) are one or more of the following pathways: interleukin-1 receptor-associated kinases (IRAKs) (e.g., IRAK1, IRAK2, IRAK4), TNF receptor (TNFR)-associated factors (TRAFs) (e.g., TRAF6), tank-binding kinases (TBKs) (e.g., TBK1), mitogen-activated protein kinases (MAPKs), protein kinase B or AKT (PKB / AKT), and / or phosphatidylinositol-3-kinase (PI3K) pathways (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).

[0165] In some embodiments, the signaling region may be derived from a cytokine receptor, or a portion or variant thereof. In some embodiments, the signaling region may be derived from a native homodimeric cytokine receptor. In some embodiments, the signaling region may be derived from a non-native homodimeric cytokine receptor.

[0166] In some embodiments, 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 β-subunit (IL-10R2), IL-6β chain (gp130), IL-2Rβ, IL-18Rα, or IL-18Rβ.

[0167] In some embodiments, the signaling region may be derived from unnatural homodimers such as the interleukin-23 receptor (IL-23R) (see, e.g., Engelowski et al., 2018

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

[15] ).

[0168] In some embodiments, the signaling region may originate from the IL-6β 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)).

[0169] In some embodiments, the signaling region may be derived from a native Janus kinase (JAK) / signaling and transcription activator (STAT) homodimer, including, but not limited to, the following: 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)).

[0170] In some embodiments, the signaling region may originate from EpoR, or a portion or variant thereof. In further embodiments, the signaling region derived from EpoR does not include a Src homology region 2 domain-containing phosphatase-1 (SHP1) binding site.

[0171] In some embodiments, the signaling region derived from EpoR may include 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 signaling region derived from EpoR includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46, or a nucleotide sequence encoding 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 signaling region derived from EpoR may be encoded by the nucleotide sequence of SEQ ID NO: 45, or by 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 signaling region derived from EpoR may include the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding the signaling region derived from EpoR includes the nucleotide sequence shown in SEQ ID NO: 45.

[0172] In some embodiments, the signaling region derived from EpoR may include 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 signaling region derived from EpoR includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 49, or a nucleotide sequence encoding 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 signaling region derived from EpoR may be encoded by the nucleotide sequence of SEQ ID NO: 154, or by 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 signaling region derived from EpoR may include the amino acid sequence of SEQ ID NO: 49.In some embodiments, the nucleotide sequence encoding the signaling region derived from EpoR includes the nucleotide sequence shown in SEQ ID NO: 154.

[0173] In some embodiments, the signaling region may originate from the GHR, or a portion or variant thereof.

[0174] In some embodiments, the signaling region derived from GHR may include 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 signaling region derived from GHR includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44, or a nucleotide sequence encoding 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 signaling region derived from GHR may be encoded by the nucleotide sequence of SEQ ID NO: 43, or by 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 signaling region derived from GHR may include the amino acid sequence of SEQ ID NO: 44. In some embodiments, the nucleotide sequence encoding the signaling region derived from EpoR includes the nucleotide sequence shown in SEQ ID NO: 43.

[0175] In some embodiments, the signaling region may be derived from a receptor tyrosine kinase, or a portion or variant thereof. For example, the receptor tyrosine kinase may be the epidermal growth factor receptor (EGFR) (see, for example, Kourouniotis et al., 2016

[12] (the contents thereof are incorporated herein by reference in their entirety for all purposes)).

[0176] In some embodiments, the signaling region may be derived from a Toll-like receptor, or a portion or 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 a 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).

[0177] In some embodiments, the signaling region may originate from a signaling adapter protein, or a portion or variant thereof. In some embodiments, the signaling adapter protein may be MyD88.

[0178] In some embodiments, the signaling region derived from MyD88 may include 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 signaling region derived from MyD88 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 38 or 220, or a nucleotide sequence encoding 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 signaling region derived from MyD88 may be encoded by the nucleotide sequence of SEQ ID NO: 37 or 219, or by 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 signaling region derived from MyD88 may include the amino acid sequence of SEQ ID NO: 38 or 220.In some embodiments, the nucleotide sequence encoding the signaling region derived from MyD88 includes the nucleotide sequence shown in SEQ ID NO: 37 or 219.

[0179] Non-limiting examples of chimeric receptors In some embodiments, the Disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, where each polypeptide monomer is 1) Extracellular region containing a homodimerized leucine zipper motif; 2) Transmembrane regions derived from EpoR, or a portion or variant thereof; 3) A signaling region derived from EpoR, or a portion or variant thereof, and optionally an intracellular region containing a signaling region derived from MyD88, or a portion or variant thereof. Includes.

[0180] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif includes 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 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9, or a nucleotide sequence encoding 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 nucleotide sequence of SEQ ID NO: 3; or by 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 includes the nucleotide sequence shown in SEQ ID NO: 3.

[0181] In some embodiments, the transmembrane region derived from EpoR includes 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 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 includes a nucleotide sequence encoding 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 nucleotide sequence encoding 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 transmembrane region derived from EpoR can be coded 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 by 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 includes 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 includes 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.

[0182] In some embodiments, the signaling region derived from EpoR does not contain a phosphatase-1 (SHP1) binding site containing two Src homologous domains.

[0183] In some embodiments, the signaling region derived from EpoR includes 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 signaling region derived from EpoR includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46, or a nucleotide sequence encoding 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 signaling region derived from EpoR may be encoded by the nucleotide sequence of SEQ ID NO: 45, or by 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 signaling region derived from EpoR may include the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleotide sequence encoding the signaling region derived from EpoR includes the nucleotide sequence shown in SEQ ID NO: 45.

[0184] In some embodiments, each polypeptide monomer of the chimeric receptor may contain the 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 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, or SEQ ID NO: 118, or a nucleotide sequence encoding 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, SEQ ID NO: 114, SEQ ID NO: 116, or SEQ ID NO: 118.In some embodiments, each polypeptide monomer may be encoded by the nucleotide sequence of SEQ ID NO: 111, 113, 115, or 117, or by 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.

[0185] In some embodiments, the Disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, where each polypeptide monomer is 1) Extracellular region containing a homodimerized leucine zipper motif; 2) Transmembrane regions derived from GHR, or a portion or variant thereof, and 3) A signaling region derived from GHR, or a portion or variant thereof, and optionally an intracellular region containing a signaling region derived from MyD88, or a portion or variant thereof. Includes.

[0186] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif may include the amino acid sequence of SEQ ID NO: 9, 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 with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9, or a nucleotide sequence encoding 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 nucleotide acid sequence of SEQ ID NO: 3, or by 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 includes the amino acid sequence described in SEQ ID NO: 9.In some embodiments, the nucleotide sequence encoding the leucine zipper motif includes the nucleotide sequence shown in SEQ ID NO: 3.

[0187] In some embodiments, the transmembrane region derived from GHR may include 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 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 includes a nucleotide sequence encoding 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 nucleotide sequence encoding 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: 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 transmembrane region derived from GHR can be coded 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 by 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: 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, the transmembrane region derived from GHR includes 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 includes 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.

[0188] In some embodiments, the signaling region derived from GHR includes 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 signaling region derived from GHR includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44, or a nucleotide sequence encoding 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 signaling region derived from GHR may be encoded by the nucleotide sequence of SEQ ID NO: 43, or by 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 signaling region derived from GHR may include the amino acid sequence of SEQ ID NO: 44. In some embodiments, the nucleotide sequence encoding the signaling region derived from GHR includes the nucleotide sequence shown in SEQ ID NO: 43.

[0189] In some embodiments, the signaling region derived from MyD88 may include 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 signaling region derived from MyD88 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 220, or a nucleotide sequence encoding 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 signaling region derived from MyD88 may be encoded by the nucleotide sequence of SEQ ID NO: 219, or by 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 signaling region derived from MyD88 may include the amino acid sequence of SEQ ID NO: 220.In some embodiments, the nucleotide sequence encoding the signaling region derived from MyD88 includes the nucleotide sequence shown in SEQ ID NO: 219.

[0190] In some embodiments, each polypeptide monomer of the chimeric receptor may contain 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 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 120, or a nucleotide sequence encoding 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 by 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 contain 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.

[0191] 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 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 221, or a nucleotide sequence encoding 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 by 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 contain 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.

[0192] In some embodiments, each polypeptide monomer of the chimeric receptor may contain 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 to 611 of SEQ ID NO: 221. In some embodiments, the nucleotide sequence encoding each polypeptide monomer includes a nucleotide sequence encoding amino acids 28-611 of the amino acid sequence of SEQ ID NO: 221, or a nucleotide sequence encoding 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 by 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 SEQ ID NO: 222. In some embodiments, each polypeptide monomer of the chimeric receptor may contain 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.

[0193] In some embodiments, the Disclosure provides a chimeric receptor that is a homodimer comprising two polypeptide monomers, where each polypeptide monomer is 1) Extracellular region containing a homodimerized leucine zipper motif; 2) Transmembrane regions derived from IL-18Rα, IL-18Rβ, or CD28, or a portion or variant thereof; and 3) Intracellular regions containing signal transduction regions derived from MyD88, or a portion thereof or a variant thereof. Includes.

[0194] In some embodiments, the leucine zipper motif may be derived from the transcription factor c-Jun. In some embodiments, the leucine zipper motif may include the amino acid sequence of SEQ ID NO: 9, 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 with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the leucine zipper motif includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9, or a nucleotide sequence encoding 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 nucleotide sequence of SEQ ID NO: 3, or by 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 includes the amino acid sequence described in SEQ ID NO: 9.In some embodiments, the nucleotide sequence encoding the leucine zipper motif includes the nucleotide sequence shown in SEQ ID NO: 3.

[0195] In some embodiments, the transmembrane region may include the 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 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 42, or a nucleotide sequence encoding 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 can be coded by the nucleotide sequence of SEQ ID NO: 39, SEQ ID NO: 35, or SEQ ID NO: 41, or by 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 transmembrane region includes the amino acid sequence shown in SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 42. In some embodiments, the nucleotide sequence encoding the transmembrane region includes the nucleotide sequence shown in SEQ ID NO: 39, SEQ ID NO: 35, or SEQ ID NO: 41.

[0196] In some embodiments, the signaling region derived from MyD88 may include 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 signaling region derived from MyD88 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 38 or 220, or a nucleotide sequence encoding 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 signaling region derived from MyD88 may be encoded by the nucleotide sequence of SEQ ID NO: 37 or 219, or by 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 signaling region derived from MyD88 may include the amino acid sequence of SEQ ID NO: 38 or 220.In some embodiments, the nucleotide sequence encoding the signaling region derived from MyD88 includes the nucleotide sequence shown in SEQ ID NO: 37 or 219.

[0197] In some embodiments, each polypeptide monomer of the chimeric receptor may contain the amino acid sequence of SEQ ID NO: 122, 124, or 126, 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: 122, SEQ ID NO: 124, or SEQ ID NO: 126. In some embodiments, the nucleotide sequence encoding each polypeptide monomer includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID NO: 126, or a nucleotide sequence encoding 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 nucleotide sequence of SEQ ID NO: 121, 123, or 125, or by 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 the nucleotide sequence of SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 125.

[0198] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 111, or an amino acid sequence encoded by 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.

[0199] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 113, or an amino acid sequence encoded by 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.

[0200] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 115, or an amino acid sequence encoded by 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.

[0201] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 117, or an amino acid sequence encoded by 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.

[0202] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 119, or an amino acid sequence encoded by 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.

[0203] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 121, or an amino acid sequence encoded by 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.

[0204] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 123, or an amino acid sequence encoded by 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.

[0205] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 125, or an amino acid sequence encoded by 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.

[0206] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 147, or an amino acid sequence encoded by 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.

[0207] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 149, or an amino acid sequence encoded by 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.

[0208] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 the nucleic acid sequence of SEQ ID NO: 222, or an amino acid sequence encoded by 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.

[0209] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer may include 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 amino acid sequences encoded by nucleotides 82-1833 of the nucleic acid sequence of SEQ ID NO: 222, or by 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 nucleotides 82-1833 of SEQ ID NO: 222.

[0210] In some embodiments, the chimeric receptor comprises a homodimer containing two polypeptide monomers, each polypeptide monomer comprising: 1) an extracellular region containing at least one homodimerization motif; 2) a transmembrane region; 3) an intracellular region containing at least one signaling region derived from a cell surface receptor or signaling adapter protein, or a portion or variant thereof; and optionally, 4) at least one additional gene.

[0211] Table 2 shows non-limiting exemplary constructs that may include: 1) an extracellular region containing at least one homodimerized motif; 2) a transmembrane region; 3) an intracellular region containing at least one signaling region derived from a cell surface receptor or signaling adapter protein, or a portion or variant thereof; and optionally, 4) at least one additional gene. [Table 2] TIFF2026514805000006.tif255162TIFF2026514805000007.tif255169TIFF202 6514805000008.tif255166TIFF2026514805000009.tif255169TIFF20265148050 00010.tif254170TIFF2026514805000011.tif255167TIFF2026514805000012.t if241170TIFF2026514805000013.tif253170TIFF2026514805000014.tif148169

[0212] Table 3 shows the amino acid sequences of the domains of candidate intracellular receptors GHR and hEpoR. GHR and hEpoR are non-limited examples of sources for intracellular signaling regions. [Table 3] TIFF2026514805000016.tif187170

[0213] Polynucleotides In one embodiment, the disclosure provides a polynucleotide capable of encoding any of the chimeric receptors disclosed herein. In some embodiments, the polynucleotide may be a DNA molecule. In some embodiments, the polynucleotide may be an RNA molecule.

[0214] In some embodiments, the polynucleotide may comprise a nucleotide sequence capable of encoding any homodimeric polypeptide disclosed herein.

[0215] In some embodiments, the polynucleotide is at least 50% of any of the nucleotide sequences of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 147, 149, or 222 (or nucleotides 82-1833 of SEQ ID NOs: 222), or at least 50% of any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 147, 149, or 222 (or nucleotides 82-1833 of SEQ ID NOs: 222). It contains nucleotide sequences having 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.

[0216] In some embodiments, a nucleotide sequence encoding a chimeric receptor polypeptide can be operably linked to one or more further nucleotide sequences encoding one or more further polypeptide sequences via a sequence encoding a self-cleaving peptide and / or an internal ribosome entry site (IRES).

[0217] In some embodiments, the self-cleaving peptide may be a 2A peptide. Non-limiting examples of self-cleaving peptide sequences include: Thosea asigna virus 2A (T2A; AEGRGSLLTCGDVEENPGP (SEQ ID NO: 155), EGRGSLLTCGDVEENPGP (SEQ ID NO: 156), or GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 157)); foot and mouth disease virus (FMDV) 2A sequence (F2A; GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 158)); sponge (Amphimedon queenslandica) 2A sequence (LLCFLLLLLSGDVELNPGP (SEQ ID NO: 159); or HHFMFLLLLLAGDIELNPGP (SEQ ID NO: 160)); acorn worm 2A sequence (Saccoglossus Examples include the kowalevskii (WFLVLLSFILSGDIEVNPGP (SEQ ID NO: 161)); the amphioxus (Branchiostoma floridae) 2A sequence (KNCAMYMLLLSGDVETNPGP (SEQ ID NO: 162); or MVISQLMLKLAGDVEENPGP (SEQ ID NO: 163)); the porcine teschovirus-1 2A sequence (P2A; GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 11); and the equine rhinitis A virus 2A sequence (E2A); GSGQCTNYALLKLAGDVESNPGPG (SEQ ID NO: 164)). In some embodiments, the isolate sequence is a naturally occurring sequence or a synthetic sequence. In certain embodiments, the isolate sequence includes the 2A consensus sequence DXEX-NPGP (SEQ ID NO: 165) (wherein X is any amino acid residue).

[0218] In some embodiments, the 2A peptide may be a P2A peptide.

[0219] In some embodiments, the P2A peptide includes 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 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11, or a nucleotide sequence encoding 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 includes 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 includes the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the P2A peptide includes the nucleotide sequence shown in SEQ ID NO: 5.

[0220] In some embodiments, nucleotide sequences may be expressed in an inducible manner, which can be achieved, for example, using inducible promoters, inducible expression systems, artificial signaling pathways, and / or drug-inducible splicing.

[0221] In some embodiments, nucleotide sequences may be expressed in an inducible manner, such as by using i) an inducible promoter, e.g., a promoter that can be activated by T cell activation (e.g., NFAT, Nur66, IFNg) or hypoxia; ii) an inducible expression system, e.g., a doxycycline or tamoxifen inducible expression system; iii) an artificial signaling circuit, e.g., SynNotch; and / or iv) drug-inducible splicing. As a non-limiting example, drug-inducible splicing methods and / or compositions useful in the practice of this disclosure are based, for example, on those described in Monteys et al., 2021

[44] (the contents of which are incorporated herein by reference in their entirety for any purpose).

[0222] In some embodiments, the nucleotide sequence may encode a moiety, including but not limited to “SWIFF” technology, such as those described in Juillerat et al., 2019

[17] , Carbonneau et al., 2021

[18] , and Jan et al., 2021

[19] (each of which is incorporated herein by reference in whole for all purposes), or an immunomodulatory drug (IMiD)-inducible degron, so that the stability of the chimeric receptor can be regulated by a small molecule.

[0223] In some embodiments, a nucleotide sequence encoding the chimeric receptor polypeptide may be operably linked to at least one regulatory element. The regulatory element may be capable of mediating the expression of the chimeric receptor polypeptide. The regulatory element includes, but is not limited to, promoters, enhancers, start sites, polyadenylated (polyA) tails, IRES elements, response elements, and termination signals. In some embodiments, the regulatory element modulates the expression of the chimeric receptor. In certain embodiments, the regulatory element increases the expression of the chimeric receptor construct. In certain embodiments, the regulatory element increases the expression of the chimeric receptor construct when the host cell is activated. In certain embodiments, the regulatory element decreases the expression of the chimeric receptor construct. In certain embodiments, the regulatory element decreases the expression of the chimeric receptor construct when the host cell is activated.

[0224] In some embodiments, the promoter is an inducible promoter. Non-limiting examples of inducible promoters include promoters derived from lac, sp6, T7, and Hsp70 and Hsp90.

[0225] In some embodiments, the inductive promoter is a tetracycline (Tc) inductive promoter.

[0226] In some embodiments, the promoter may be a T cell-specific promoter or an NK cell-specific promoter.

[0227] additional genes In addition to the chimeric receptor construct, the polynucleotide may further include at least one additional gene encoding an additional peptide. Examples of additional genes include selection markers for transduced host cells, in vivo tracking markers, cytokines, suicide genes, or several other functional genes. In certain embodiments, the additional functional gene may induce the expression of another molecule. In certain embodiments, the additional functional gene may enhance the safety of the chimeric receptor. For example, the chimeric receptor construct may include an additional gene that is truncated CD19 (tCD19). tCD19 can be used as a tag. The expression of tCD19 may also be useful in determining the transduction efficiency. In some embodiments, the additional functional gene may allow for the modulation of the stability of the chimeric receptor.

[0228] Non-limiting examples of additional genes that can be used to enhance the effector function of modified host cells include: (a) secretory cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, but not limited to these), (b) membrane-bound cytokines (e.g., IL-15, but not limited to these), (c) other chimeric receptors (e.g., IL-2 / IL-7, IL-4 / IL-7, but not limited to these), (d) constitutively active cytokine receptors (e.g., C7R, but not limited to these), and (e) dominant-negative receptors (DNR; e.g., TGFRII). Examples include (f) DNRs (but not limited to these), (g) ligands for co-stimulatory molecules (e.g., CD80, 4-1BBL, but not limited to these), (h) nuclear factors (NFATs) of activated T cells (e.g., NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5), (i) antibodies (including their fragments and bispecific antibodies) (e.g., bispecific T cell engagers (BiTEs), but not limited to these), (j) chimeric antigen receptors (CARs), or (c) safety switches or suicide genes (e.g., CD20, truncated EGFR or HER2, inducible caspase 9 molecules).

[0229] In certain 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.

[0230] In certain embodiments, the chimeric receptor construct may include, for example, at least one additional gene encoding one or more cell markers, epitope tags, cytokines, safety switches, dimerization moieties, or degradation moieties.

[0231] In some embodiments, the additional gene is an epitope tag. In a non-limiting example, the epitope tag is FLAG. In another non-limiting example, the epitope tag is Myc.

[0232] In some embodiments, the additional gene is a cell marker. In a non-limiting example, the cell marker is mClover3. In another non-limiting example, the cell marker is mRuby.

[0233] In some embodiments, additional genes may encode portions such that the stability of the chimeric receptor can be regulated by small molecules (including, for example, the “SWIFF” technology described 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) or immunomodulatory drug (IMiD)-inducible degron).

[0234] In certain embodiments, the additional functional gene may be a suicide gene. A suicide gene is a recombinant gene that causes the host cell on which the gene is expressed to undergo programmed cell death or antibody-mediated clearance at a desired time. Suicide genes may function to enhance 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 targetable with clinical-grade monoclonal antibodies, including CD20, EGFR or its fragments, HER2 or its fragments, and ii) inducible suicide genes (e.g., inducible caspase 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; U.S. Patent Application Publication No. 2011 / 0286980, each incorporated herein by reference in its entirety for all purposes)).

[0235] In certain embodiments, the chimeric receptors of this disclosure may be modulated by a safety switch. As used herein, the term “safety switch” refers to any mechanism that can remove or inhibit the effects of the chimeric receptor from a system (e.g., a culture or subject). The safety switch may function to enhance the safety of the chimeric receptor.

[0236] The function of the safety switch may be inducible. Non-limiting examples of safety switches include (a) molecules expressed on the cell surface and targetable with clinical-grade monoclonal antibodies, including CD20, EGFR or fragments thereof, and HER2 or fragments thereof; and (b) inducible suicide genes (e.g., herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; U.S. Patent Application Publication No. 2011 / 0286980, each incorporated herein by reference in whole for all purposes)).

[0237] In some embodiments, the safety switch may be a CD20 polypeptide. Expression of human CD20 on the cell surface presents an attractive strategy for a safety switch. We and other researchers have shown that cells expressing CD20 can be rapidly eliminated using the FDA-approved monoclonal antibody rituximab via complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. (See, for example, Griffioen, M., et al. Haematologica 94, 1316-1320 (2009) (the whole is incorporated herein by reference for all purposes)). Rituximab is an FDA-approved anti-CD20 monoclonal antibody for chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL), among other things (Storz, U. MAbs 6, 820-837 (2014) (the whole is incorporated herein by reference for all purposes)). The CD20 safety switch is non-immunogenic and can function 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 of these is incorporated herein by reference in whole for all purposes).

[0238] In some embodiments, chimeric receptors may be expressed in an inducible manner, such as by using i) an inducible promoter, e.g., a promoter that can be activated by T cell activation (e.g., NFAT, Nur66, IFNγ) or hypoxia; ii) an inducible expression system, e.g., a doxycycline or tamoxifen inducible expression system; iii) an artificial signaling circuit, including but not limited to SynNotch; and / or iv) drug-inducible splicing. As a non-limiting example, drug-inducible splicing methods and / or compositions useful in the practice of this disclosure are based, for example, on those described in Monteys et al., 2021

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

[0239] In certain embodiments, the chimeric receptor may include at least one additional gene (i.e., a second gene). In certain embodiments, the chimeric receptor may include one second gene. In other embodiments, the chimeric receptor may include two additional genes (i.e., a third gene). In yet another embodiment, the chimeric receptor may include three additional genes (i.e., a fourth gene). In certain embodiments, the additional genes may be separated from each other and from the chimeric receptor construct. For example, they may be separated by the 2A sequence and / or internal ribosome entry sites (IRES), as described above. In certain examples, the chimeric receptor may be located at any position on the polynucleotide chain.

[0240] Recombinant vectors This disclosure provides a recombinant vector which may contain a polynucleotide encoding a chimeric receptor. Such a recombinant vector may contain a polynucleotide encoding the protein disclosed above. In one embodiment, the polynucleotide may be operably linked to at least one regulatory element for the expression of the chimeric receptor.

[0241] In certain embodiments, the vector may be a viral vector. In certain embodiments, the viral vector may be, but is not limited to, a retroviral vector, a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, an alphavirus vector, a herpesvirus vector, a baculovirus vector, or a vacciniavirus vector.

[0242] In some embodiments, the viral vector may be a retroviral vector.

[0243] In some embodiments, the vector may be a nonviral vector. Suitable nonviral vectors for use in the present invention include, but are not limited to, minicircle plasmids, transposon systems (e.g., Sleeping Beauty transposons, piggyBac transposons), or single-stranded or double-stranded DNA molecules used as templates for homologous recombination repair (HDR)-based gene editing.

[0244] In some embodiments, the vector includes 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 any of the nucleotide sequences 111, 113, 115, 117, 119, 121, 123, 125, 147, 149, or at least 92%.

[0245] Isolated host cells In another aspect, provided herein is an isolated host cell that can contain any of the various polynucleotides described herein or a recombinant vector described herein.

[0246] In a further aspect, provided herein is an isolated host cell that can contain a chimeric receptor encoded by a polynucleotide described herein.

[0247] In certain embodiments, the host cell can be an immune cell. In various embodiments, the host cell is a T cell.

[0248] T cells can 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 can be helper T (Th) cells, such as helper T1 (Th1) or helper T2 (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 exemplary populations of T cells suitable for use in certain embodiments include naive T cells, memory T cells, NKT cells, and iNKT cells.

[0249] In some embodiments, the host cell can be a T cell, natural killer (NK) cell, mesenchymal stem cell (MSC), or macrophage. In some embodiments, the host cell can be a T cell.

[0250] In some embodiments, the T cell is selected from 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, stem cell-like memory T cell (T SCM scm). naive T cell, effector T cell, helper T cell. or regulatory T cell (Treg).

[0251] In various embodiments, the host cell can be a natural killer (NK) cell. NK cells refer to differentiated lymphocytes having a CD3−CD16+, CD3−CD56+, CD16+CD56+ and / or CD57+ TCR− phenotype.

[0252] In some embodiments, the host cell can be an NK cell derived from peripheral blood, umbilical cord blood, IPC, induced pluripotent stem (iPS) cells (iPSC), and / or cell lines (e.g., NK-92 cells).

[0253] In some embodiments, the host cell can be an immune cell. In some embodiments, the immune cell can be derived from induced pluripotent stem (iPS) cells (iPSC). Non-limiting examples of iPSCs that can be used according to the present disclosure can be any such cells as described, for example, in Zhu et al., 2019

[21] and Iriguchi et al., 2021

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

[0254] In various embodiments, other host immune cells may be selected, for example, without limitation, macrophages can be selected. In various embodiments, the host immune cell can be a dendritic cell, a Langerhans cell, or a B cell. In various embodiments, the host immune cell can be a professional antigen-presenting cell (APC). In various embodiments, the host immune cell can be a non-professional antigen-presenting cell (APC).

[0255] In some embodiments, host cells may further express one or more antigen-recognizing molecules. In some embodiments, one or more antigen-recognizing 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 engagers, native or transgenic T cell receptors, and antibodies, or combinations thereof.

[0256] In some embodiments, host cells may be further genetically modified to enhance their function by using gene editing techniques (e.g., CRISPR-Cas9 or transcriptional activator-like effector nucleases (TALENs)) to a) express one or more additional genes (e.g., transcription factor (c-Jun)) or b) delete 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 are described, for example, in WO2020 / 219682 and WO2020 / 210365, which are incorporated herein by reference in their entirety.

[0257] In various embodiments, host cells are activated and / or enlarged ex vivo.

[0258] In various embodiments, the host cells may be allogeneic cells. In various embodiments, the host cells may be autologous cells.

[0259] In certain embodiments, host cells may be isolated from a subject having cancer. In some embodiments, host cells may be isolated from a subject having a tumor. In various embodiments, cancer may be a solid tumor, a brain tumor, or a leukemia. In some embodiments, tumors may be found in, but are not limited to, breast tissue, prostate tissue, bladder tissue, oral and / or dental tissue, head and / or cervical tissue, stomach tissue, liver tissue, colon tissue, lung tissue, brain tissue, ovaries, cervix, esophagus, skin, lymph nodes, and / or bone. In some embodiments, tumors may be cancer. In some embodiments, cancer can be, for example, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma and other Ewing sarcoma family tumors, neuroblastoma, ganglioneuroblastoma, fibroplastic round cell tumor, malignant peripheral nerve sheath tumor, synovial sarcoma, undifferentiated sarcoma, adrenocortical carcinoma, hepatoblastoma, Wilms' tumor, rhabdoid tumor, high-grade glioma (glioblastoma multiforme), medulloblastoma, astrocytoma, glioma, ependymoma, atypical teratomatous rhabdoid tumor, meningioma, craniopharyngioma, primitive neuroectodermal tumor, diffuse intrinsic pontine glioma. This may include, but is not limited to, glioma and other brain tumors, acute myeloid leukemia, multiple myeloma, lung cancer, mesothelioma, breast cancer, bladder cancer, stomach 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, and malignant melanoma.

[0260] In certain embodiments, host cells may be isolated from a subject having a tumor.

[0261] In some embodiments, the host cells may be derived from blood, bone marrow, tissue, or tumor samples.

[0262] In certain embodiments, this disclosure provides a method for generating isolated host cells as described herein. The method comprises genetically modifying host cells with polynucleotides or recombinant vectors as described herein. In some embodiments, the method may further comprise genetically modifying host cells to express one or more antigen-recognizing molecules. One or more antigen-recognizing 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 engagers, native or transgenic T cell receptors, and antibodies, or combinations thereof.

[0263] In some embodiments, the gene modification step may be carried out via viral gene delivery. In some embodiments, the gene modification step may be carried out via non-viral gene delivery. In some embodiments, the gene modification step may be carried out ex vivo. In some embodiments, the method may further include activation and / or expansion of host cells ex vivo before, after, and / or during the gene modification. In some embodiments, the method may further include activation and / or expansion of host cells ex vivo before, after, and / or during the gene modification step.

[0264] In addition to genetically modifying immune cells ex vivo, polynucleotides or recombinant vectors encoding the chimeric receptors described herein may be prepared ex vivo and injected directly into patients using non-viral or viral delivery approaches (i.e., in vivo gene delivery) (see, for example, Rurik et al., Science. 2022 January 07; 375(6576): 91-96 (the entire text of which is incorporated herein by reference)).

[0265] Isolation / Enrichment The host cells may be autologous / autogeneic ("self") or non-autologous ("non-self," e.g., allogeneic, syngeneic, or xenogeneic). In certain embodiments, the host cells are obtained from mammalian subjects. In other embodiments, the host cells are obtained from primate subjects. In certain embodiments, the host cells are obtained from human subjects.

[0266] Lymphocytes can be obtained from sources such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, and tumors, but are not limited to these sources. Lymphocytes can also be produced by the differentiation of stem cells. In certain embodiments, lymphocytes can be obtained from blood collected from a subject using techniques commonly known to those skilled in the art, such as sedimentation, e.g., FICOLL® separation.

[0267] In certain embodiments, cells derived from the circulating blood of interest are obtained by apheresis. The apheresis apparatus typically contains leukocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated leukocytes, as well as erythrocytes and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or medium for further processing. The cells may be washed with PBS or another suitable solution lacking calcium, magnesium, and most, if not all, other divalent cations. The washing step can be achieved by methods known to those skilled in the art, including, but not limited to, using a semi-automatic flow-through centrifuge (e.g., a Cobe 2991 cell processor or a Baxter CytoMate). After washing, the cells may be resuspended in a variety of biocompatible buffers, cell culture media, or other salines, with or without buffers.

[0268] In certain embodiments, host cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing erythrocytes and depleting monocytes. For example, cells can be sorted by centrifugation using a PERCOLL® gradient. In certain embodiments, after isolation of PBMCs, both cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive T cells, memory T cells, and effector T cell subpopulations, either before or after activation, expansion, and / or genetic modification.

[0269] In certain embodiments, T lymphocytes can be enriched. For example, using either positive or negative selection techniques, a specific subpopulation of T lymphocytes expressing one or more markers such as 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 a combination thereof, can be enriched. In certain embodiments, T lymphocytes for use in the compositions of the present 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.

[0270] In certain embodiments, NK cells can be enriched. For example, using either positive or negative selection techniques, a specific subpopulation of T lymphocytes expressing one or more markers such as CD2, CD16, CD56, CD57, CD94, or CD122, or a combination thereof, can be enriched.

[0271] stimulation / activation To reach a sufficient therapeutic dose of the host cell composition, the host cells are often subjected to one or more stimulations / activations. In certain embodiments, a method of generating host cells for administration to a subject comprises stimulating and activating the host cells in the presence of one or more stimulating signals or agents (e.g., compounds, small molecules (e.g., organic small molecules), nucleic acids, polypeptides, or fragments, isoforms, variants, analogs, or derivatives thereof). In certain embodiments, a method of generating host cells for administration to a subject comprises stimulating, activating, and further proliferating the host cells in the presence of one or more stimulating signals or agents.

[0272] Host cells (e.g., T lymphocytes and NK cells) can be activated by inducing changes in their biological state such that the cells express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All of these changes can be caused by primary stimulating signals. Costimulatory signals amplify the magnitude of the primary signal, suppress cell death after initial stimulation, and result in a more durable activated state and thus higher cytotoxic capacity.

[0273] T cells can generally be activated using methods such as those described in, for example, U.S. Patent Nos. 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.

[0274] In certain embodiments, T cell-based host cells can be activated by binding to an agent that activates CD3ζ.

[0275] In other embodiments, CD2-binding agents may be used to provide primary stimulatory signals to T cells. For example, CD2-binding agents include, but are not limited to, T1 1.3 antibodies combined with CD2 ligands and anti-CD2 antibodies, such as T1 1.1 or T1 1.2 antibodies (Meuer, SC et al. (1984) Cell 36:897-90), and 9.6 antibodies (which recognize the same epitope as T1 1.1) combined with 9-1 antibodies (Yang, SY et al. (1986) J. Immunol. 137:1097-1100). Other antibodies that bind to the same epitopes as any of the above antibodies may also be used.

[0276] In certain embodiments, host cells are activated by administering phorbol myristate acetate (PMA) and ionomycin. In certain embodiments, host cells are activated by administering a suitable antigen that induces activation and then proliferation (expansion). In certain embodiments, PMA, ionomycin, and / or a suitable antigen are administered along with CD3-inducible activation and / or expansion.

[0277] Generally, activating agents used in this disclosure include, but are not limited to, antibodies, their fragments, and protein-binding molecules having antibody-like function. Examples of (recombinant) antibody fragments include Fab fragments, Fv fragments, single-chain Fv fragments (scFv), bivalent antibody fragments (e.g., (Fab)2'-fragments), diabodies, 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). The bivalent antibody fragment may be a (Fab)2' fragment or a bivalent single-stranded Fv fragment, and the monovalent antibody fragment may be selected from the group consisting of Fab fragments, Fv fragments, and single-stranded Fv fragments (scFv).

[0278] In certain embodiments, one or more binding sites of the CD3ζ drug may be a bivalent proteinoid artificial binding molecule, such as dimeric lipocalin mutein (i.e., duocalin). In certain embodiments, the receptor binding reagent may have a single second binding site (i.e., monovalent). Examples of monovalent drugs include, but are not limited to, monovalent antibody fragments, proteinoid 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).

[0279] Drugs that specifically bind to CD3 include, but are not limited to, anti-CD3 antibodies, bivalent anti-CD3 antibody fragments, monovalent anti-CD3 antibody fragments, and protein-based CD3 binding molecules with antibody-like binding properties. Protein-based CD3 binding molecules with antibody-like binding properties may include aptamers, muteins based on lipocalin family polypeptides, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adonectins, and avimers. These may be conjugated to beads.

[0280] In certain embodiments, the activator (e.g., CD3-binding substance) can be present at concentrations of about 0.1 to about 10 μg / ml. In certain embodiments, the activator (e.g., CD3-binding substance) can 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 certain embodiments, the activator (e.g., CD3-binding substance) is administered at concentrations of approximately 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, or 10 μg / ml. In certain embodiments, the CD3-binding substance may be present at a concentration of 1 μg / ml.

[0281] NK cells are generally referred to, for example, in U.S. Patent Nos. 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 Application No. 2004 / 0058445. They can be activated using methods such as those described in International Patent Publication Nos. 2007 / 0160578, 2013 / 0011376, 2015 / 0118207, 2015 / 0037887; and International Patent Publication WO2016 / 122147, each of which is incorporated herein by reference in whole for any purpose.

[0282] In certain embodiments, NK-based host cells may be activated by inhibition of inhibitory receptors on NK cells (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E, or LILRB5 receptors), for example.

[0283] In certain embodiments, NK-based host cells may be activated by, for example, but not limited to, feeder cells (e.g., native K562 cells, or K562 cells genetically modified to express 4-1BBL and cytokines (e.g., IL-15 or IL-21)).

[0284] In other embodiments, NK cells can be activated using interferons or macrophage-derived cytokines. For example, such interferons include, but are not limited to, interferon-α and interferon-γ, and such cytokines include, but are not limited to, IL-15, IL-2, and IL-21.

[0285] In certain embodiments, the NK activator can be present at concentrations of approximately 0.1 to approximately 10 μg / ml. In certain embodiments, the NK activator can be present at concentrations of approximately 0.2 μg / ml to approximately 9 μg / ml, approximately 0.3 μg / ml to approximately 8 μg / ml, approximately 0.4 μg / ml to approximately 7 μg / ml, approximately 0.5 μg / ml to approximately 6 μg / ml, approximately 0.6 μg / ml to approximately 5 μg / ml, approximately 0.7 μg / ml to approximately 4 μg / ml, approximately 0.8 μg / ml to approximately 3 μg / ml, or approximately 0.9 μg / ml to approximately 2 μg / ml. In certain embodiments, the NK activator is administered at concentrations of approximately 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, or 10 μg / ml. In certain embodiments, the NK activator may be present at a concentration of 1 μg / ml.

[0286] In certain embodiments, the activator may be bound to a solid support such as beads, an absorbent polymer present in a culture plate or well, or other matrix such as Sepharose or glass, but not limited to those skilled in the art; or it may be expressed on the cell surface of a natural or recombinant cell line (in a natural or recombinant form, etc.) by means known to those skilled in the art.

[0287] Polynucleotide transcription In certain embodiments, host cells are genetically modified to express the chimeric receptor described above. In certain embodiments, host cells are further genetically modified to express the CAR, TCR, antibody, or a fragment or derivative thereof disclosed herein. Host cells may be genetically modified after stimulation / activation. In certain embodiments, host cells are modified within 12, 16, 24, 36, or 48 hours after stimulation / activation. In certain embodiments, cells are modified within 16-24 hours after stimulation / activation. In certain embodiments, host cells are modified within 24 hours.

[0288] To genetically modify host cells to express chimeric receptors or other related molecules (e.g., CARs, TCRs, antibodies, their fragments, or derivatives), polynucleotide constructs must be introduced into host cells. Polynucleotide introduction may be via viral or non-viral genetic methods. Suitable methods for polynucleotide delivery for use with this method include any methods known to those skilled in the art that can introduce polynucleotides into organelles, cells, tissues, or organisms.

[0289] In some embodiments, polynucleotides are transferred into cells via non-viral vectors. Suitable non-viral vectors for use in the present invention include, but are not limited to, minicircle plasmids, transposon systems (e.g., Sleeping Beauty, piggyBac), or single-stranded or double-stranded DNA molecules used as templates for homologous recombination repair (HDR)-based gene editing.

[0290] Nucleic acid vaccines can be used to introduce 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.

[0291] In some embodiments, host cells can be genetically modified by methods commonly used by those skilled in the art. In some embodiments, host cells can be transduced via retroviral transduction. References describing retroviral transduction of genes include U.S. Patent No. 5,399,346 by Anderson et al., Cell 33:153 (1983); U.S. Patent No. 4,650,764 by Temin et al., U.S. Patent No. 4,980,289 by Temin et al., Markowitz et al., J. Virol. 62:1120 (1988); U.S. Patent No. 5,124,263 by Temin et al., International Publication No. 95 / 07358, published March 16, 1995, by Dougherty et al., and Kuo et al., Blood 82:845 (1993), each of which is incorporated herein by reference in its entirety.

[0292] One method of genetic modification involves ex vivo modification. Various methods are available for transfecting cells and tissues isolated from a subject via ex vivo modification. For example, cells isolated from a subject can be genetically modified using in vitro retroviral gene transfer, and then these cells can be returned to the subject. See, for example, Wilson et al., Science, 244:1344-1346, 1989 and Nabel et al., Science, 244(4910):1342-1344 (both of which are incorporated herein by reference in their entirety). In certain embodiments, host cells can be isolated from a subject and transfected ex vivo using the polynucleotides (e.g., expression vectors) of the Disclosure. In certain embodiments, host cells obtained from a subject can be transfected or transduced with the polynucleotides (e.g., expression vectors) of the Disclosure and then administered back to the subject.

[0293] Another method of gene transfer is injection. In some embodiments, cells or polynucleotides or viral vectors may be delivered to cells, tissues or organisms via one or more injections (e.g., needle injection). Non-limiting methods of injection include injection of compositions (e.g., saline-based compositions). Polynucleotides can also be introduced by direct microinjection. Non-limiting sites of injection include subcutaneous, intradermal, intramuscular, intranodular (allowing direct delivery of antigens to lymphoid tissue), intravenous, intraprostatic, intratumoral, intralymphatic (allowing direct administration of DCs), and intraperitoneal. It is understood that preparation of the appropriate injection site (e.g., shaving the injection site to observe proper needle placement) is necessary.

[0294] Electroporation is another method of 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 certain embodiments, cell wall-degrading enzymes, such as pectinases, can be used to make host cells more sensitive to electroporation-induced genetic modification than untreated cells. See, for example, U.S. Patent No. 5,384,253 (in its entirety for all purposes).

[0295] In vivo electroporation involves a basic injection technique that involves intradermal injection of a vector. The electrode then applies an electrical pulse to the intradermal site, causing localized cells (e.g., resident skin dendritic cells) to take up the vector. These tumor antigen-expressing dendritic cells, activated by local inflammation, can then migrate to lymph nodes.

[0296] Examples of electroporation methods for use in conjunction with the present invention include 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.

[0297] Further methods for polynucleotide transfer include liposome-mediated transfection (e.g., polynucleotides captured 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). Polynucleotides complexed with Lipofectamine or Superfect are also considered; DEAE-dextran (e.g., polynucleotides are delivered to cells using DEAE-dextran followed by polyethylene glycol; see, e.g., Gopal, TV, Mol Cell Biol. 1985 May; 5(5):1188-90); calcium phosphate (e.g., polynucleotides are introduced into cells using calcium phosphate precipitation; see, e.g., 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 (introduction of polynucleotides by direct sonic loading; see, e.g., Fechheimer et al., (1987) Proc. Nat'l Acad. Sci. See USA, 84, 8463-8467); Microprojectile bombardment (for example, one or more particles coated with at least one polynucleotide can be delivered to 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 publication WO94 / 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., selective uptake of macromolecules by receptor-mediated endocytosis occurring in target cells using cell-type specific distributions of various receptors; e.g., Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; See also 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 each reference cited herein is incorporated in its entirety by reference for all purposes.

[0298] In further embodiments, host cells are genetically modified using gene editing with homologous recombination repair (HDR). Homologous recombination repair (HDR) is a mechanism used by cells to repair double-strand DNA breaks. In HDR, a donor polynucleotide homologous to the double-strand DNA break site is used as a template to repair the broken DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the DNA. Thus, new nucleic acid material can be inserted or copied into the target DNA break site. Double-strand DNA breaks in host cells can be induced by site-specific nucleases. As used herein, the term “site-specific nuclease” refers to a nuclease that can specifically recognize and cleave a nucleic acid (DNA or RNA) sequence. Suitable site-specific nucleases for use in the present 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 is introduced into host cells together with the chimeric receptor of this disclosure and, optionally, a donor polynucleotide encoding an additional protein (e.g., a CAR, TCR, antibody, or a fragment or derivative thereof).

[0299] Expansion / Proliferation After activating and transducing host cells, the cells are cultured and proliferated. 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, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more expansion rounds.

[0300] Drugs that can be used to enlarge 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 exemplary examples of drugs that may be used for T cell enlargement are drugs that bind to CD8, CD45, or CD90, such as αCD8 antibodies, αCD45 antibodies, or αCD90 antibodies. Exemplary examples of T cell populations include antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells (an exemplary example of memory T cells is CD62L / CD8+ specific central memory T cells), or regulatory T cells (an exemplary example of Treg cells is CD4+CD25+CD45RA+ Treg cells).

[0301] Further agents that may be used to proliferate T lymphocytes include, for example, methods described in U.S. Patent Nos. 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 whole.

[0302] In certain embodiments, the agent used for proliferation (e.g., IL-2) is administered at approximately 20 units / ml to approximately 200 units / ml. In certain embodiments, the agent used for expansion (e.g., IL-2) is administered at approximately 25 units / ml to approximately 190 units / ml, approximately 30 units / ml to approximately 180 units / ml, approximately 35 units / ml to approximately 170 units / ml, approximately 40 units / ml to approximately 160 units / ml, approximately 45 units / ml to approximately 150 units / ml, approximately 50 units / ml to approximately 140 units / ml, approximately 55 units / ml to approximately 130 units / ml, approximately 60 units / ml to approximately 120 units / ml, approximately 65 units / ml to approximately 110 units / ml, approximately 70 units / ml to approximately 100 units / ml, approximately 75 units / ml to approximately 95 units / ml, or approximately 80 units / ml to approximately 90 units / ml. In certain embodiments, the agent used for expansion (e.g., IL-2) is 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, approximately 105 units / ml, and approximately 11 It is administered at 0 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. In certain embodiments, the agent used for expansion (e.g., IL-2) is administered at approximately 5 mg / ml to approximately 10 ng / ml. In certain embodiments, the drug used for expansion (e.g., IL-2) is administered at approximately 5.5 ng / ml to approximately 9.5 ng / ml, approximately 6 ng / ml to approximately 9 ng / ml, approximately 6.5 ng / ml to approximately 8.5 ng / ml, or approximately 7 ng / ml to approximately 8 ng / ml. In certain embodiments, the drug used for expansion (e.g., IL-2) is administered at approximately 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml.

[0303] The host cells are activated and transduced, then cultured and proliferated. 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, in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more expansion rounds.

[0304] Drugs that can be used to enlarge NK cells may include drugs that bind to CD16 or CD56, such as αCD16 antibodies or αCD56 antibodies. In certain embodiments, the binding agent includes an antibody (see, e.g., Hoshino et al, Blood. 1991 Dec. 15; 78(12):3232-40). Another drug that may be used for enlarge NK cells may be IL-15 (see, e.g., Vitale et al. 2002. The Anatomical Record. 266:87-92 (which is incorporated herein by reference in its entirety for all purposes)).

[0305] Suitable conditions for culturing T cells include appropriate culture media (e.g., minimal essential medium (MEM), RPMI medium 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM-V, DMEM, a-MEM, F-12, TexMACS, X-Vivo15, and X-Vivo20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free, or a defined set of appropriate amounts of serum (or plasma) or hormones, and / or sufficient amounts of cytokines for growth and expansion).

[0306] Other additives for host cell enlargement include, but are not limited to, surfactants, piasmanates, pH buffers such as HEPES, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental culture and not in the cell culture to be injected into the target. The target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0307] In certain embodiments, the host cells of this disclosure may be modified to reduce or eliminate the expression of endogenous TCRs, MHC molecules, or other immunogenic molecules. When using allogeneic cells, there is a concern that therapeutic cell rejection may occur, as this can lead to serious complications such as graft-versus-host disease (GvHD). While we do not wish to be bound by theory, immunogenic molecules (e.g., endogenous TCRs and / or MHC molecules) are typically expressed on the cell surface and are involved in self-versus-non-self recognition. Reducing or eliminating the expression of such molecules may reduce or eliminate the ability of therapeutic cells to cause GvHD.

[0308] In certain embodiments, the expression of endogenous TCRs in host cells is reduced or eliminated. In certain embodiments, the expression of endogenous TCRs (e.g., αβTCRs) in host cells is reduced or eliminated. The expression of endogenous TCRs may be reduced or eliminated by disrupting the TRAC locus, the TCR beta constant locus, and / or the CD3 locus. In certain embodiments, the expression of endogenous TCRs may be reduced or eliminated by disrupting one or more of the TRAC, TRBC1, TRBC2, CD3E, CD3G, and / or CD3D loci.

[0309] In certain embodiments, the expression of one or more endogenous MHC molecules in host cells is reduced or eliminated. The modified MHC molecules may be MHC class I or class II molecules. In certain 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.

[0310] The expression of endogenous TCRs, MHC molecules, and / or any other immunogenic molecules in host cells can be disrupted using genome editing technologies such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and meganucleases. These genome editing methods can disrupt target genes by completely knocking out all of their output or by partially knocking down their expression. In certain embodiments, the expression of endogenous TCRs, MHC molecules, and / or any other immunogenic molecules in host cells is disrupted using CRISPR / Cas technology.

[0311] Pharmaceutical composition In some embodiments, the composition includes one or more polypeptides of a chimeric receptor and other related molecules (e.g., CARs, TCRs, or antibodies, or fragments or derivatives thereof), polynucleotides, vectors containing the same, and cellular compositions, as disclosed herein. The compositions of this disclosure include, but are not limited to, pharmaceutical compositions.

[0312] In some embodiments, the pharmaceutical composition comprises one of the host cells disclosed herein and a pharmaceutically acceptable carrier and / or excipient.

[0313] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a polynucleotide or recombinant vector encoding a chimeric receptor as described herein and a pharmaceutically acceptable carrier and / or excipient.

[0314] In another aspect, the disclosure provides a pharmaceutical composition comprising a modified host cell containing the chimeric receptor described herein and a pharmaceutically acceptable carrier and / or excipient.

[0315] Examples of pharmaceutical carriers include, but are not limited to, water and sterile liquids such as oil (e.g., petroleum, animal, plant, or synthetic sources such as peanut oil, soybean oil, mineral oil, and sesame oil). Water or saline solutions, as well as aqueous dextrose and glycerol solutions, are particularly preferred as carriers for injectable solutions.

[0316] Compositions comprising modified host cells disclosed herein may include buffers (e.g., neutral buffered saline, phosphate-buffered saline, etc.); carbohydrates (e.g., glucose, mannose, sucrose or dextran, mannitol, etc.); proteins; polypeptides or amino acids (e.g., glycine, etc.); antioxidants; chelating agents (e.g., EDTA or glutathione, etc.); adjuvants (e.g., aluminum hydroxide, etc.); and preservatives.

[0317] A composition comprising modified host cells disclosed herein may comprise one or more of the following: sterile diluents (e.g., water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides that can function as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol or other solvents); antimicrobial agents (e.g., benzyl alcohol or methylparaben); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid); buffers (e.g., acetates, citrates or phosphates); and agents for adjusting tonicity (e.g., sodium chloride or dextrose).

[0318] In some embodiments, the composition is formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural, or intramuscular administration. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Pharmaceutical compositions for injection are preferably sterile. In some embodiments, the composition is reconstituted from a lyophilized preparation before administration.

[0319] In some embodiments, modified host cells may be mixed with substances that adhere to or penetrate them before administration, such as, but not limited to, nanoparticles.

[0320] Treatment method In one embodiment, the present disclosure provides a method for enhancing the effector function of immune cells, which may include genetically modifying cells with a polynucleotide or recombinant vector encoding a chimeric receptor. In some embodiments, immune cells may express one or more antigen-recognizing molecules selected from: one or more antigen-recognizing 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 engagers, native or transgenic T cell receptors, and antibodies, or combinations thereof.

[0321] In some embodiments, the Disclosure provides methods for enhancing the effector function of immune cells capable of expressing chimeric antigen receptors (CARs), which include genetically modifying cells with polynucleotides or recombinant vectors disclosed herein.

[0322] In some embodiments, the effector function may be one or more of the following: amplification, persistence, and / or cytotoxicity (e.g., antitumor activity).

[0323] The terms “expand” or “expansion,” when used in relation to immune cells, refer to the ability of immune cells to undergo cell proliferation (i.e., to increase in number). As used herein, the terms encompass the expansion of immune cells both in vivo and in vitro.

[0324] The terms “persist” or “persistence,” when used in relation to immune cells, refer to the ability of immune cells (and / or their offspring) to be maintained in a recipient (e.g., a subject) over a period of time. As used herein, the terms encompass the persistence of immune cells both in vivo and in vitro.

[0325] As used herein, 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).

[0326] In one embodiment, the present disclosure provides a method for treating a disease, comprising targeting an effective amount of host cells containing the chimeric receptor described herein, or a pharmaceutical composition containing host cells. In some embodiments, the disease may be cancer, such as a solid tumor, but is not limited. In some embodiments, the disease may be an infectious disease. In some embodiments, the disease may be an autoimmune disease.

[0327] In one embodiment, the present disclosure provides a method for treating a tumor in a subject where it is needed. A therapeutically effective amount of modified host cells containing the chimeric receptor described herein, or a pharmaceutical composition containing host cells, is administered to the subject.

[0328] The term “tumor” refers to the benign or malignant abnormal growth of tissue. The term “tumor” includes cancer. Examples of tumors include, but are not limited to, solid tumors, which are soft tissue tumors (e.g., lymphoma), tumors of the blood and hematopoietic organs (e.g., leukemia), and tumors that grow in anatomical sites 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 lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell cancer), adenosquamous cell carcinoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (e.g., gastrointestinal cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, and hepatic This includes, but is not limited to, carcinoma, anal cancer, penile cancer, primary or metastatic malignant melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, brain cancer (e.g., high-grade glioma, diffuse pontine glioma, ependymoma, neuroblastoma, or glioblastoma), and head and neck cancers and associated metastases.Further 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) (digital online version on the 2018 Merck Manual website); and the SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.

[0329] 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, ovarian, uterine serous endometrial carcinoma, colon, bladder, lung, cervix, head and neck, sarcoma, bone tumor, or esophageal cancer.

[0330] 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 cancer, prostate cancer, bladder cancer, skin cancer, lung cancer, ovarian cancer, sarcoma, bone tumor, or brain cancer.

[0331] In some embodiments, the cancer treated by the method of the present invention is a B7-H3 positive cancer. In some embodiments, B7-H3 positive cancers include osteosarcoma, rhabdomyosarcoma, Ewing sarcoma and other Ewing sarcoma family tumors, neuroblastoma, ganglioblastoma, fibroplastic round cell tumor, malignant peripheral nerve sheath tumor, synovial sarcoma, undifferentiated sarcoma, adrenocortical carcinoma, hepatoblastoma, Wilms' tumor, rhabdoid tumor, and high-grade glioma (glioblastoma multiforme). multiforme)) may be, but is not limited to, medulloblastoma, astrocytoma, glioma, ependymoma, atypical teratomatous rhabdoid tumor, meningioma, craniopharyngioma, primitive neuroectodermal tumor, diffuse pontine glioma and other brain tumors, acute myeloid leukemia, multiple myeloma, lung cancer, mesothelioma, breast cancer, bladder cancer, stomach 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 cancer, leiomyosarcoma, or malignant melanoma.

[0332] In some embodiments, a therapeutic method of the present disclosure may comprise one or more of the following steps: a) isolating immune cells (e.g., T cells or NK cells) from a subject or donor; b) modifying the immune cells (e.g., T cells or NK cells) ex vivo with a polynucleotide or recombinant vector encoding a chimeric receptor as described herein; c) optionally modifying the immune cells (e.g., T cells or NK cells) ex vivo to express a chimeric antigen receptor (CAR), an antigen-specific T cell receptor (TCR) and / or an antibody or a fragment or derivative thereof, wherein the CAR, TCR or antibody is capable of specifically binding to an antigen associated with the disease; d) optionally expanding and / or activating the modified immune cells (e.g., T cells or NK cells) before, after, and / or between steps 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 upon activation. In some aspects, immune cells are αβTCR T cells, γδT cells, or iNKT cells.

[0333] In some embodiments, one or more antigen-recognizing 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 engagers, native or transgenic T cell receptors, and antibodies, or combinations thereof.

[0334] In some embodiments, a therapeutic method of the present disclosure may comprise one or more of the following steps: a) isolating T cells or NK cells from a subject or donor; b) modifying the T cells or NK cells ex vivo with a polynucleotide encoding a chimeric receptor as described herein or a recombinant vector encoding the polynucleotide; c) optionally modifying the T cells or NK cells ex vivo 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 between steps b) or c); and e) introducing a therapeutically effective amount of the modified T cells or NK cells into a subject.

[0335] In some embodiments, the modified host cells are autologous cells. In some embodiments, the modified host cells are allogeneic cells. If the host cells are isolated from a donor, the method may further include a method for preventing graft-versus-host disease (GVHD) and host cell rejection.

[0336] In some embodiments, the modified host cells may also express the CD20 polypeptide as a safety switch. Therefore, the method may further include administering an anti-CD20 antibody to the target to remove the isolated host cells. The anti-CD20 antibody is administered in an amount effective for sufficient removal of the isolated host cells from the target. In some embodiments, the anti-CD20 antibody is administered in an amount effective for removing more than 50% of the isolated host cells from the target. 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 the isolated host cells from the target. 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 the isolated host cells from the target.

[0337] Non-limiting examples of anti-CD20 antibodies that can be used to remove isolated host cells include Rituximab, Ibritumomab tiuxetan, Tositumomab, Ofatumumab, Ocrelizumab, TRU-015, Veltuzumab, AME-133v, PRO131921, and Obinutuzumab. In some embodiments, the anti-CD20 antibody is Rituximab.

[0338] In some embodiments of any of the above treatment methods, the composition is administered in a therapeutically effective amount. The dosage of the composition administered in the methods of the present invention can vary widely depending on the subject's physical parameters, frequency of administration, mode of administration, clearance rate, etc. The initial dosage may be higher, and a lower maintenance dosage may follow. The dosage may be administered at a low frequency, such as weekly or bi-weekly, or divided into lower dosages and administered daily, twice a week, etc., in order to maintain an effective dosage level. Various dosages are believed to be effective in achieving the in vivo persistence of the modified host cells. It is also contemplated that various dosages are effective in improving the effector function of the modified host cells in vivo.

[0339] In some embodiments, a composition comprising modified host cells produced by the methods described herein is 10 2 ~10 10 cells / kg body weight, 10 5 ~10 9 cells / kg body weight, 10 5 ~10 8 cells / kg body weight, 10 5 ~10 7 cells / kg body weight, 10 7 ~10 9 cells / kg body weight, or 10 7 ~10 8It can be administered in doses of cells / kg body weight (including all integer values ​​within these ranges). The number of modified host cells depends on the intended therapeutic use of the composition.

[0340] Modified host cells can be administered multiple times at the doses listed above. Modified host cells may be allogeneic, syngeneic, heterogeneic, or autologous to the patient receiving treatment.

[0341] The compositions and methods described herein may be used in conjunction with other types of treatment for tumors, such as chemotherapy, surgery, radiation, and gene therapy.

[0342] When used to treat a variety of diseases / disorders, the compositions and methods of this disclosure are intended to be used in conjunction with other therapeutic methods / agents suitable for the same or similar diseases / disorders. Such other therapeutic methods / agents may be coadministrated (simultaneously or sequentially) to produce additive or synergistic effects. The appropriate therapeutically effective dose of each agent may be reduced by the additive or synergistic effect.

[0343] In some embodiments of any of the above-described therapeutic methods, the method further comprises administering to a target one or more additional compounds selected from the group consisting of immunosuppressants, biological agents, probiotics, prebiotics, and cytokines (e.g., GM-CSF, IFN, or IL-2).

[0344] In some embodiments, the methods described herein further include providing exogenous GM-CSF in addition to GM-CSF produced by immune cells to enhance the function of immune cells expressing the chimeric receptor of this disclosure. Exogenous GM-CSF may be provided, for example, by i) injection of the FDA-approved GM-CSF drug Sargramostin (Leukine®), or by ii) the use of a non-viral or viral vector for expressing GM-CSF (e.g., the FDA-approved GM-CSF expressing oncolytic virus talimogene laherparepvec [TVEC, Imlygic®]). These drugs may be administered before, concurrently with, or after the administration (e.g., by infusion) of immune cells expressing the chimeric receptor of this disclosure to a patient.

[0345] As a non-limiting example, the compositions of this disclosure may be combined with other therapies that block inflammation (e.g., by blocking IL-1, IFNα / β, IL-6, TNF, IL-23, etc.).

[0346] The methods and compositions of this disclosure can be combined with other immunomodulatory treatments, such as 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 may also be combined with other treatments that have the ability to modulate NKT function or stability, including, but not limited to, unloaded or loaded CD1d, CD1d fusion proteins, CD1d dimers or larger polymers of CD1d, CD1d chimeric antigen receptors (CD1d-CAR), or any other of the five known CD1 isomers present in humans (CD1A, CD1B, CD1c, CD1e). The methods of the present invention can also be combined with other treatments, such as midostaurin or enasidenib, or a combination thereof.

[0347] The therapeutic methods disclosed herein may be combined with additional immunotherapies and therapies. For example, when used to treat tumors, the compositions of the present invention may be used in combination with conventional therapies, such as surgery, radiotherapy, chemotherapy, or a combination thereof, depending on the type of tumor, the patient's condition, other health problems, and various other factors. In certain embodiments, other therapeutic agents useful in combination tumor therapy with the inhibitors of the present invention include anti-angiogenic agents. Many angiogenesis inhibitors have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, thrombospondin-1, tissue metalloprotease inhibitors (TIMP1 and TIMP2), prolactin (16Kd fragment), angiostatin (38Kd fragment of plasminogen), endostatin, bFGF-soluble receptor, transforming growth factor β, interferon α, soluble KDR and FLT-1 receptors, placental proliferin-related protein, and those listed in Carmeliet and Jain (2000). In one embodiment, the modified host cells of the present invention can be used in combination with VEGF antagonists or VEGF receptor antagonists, such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, anti-VEGFR neutralizing antibodies, VEGFR tyrosine kinase inhibitors, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab, or ranibizumab).

[0348] Non-limiting examples of chemotherapeutic compounds that may be used in the combination therapies of this disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, BCG, bicalutamide, bleomycin, buserelin, busulfan, campotecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, and cyprotein. Lon, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, Goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ileotecan, letrozole, leucovorin, leuprolide, levamisol, lomustine, mechloretamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxy Examples include saliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, larcitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocenedic chloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0349] These chemotherapeutic compounds can be classified into the following groups, for example, based on their mechanism of action: antimetabolites / antinomatal agents, e.g., pyrimidine analogs (5-fluorouracil, phloxuridine, capecitabine, gemcitabine, and cytarabine) as well as purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, e.g., bin Natural products such as alkaloids (vinblastine, vincristine, and vinorelbine), taxanes (paclitaxel, docetaxel), microtubule disruptors such as vincristine, vinblastine, nocodazole, epothiron, and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrin, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambushi) Cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyneneamine, oxaliplatin, ifosfamide, melphalan, mechloretamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide Includes etoposide (VP16); antibiotics, such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mitramycin), and mitomycin; enzymes (L-asparaginase, which systematically metabolizes L-asparagine and depletes cells that lack the ability to synthesize asparagine itself); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, e.g., nitrogen mustard (mechloretamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosourea (carmustine (BCNU) and analogs, streptozosin), trazene-dacarbadinine (DTIC), etc.; antiproliferative / antimitotic antimetabolites, e.g., folic acid analogs (methotrexate), etc.; platinum complexes (cisplatin, carboplatin) ), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytic agents (tissue plasminogen activator, streptokinase, and urokinase, etc.), aspirin, dipyridamole, ticlopidine, clopidogrel, absiximab; antimotaxis agents; Antisecretorts (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); 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 (tretinopropyl alcohol). Ingredients; mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrin, camptothecin, daunorubicin, dactinomycin, geniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, and caspase activators;Also, a chromatin-destroying agent.

[0350] In various embodiments of the methods described herein, the subjects are human beings. The subjects may be young or adult, having any age or sex.

[0351] According to the present invention, there may be numerous tools and techniques within the scope of the art, such as those commonly used in molecular biology, pharmacology, and microbiology. Such tools and techniques are available, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Current and Protocols in Protein Science, John Wiley and Details are described in Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. [Examples]

[0352] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, and not limit, the embodiments disclosed.

[0353] Example 1. Jun.MyD88 homodimer activates NFκB signaling and improves the antitumor activity of CAR T cells. Homodimerous IL-18 ZipR constructs were created using either an IL-18Rα chain or an IL-18Rβ chain conjugated to a human cJun leucine zipper. These constructs were able to activate NFκB signaling in NFκB reporter cells only when expressed together, not as homodimer molecules (Figure 1A). IL-18R signaling involves MyD88 aggregation to initiate downstream signal activation; therefore, human MyD88 was then fused to cJun via a CD28 transmembrane (TM) domain (Jun.CD28TM.MyD88), an IL-18Rα chain TM domain (Jun.18RαTM.MyD88), or an IL-18Rβ chain TM domain (Jun.18RβTM.MyD88). All three constructs were 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 and proliferate A673 tumor cells (Figures 1C-1D).

[0354] Example 2. Jun leucine zipper-based homodimers activate STAT5, resulting in improved T cell viability, and are inhibited by ruxolitinib. The following constructs were created and tested: i) Jun.IL-2Rβ chain constructs with 0-6 alanine insertions to examine all potential intracellular orientations; ii) Jun.EpoR constructs with 0, 1, 3, or 5 amino acid TM domain deletions to examine receptors with 0, 100, 300, or 500 degree rotations; iii) Jun. GHR; and iv) Jun.18β.MyD88 (Jun.MyD88.EpoR or Jun.MyD88.GHR) fused with EpoR or GHR.

[0355] The STAT5 signaling pathway improves the functionality of CAR T cells. When expressed in human T cells, all Jun.EpoR ZipR and Jun.GHR ZipR were able to phosphorylate (activate) STAT5 (Figure 2A). In contrast, the homodimeric Jun.IL-2Rβ ZipR and Jun.MyD88.EpoR or Jun.MyD88.GHR ZipR were unable to do so (Figure 2B).

[0356] T cells expressing Jun.EpoR ZipR or Jun.GHR ZipR were treated with increasing concentrations of ruxolitinib, an FDA-approved JAK1 / 2 inhibitor, as potential safety switches for constitutively active homodimeric ZipRs. All ZipRs were inhibited by ruxolitinib, as determined by their ability to phosphorylate STAT5 (Figures 2C-2E). The semi-maximal effective concentrations (EC50) of ruxolitinib for each ZipR ranged from 6.3 to 668.3 nM (Table 4), indicating that each ZipR has a different signaling intensity, enabling regulated STAT5 signaling in T cells.

[0357] In addition to STAT5 activation, we determined the ability of Jun ZipR to improve T cell function. 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, dead, and dying cells were quantified. ZipR signaling allowed cells to survive 7 days of cytokine starvation, which was abolished by co-treatment with ruxolitinib (Figure 2F). [Table 4]

[0358] Example 3. Jun homodimer ZipR does not alter the immunophenotype of human T cells. Finally, we evaluated the effects of ZipR signaling on T cell phenotype and differentiation. ZipR was expressed in human T cells (Figure 3A), but it did not alter the frequency of CD4+ or CD8+ T cells (Figure 3B) or their immunophenotype (Figure 3C).

[0359] In summary, the data provided in Figures 1–3 demonstrate that the constructed EpoR (ZipEpoR) and GHR (ZipGHR)-based ZipR activate STAT5 in reporter cell lines and primary human T cells.

[0360] The following is the method used in the above example.

[0361] Construction of viral vectors. The construction of RD114-pseudotyped gamma-retroviral vectors (pSFGs) encoding the second-generation EphA2-specific CAR (4H5.CD28z) has been previously described [29,30]. pSFG vectors encoding Jun.IL2Rβ ZipR, Jun.EpoR ZipR, Jun.GHR ZipR, Jun.IL18R ZipR, and Jun.MyD88 ZipR were constructed by synthesizing gene fragments (Thermo Fisher) via in-fusion cloning (Takara). These consist of (i)(ii) human cJun (amino acids 277-315) ligated to the intracellular domain of the cytokine receptor chain shown, (iii) a P2A sequence, and (iv) mClover for transduction detection. The sequence of the final construct was confirmed by sequencing (Hartwell Center, St. Jude Children's Research Hospital). RD114 pseudotyped retroviral particles were generated by transient transfection of 293T cells, as previously described. 4 The supernatant was collected after 48 hours, filtered, and rapidly frozen.

[0362] EphA2-CAR T cell generation. Human peripheral blood mononuclear cells (PBMCs) were isolated from an unidentified elution chamber of leukocyte apheresis products obtained from the St. Jude Donor Center using Lymphoprep (Abbott Laboratories). On day 0, PBMCs were stimulated overnight on 24-well non-tissue culture treated plates pre-coated with CD3 and CD28 antibodies (Miltenyi). On day 1, cytokines IL-7 (10 ng / mL) and IL-15 (5 ng / mL) (PeproTech, Rocky Hill, New Jersey, USA) were added to the cultures. On day 2, T cells were transferred to retronectin-coated (Clontech) plates containing retroviral particles and maintained for 2–4 days. On day 3, T cells were transferred to new 24-well tissue culture treated plates and subsequently expanded with IL-7 and IL-15. All experiments were performed 7–14 days after transduction. Biological replication was performed using PBMCs derived from different healthy donors.

[0363] Preparation of ZipR-transduced T cells. To transduce EphA2-CAR and ZipR into T cells, transduction was performed as described in the "Preparation of EphA2-CAR T cells" section, but a mixture of EphA2-CAR gamma-retrovirus and ZipR gamma-retrovirus vector was used. To transduce T cells with 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 the initial stimulation and maintained in culture until functional studies were performed. The cells were then seeded on retronectin-coated (Clontech) plates containing retroviral particles and maintained for 2–4 days. The T cells were transferred to new 24-well tissue culture-treated plates and subsequently expanded with IL-7 and IL-15. Non-transduced T cells were prepared similarly, except that retroviruses were not included in the retronectin wells. All experiments were performed 7–14 days after transduction. Non-transduced (NT) T cells were prepared similarly, except that retroviruses were not included in the retronectin wells. Biological replication was performed using PBMCs derived from different healthy donors.

[0364] Cell line A673 (Ewing's sarcoma) was purchased from the American Type Culture Collection (ATCC). The cell line was certified by the ATCC Human Short Tandem Repeat Profiling Cell Certification Service and routinely checked for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza). After thawing, the cell line was cultured for up to 3 months, after which a new reference vial was thawed. The cell line was maintained and expanded in Dulbecco's 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).

[0365] Flow cytometry. Flow cytometry data was 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 in the solution. For all experiments, matched isotypes or known negatives (e.g., NT T cells) were used as gating controls. CAR detection was performed using F(ab')2 fragment-specific antibody (polyclonal, Jackson Immuno Research, West Grove, Pennsylvania, USA). T cells were stained with fluorescent dye conjugated antibodies using the following marker combinations: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone 2-L1-A, BD Biosciences), and CD45RO (clone HI100, BD Biosciences).

[0366] Intracellular staining. Intracellular staining to detect pSTAT5 was performed using BD Phosflow Protocol III. Briefly, T cells were either left unstimulated or stimulated with 5 ng / mL IL-15 for 15 minutes, then lysed / fixed with 1×Lyse / Fix Buffer (BD Biosciences) at 37°C for 12 minutes. The cells were washed with PBS + 1% FBS, then permeabilized with pre-cooled Perm Buffer III (BD Biosciences). After washing the cells three times, they were stained with an antibody against pSTAT5 (clone 47, BD Biosciences).

[0367] Cytokine starvation. To measure cell viability after 7 days of cytokine withdrawal, 1 × 10⁻⁶ cells were used. 6The cells were seeded in 1 mL of complete RPMI without cytokines in a tissue culture-treated 48-well plate. After 7 days, the cells were harvested and stained for flow cytometry. To quantify apoptosis, the cells were labeled with Annexin V (BD Biosciences) and eBioscience Fixable Viability Dye (Invitrogen).

[0368] Repetitive stimulation assay. 1 x 10 6 T cells were placed in a 24-well tissue culture treatment plate, 5 × 10⁶ 5 The tumor cells were co-cultured in complete RPMI. Fresh complete RPMI was supplied to the cells, and they were divided as needed. After 7 days, the T cells were harvested, counted, and re-seeded in the same ratio as the fresh tumor cells only if they had killed the tumor cells, as determined by microscopic examination.

[0369] Example 4. Modification of homochimeric Jun.GHR ZipR for further activation of signaling pathways. Figures 2A-2B of this disclosure (see also Example 2 above) demonstrate that the homochimeric Jun.GHR ZipR is functional as determined by STAT5 phosphorylation. To demonstrate that this receptor can be modified to activate further signaling pathways, a novel molecule, Jun.GHR.MyD88 ZipR, was created by fusing the MyD88 signaling domain to Jun.GHR ZipR. The ability of this ZipR (Jun.GHR.MyD88) to activate the STAT5 and MyD88 signaling pathways was evaluated using reporter cell lines. Figure 4A shows that the STAT5 reporter cell line was successfully transduced with retroviral constructs encoding Jun.GHR and Jun.GHR.MyD88. 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 shows that Jun.GHR and Jun.GHR.MyD88 phosphorylate STAT5. Therefore, the addition of the MyD88 domain to Jun.GHR does not interfere with the GHR domain's ability to phosphorylate STAT5.

[0370] To evaluate the functionality of the MyD88 domain of Jun.GHR.MyD88 ZipR, Jun.GHR and Jun.GHR.MyD88 were transduced into Ramos Blue MyD88 signaling reporter cell lines (Figure 5A). Cells transduced with Jun.MyD88 ZipR were used as positive controls. Figure 5B shows that Jun.GHR.MyD88 activates MyD88 signaling in contrast to Jun.GHR. Thus, this embodiment demonstrates for the first time that it is possible to construct homodimeric ZipRs that activate the STAT5 and MyD88 signaling pathways. This approach is applicable to EpoR-based homodimeric ZipRs (Figure 2A), which are also described herein. References

[0371] 1. June CH, Sadelain M. Chimeric Antigen Receptor Therapy. New Engl J Med (2018) 379(1):64-73. doi: 10.1056 / NEJMra1706169. 2. Srivastava S, Riddell SR. Engineering CAR-T Cells: Design Concepts. Trends Immunol (2015) 36(8):494-502. doi: 10.1016 / j.it.2015.06.004. 3. Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and Development of Therapies Using Chimeric Antigen Receptor-Expressing T Cells. Immunol Rev (2014) 257(1):107-26. doi: 10.1111 / imr.12131. 4. Juillerat, A, Marechal, A, Filhol, JM et al. Design of chimeric antigen receptors with integrated controllable transient functions. Sci Rep 6, 18950 (2016) doi.org / 10.1038 / srep18950 5. Leung, W, Gay, J, Martin, U, A. Astrakhan et al., Sensitive, and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization. JCI Insight. 2019;4(11):e124430. doi.org / 10.1172 / jci.insight.124430. 6. Moll JR, Ruvinov SB, Pastan I, Vinson C. Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M. Protein Sci. 2001 Mar;10(3):649-55. doi: 10.1110 / ps.39401. 7. Reinke AW, Baek J, Ashenberg O, and Keating AE. Networks of bZIP protein-protein interactions diversified over a billion years of evolution. Science. 2013 May 10;340(6133):730-4. doi: 10.1126 / science.1233465. 8. Newman JRS and Keating AE. Comprehensive identification of human bZIP interactions with coiled-coil arrays. Science. 2003 Jun 27;300(5628):2097-101. doi: 10.1126 / science.1084648. Epub 2003 Jun 12. 9. Mata M, Gerken C, Nguyen P, Krenciute G, Spencer DM, Gottschalk S. Inducible Activation of MyD88 and CD40 in CAR T Cells Results in Controllable and Potent Antitumor Activity in Preclinical Solid Tumor Models. Cancer Discov. 2017 Nov;7(11):1306-1319. doi: 10.1158 / 2159-8290.CD-17-0263. Epub 2017 Aug 11. PMID: 28801306; PMCID: PMC5780189. 10. Katsarou A, Sjoestrand M, Naik J, Mansilla-Soto J, Kefala D, Kladis G, Nianias A, Ruiter R, Poels R, Sarkar I, Patankar YR, Merino E, Reijmers RM, Frerichs KA, Yuan H, de Bruijn J, Stroopinsky D, Avigan D, van de Donk NWCJ, Zweegman S, Mutis T, Sadelain M, Groen RWJ, Themeli M. Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence. Sci Transl Med. 2021 Dec 8;13(623):eabh1962. doi: 10.1126 / scitranslmed.abh1962. Epub 2021 Dec 8. PMID: 34878825; PMCID: PMC9869449. 11. Staerk J, Defour JP, Pecquet C, Leroy E, Antoine-Poirel H, Brett I, Itaya M, Smith SO, Vainchenker W, Constantinescu SN. Orientation-specific signaling by thrombopoietin receptor dimers. EMBO J. 2011 Sep 2;30(21):4398-413. doi: 10.1038 / emboj.2011.315. PMID: 21892137; PMCID: PMC3230373. 12. Kourouniotis G, Wang Y, Pennock S, Chen X, Wang Z. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor. International Journal of Molecular Sciences. 2016; 17(8):1200. doi.org / 10.3390 / ijms17081200. 13. Haifeng Zhang, Puei Nam Tay, Weiping Cao, Wei Li, Jinhua Lu. Integrin-nucleated Toll-like receptor (TLR) dimerization reveals subcellular targeting of TLRs and distinct mechanisms of TLR4 activation and signaling. Febs Letters. 2002, 532(1-2) 171-176. doi.org / 10.1016 / S0014-5793(02)03669-4. 14. Engelowski, E., Schneider, A., Franke, M. et al. Synthetic cytokine receptors transmit biological signals using artificial ligands. Nat Commun 9, 2034 (2018). doi.org / 10.1038 / s41467-018-04454-8. 15. Mossner, S., Kuchner, M., Modares, N., Knebel, B., Al-Hasani, H., Floss, D., Scheller, J. Synthetic interleukin 22 (IL-22) signaling reveals biological activity of homodimeric IL-10 receptor 2 and functional cross-talk with the IL-6 receptor gp130. Journal of Biological Chemistry, Volume 295, Issue 35, 2020, Pages 12378-12397, ISSN 0021-9258, doi.org / 10.1074 / jbc.RA120.013927. 16. Stuhlmann-Laeisz, C., Lang, S., Chalaris, A., Krzysztof, P., Enge, S., Eichler, J., Klingmueller, U., Samuel, M., Ernst, M., Rose-John, S., and Juergen Scheller. Forced Dimerization of gp130 Leads to Constitutive STAT3 Activation, Cytokine-independent Growth, and Blockade of Differentiation of Embryonic Stem Cells. Molecular Biology of the Cell 2006 Vol. 17, No. 7 19 Apr doi.org / 10.1091 / mbc.e05-12-1129. 17. Juillerat A, Tkach F, Busser BW, Temburni S, Valton J, Duclert A, Poirot L, Depil S, and Duchateau P. Modulation of chimeric antigen receptor surface expression by a small molecule switch. BMC Biotechnol. 2019; 19: 44. Published online 2019 Jul 3. doi: 10.1186 / s12896-019-0537-3. 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 provides reversible regulation for chimeric antigen receptor expression and activity. Cell Chemical Biology. Volume 28, Issue 6, 17 June 2021, Pages 802-812.e6 19. Jan M, Scarfo I, Larson RC,Walker A, Schmidts A, Guirguis AA, Gasser JA, Slabicki M, Bouffard AB, Castano AP, Kann MC, Cabral ML, Tepper A, Grinshpun DE, Sperling AS, Kyung T, Sievers QL, Birnbaum ME, Maus MV, Ebert BL. Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide. Sci Transl Med. 2021 Jan 6;13(575):eabb6295. doi: 10.1126 / scitranslmed.abb6295. 20. Monteys AM, Hundley AA, Ranum PT, Tecedor L, Muehlmatt A, Lim E, Lukashev D, Sivasankaran R, Davidson BL. Regulated control of gene therapies by drug-induced splicing. Nature. 2021 Aug;596(7871):291-295. doi: 10.1038 / s41586-021-03770-2. Epub 2021 Jul 28. 21. Zhu H and Kaufman DS. An Improved Method to Produce Clinical-Scale Natural Killer Cells from Human Pluripotent Stem Cells. Methods Mol Biol. 2019;2048:107-119. doi: 10.1007 / 978-1-4939-9728-2_12. 22. Iriguchi S, Yasui Y, Kawai Y, Arima S, Kunitomo M, Sato T, Ueda T, Minagawa A, Mishima Y, Yanagawa N, Baba Y, Miyake Y, Nakayama K, Takiguchi M, Shinohara T, Nakatsura T, Yasukawa M, Kassai Y, Hayashi A & Kaneko S. A clinically applicable and scalable method to regenerate T cells from iPSCs for off-the-shelf T-cell immunotherapy. Nature Communications volume 12, Article number: 430 (2021). 23. Musso T, Calosso L, Zucca M, Millesimo M, Ravarino D, Giovarelli M, et al. Human Monocytes Constitutively Express Membrane-Bound, Biologically Active, and Interferon-Gamma-Upregulated Interleukin-15. Blood (1999) 93(10):3531–9. doi: 10.1182 / blood.V93.10.3531.410k32_3531_3539. [ PMC free article ] [ PubMed ] 24. Trinchieri G. Interleukin-12 and the Regulation of Innate Resistance and Adaptive Immunity. Nat Rev Immunol (2003) 3(2):133–46. doi: 10.1038 / nri1001. * * *

[0372] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0373] All patents, applications, publications, test methods, documents, and other materials cited herein are incorporated herein by reference in their entirety as if they were physically present herein.

[0374] TIFF2026514805000018.tif252170TIFF2026514805000019.tif255163TIFF2026514805000020.tif25 5167TIFF2026514805000021.tif255162TIFF2026514805000022.tif255167TIFF2026514805000023.t if255163TIFF2026514805000024.tif255164TIFF2026514805000025.tif255163TIFF20265148050000 26.tif248170TIFF2026514805000027.tif255163TIFF2026514805000028.tif255170TIFF20265148050 00029.tif255170TIFF2026514805000030.tif255160TIFF2026514805000031.tif255168TIFF2026514 805000032.tif255166TIFF2026514805000033.tif255164TIFF2026514805000034.tif255170TIFF202 6514805000035.tif255166TIFF2026514805000036.tif253170TIFF2026514805000037.tif240170TIF F2026514805000038.tif255170TIFF2026514805000039.tif255163TIFF2026514805000040.tif100170

Images